R tools for MicroRNA pathway analysis

In the early 2000s, microRNAs (miRNAs) were discovered as segments of a new class of highly conserved and small non-coding RNA molecules of 20-25 nucleotides that are transcribed from DNA.
They do not translation into proteins, rather they inhibit protein expression by binding to the 3’untranslated regions (3’ UTRs) of specific mRNA targets (that is/are complementary to them) and guiding their translational repression or complete degradation and gene silencing. With this, miRNAs provide a second level of regulation beyond primary gene expression. Integrative study of cellular pathways is pivotal to understanding the functions of individual genes and proteins in terms of systems and processes that contribute to normal physiology and to disease. "WikiPathways":http://wikipathways.org is an open, collaborative platform dedicated to the curation of biological pathways by and for the scientific community. The collection of pathways is publicly available to the researchers. The miRNA’s predicted by TargetScan in cardiomyocytes hypertrophy pathway has already been visualized on WikiPathways (WP1560). Since more studies investigate miRNAs using microarray technologies it would be desirable to be able to use information about miRNA’s in that analysis. One way to do that is to add the miRNA’s to all pathways. Therefore, we are integrating both validated and predicted miRNA information into biological pathways and making them available in WikiPathways. Initially, we focused on pathways related to the heart because miRNAs created a true revolution in the cardiovascular research field. The validated miRNAs have been downloaded from miRNA databases such as TarBase or miRTarbase. In order to link the validated miRNA targets to the genes in the pathways of our interest, we use "BridgeDb":http://www.bridgedb.org for identifier mapping. BridgeDb is a middleware between the relational databases, files and mapping services. BridgeDb is available in two forms. The first is a framework suitable for integration in Java applications. The other is based on Representational State Transfer (REST) webservices and is suitable for all other programming languages. The identifier mapping has been done in the R statistical environment as the connected Bioconductor repository has many pre-existing packages for microarray data analysis. For now we used the REST interface from R but we will also submit BridgeDb R package to Bioconductor.
Predicted miRNA targets by different prediction algorithms were verified by co evaluating miRNA and mRNA expression using microarray analysis. Quality control and normalization of the microarray datasets was done using the current functionality of the arrayanalysis.org web portal. Statistical analysis was done using Limma and the miRNAs were visualized in the pathways of interest using "PathVisio":http://www.pathvisio.org. Modules for statistical and pathway analysis have been developed which will be added to the "arrayanalysis.org":http://www.arrayanalysis.org portal. This also required connecting R to PathVisio, for which a new XMLRPC interface was developed. Through this PathVisio can be controlled by R scripts.
In conclusion, these R tools can help to integrate information about miRNAs with other knowledge about biological pathways and used for research purposes

Fine needle aspiration cytology diagnosis of caesarean scar endometriosis with histopathological correlation: a case report

Endometriosis is defined as the presence of endometrial tissue outside the endometrium or myometrium. It occurs in 8-15% of women of reproductive age group with abdominal wall endometriosis accounting for only 0.5-1% of all pelvic endometriosis. We present a case of a 25 year old lady who presented with a mass in the anterior abdominal wall over a previous caesarean section scar. Endometriosis was diagnosed on Fine Needle Aspiration Cytology (FNAC) based on the findings of monolayered sheets of endometrial cells, few spindle shaped stromal cells and hemosiderin laden macrophages in the background. Histopathological examination confirmed the cytological diagnosis. Endometriosis in scar tissue is a rare disease which might be difficult to diagnose and should always be considered in evaluation of painful abdominal masses in women. Fine needle aspiration cytology is economical, fast and accurate method to make the diagnosis of scar endometriosis and to plan better surgical approach

MiR-4521 perturbs FOXM1-mediated DNA damage response in breast cancer

Introduction: Forkhead (FOX) transcription factors are involved in cell cycle control, cellular differentiation, maintenance of tissues, and aging. Mutation or aberrant expression of FOX proteins is associated with developmental disorders and cancers. FOXM1, an oncogenic transcription factor, is a promoter of cell proliferation and accelerated development of breast adenocarcinomas, squamous carcinoma of the head, neck, and cervix, and nasopharyngeal carcinoma. High FOXM1 expression is correlated with chemoresistance in patients treated with doxorubicin and Epirubicin by enhancing the DNA repair in breast cancer cells.Method: miRNA-seq identified downregulation of miR-4521 in breast cancer cell lines. Stable miR-4521 overexpressing breast cancer cell lines (MCF-7, MDA-MB-468) were developed to identify miR-4521 target gene and function in breast cancer.Results: Here, we showed that FOXM1 is a direct target of miR-4521 in breast cancer. Overexpression of miR-4521 significantly downregulated FOXM1 expression in breast cancer cells. FOXM1 regulates cell cycle progression and DNA damage response in breast cancer. We showed that miR-4521 expression leads to increased ROS levels and DNA damage in breast cancer cells. FOXM1 plays a critical role in ROS scavenging and promotes stemness which contributes to drug resistance in breast cancer. We observed that breast cancer cells stably expressing miR-4521 lead to cell cycle arrest, impaired FOXM1 mediated DNA damage response leading to increased cell death in breast cancer cells. Additionally, miR-4521-mediated FOXM1 downregulation perturbs cell proliferation, invasion, cell cycle progression, and epithelial-to-mesenchymal progression (EMT) in breast cancer.Discussion: High FOXM1 expression has been associated with radio and chemoresistance contributing to poor patient survival in multiple cancers, including breast cancer. Our study showed that FOXM1 mediated DNA damage response could be targeted using miR-4521 mimics as a novel therapeutic for breast cancer

Mitochondrial DNA variation analysis in cervical cancer

This study was undertaken to investigate the mitochondrial DNA (mtDNA) variation in non-malignant and malignant cervical tissue samples. We have identified 229 and 739 variations non-malignant and malignant tissues respectively distributed over 321 locations in the D-loop (50 in non-malignant and 166 in malignant; 216 variations), coding region (139 in non-malignant and 455 in malignant; 594 variations) tRNA and rRNA genes (39 in non-malignant and 119 in malignant; 158 variations). Besides, 77 novel and 34 various other disease associated variations were identified in non-malignant and malignant samples. A total of 236 tumor specific variations in 201 locations representing 30.1% in D-loop, 59.3% in coding regions and 10.6% in RNA genes were also identified. Our study shows that D loop (in 67 locations) is highly altered followed by ND5 (35 locations) region. Moreover, mtDNA alterations were significantly higher in malignant samples by two tailed Fisher's exact test (P ≤ 0.05) with decreased mtDNA copy numbers. Bioinformatic analysis of 59 non-synonymous changes predicted several variations as damaging leading to decreased stability of the proteins. Taken together, mtDNA is highly altered in cervical cancer and functional studies are needed to be investigated to understand the consequence of these variations in cervical carcinogenesis and their potential application as biomarkers

DataSheet1_MiR-4521 perturbs FOXM1-mediated DNA damage response in breast cancer.docx

Introduction: Forkhead (FOX) transcription factors are involved in cell cycle control, cellular differentiation, maintenance of tissues, and aging. Mutation or aberrant expression of FOX proteins is associated with developmental disorders and cancers. FOXM1, an oncogenic transcription factor, is a promoter of cell proliferation and accelerated development of breast adenocarcinomas, squamous carcinoma of the head, neck, and cervix, and nasopharyngeal carcinoma. High FOXM1 expression is correlated with chemoresistance in patients treated with doxorubicin and Epirubicin by enhancing the DNA repair in breast cancer cells.Method: miRNA-seq identified downregulation of miR-4521 in breast cancer cell lines. Stable miR-4521 overexpressing breast cancer cell lines (MCF-7, MDA-MB-468) were developed to identify miR-4521 target gene and function in breast cancer.Results: Here, we showed that FOXM1 is a direct target of miR-4521 in breast cancer. Overexpression of miR-4521 significantly downregulated FOXM1 expression in breast cancer cells. FOXM1 regulates cell cycle progression and DNA damage response in breast cancer. We showed that miR-4521 expression leads to increased ROS levels and DNA damage in breast cancer cells. FOXM1 plays a critical role in ROS scavenging and promotes stemness which contributes to drug resistance in breast cancer. We observed that breast cancer cells stably expressing miR-4521 lead to cell cycle arrest, impaired FOXM1 mediated DNA damage response leading to increased cell death in breast cancer cells. Additionally, miR-4521-mediated FOXM1 downregulation perturbs cell proliferation, invasion, cell cycle progression, and epithelial-to-mesenchymal progression (EMT) in breast cancer.Discussion: High FOXM1 expression has been associated with radio and chemoresistance contributing to poor patient survival in multiple cancers, including breast cancer. Our study showed that FOXM1 mediated DNA damage response could be targeted using miR-4521 mimics as a novel therapeutic for breast cancer.</p

Hypoxia increases mutational load of breast cancer cells through frameshift mutations

Tumor hypoxia-induced downregulation of DNA repair pathways and enhanced replication stress are potential sources of genomic instability. A plethora of genetic changes such as point mutations, large deletions and duplications, microsatellite and chromosomal instability have been discovered in cells under hypoxic stress. However, the influence of hypoxia on the mutational burden of the genome is not fully understood. Here, we attempted to elucidate the DNA damage response and repair patterns under different types of hypoxic stress. In addition, we examined the pattern of mutations exclusively induced under chronic and intermittent hypoxic conditions in two breast cancer cell lines using exome sequencing. Our data indicated that hypoxic stress resulted in transcriptional downregulation of DNA repair genes which can impact the DNA repair induced during anoxic as well as reoxygenated conditions. In addition, our findings demonstrate that hypoxic conditions increased the mutational burden, characterized by an increase in frameshift insertions and deletions. The somatic mutations were random and non-recurring, as huge variations within the technical duplicates were recognized. Hypoxia also resulted in an increase in the formation of potential neoantigens in both cell lines. More importantly, these data indicate that hypoxic stress mitigates DNA damage repair pathways and causes an increase in the mutational burden of tumor cells, thereby interfering with hypoxic cancer cell immunogenicity

A Microfluidic Cancer-on-Chip Platform Predicts Drug Response Using Organotypic Tumor Slice Culture

Optimal treatment of cancer requires diagnostic methods to facilitate therapy choice and prevent ineffective treatments. Direct assessment of therapy response in viable tumor specimens could fill this diagnostic gap. Therefore, we designed a microfluidic platform for assessment of patient treatment response using tumor tissue slices under precisely controlled growth conditions. The optimized Cancer-on-Chip (CoC) platform maintained viability and sustained proliferation of breast and prostate tumor slices for 7 days. No major changes in tissue morphology or gene expression patterns were observed within this time frame, suggesting that the CoC system provides a reliable and effective way to probe intrinsic chemotherapeutic sensitivity of tumors. The customized CoC platform accurately predicted cisplatin and apalutamide treatment response in breast and prostate tumor xenograft models, respectively. The culture period for breast cancer could be extended up to 14 days without major changes in tissue morphology and viability. These culture characteristics enable assessment of treatment outcomes and open possibilities for detailed mechanistic studies. SIGNIFICANCE: The Cancer-on-Chip platform with a 6-well plate design incorporating silicon-based microfluidics can enable optimal patient-specific treatment strategies through parallel culture of multiple tumor slices and diagnostic assays using primary tumor material.Electronic Components, Technology and Material

Immunophenotyping of normal individuals classified on the basis of human dosha prakriti

Background:Human variations related to immune response and disease susceptibility is well-documented in Ayurveda. Prakriti (body constitution) is the basic constitution of an individual established at the time of birth and distinguishes variations, into three broad phenotype categories such as vata, pitta and kapha. Variation in immune response is often attributed to and measured from the difference in cluster differentiation (CD) markers expressed in lymphocytes. Currently, there are no reports available on the expression of CD markers related to prakriti. Objective: This is a pilot study performed to evaluate a panel of lymphocyte subset CD markers in dominant prakriti individuals. Materials and Methods: Immunophenotyping was carried out using whole blood from a total of healthy 222 subjects, who are grouped into kapha (n = 95), pitta (n = 57) and vata (n = 70) prakritis. CD markers such as CD3, CD4, CD8, CD14, CD25, CD56, CD69, CD71 and HLA-DR were analyzed using flow cytometry method. Differences between groups were analyzed using one-way ANOVA or Kruskal-Wallis analysis of variance (ANOVA) and multiple comparisons between groups were performed by Bonferroni or Mann-Whitney U test with corrections for type I error respectively. Significance was evaluated by ANOVA and Pearson′s correlation. Results: We observed a significant difference (P < 0.05) in the expression of CD markers such as CD14 (monocytes), CD25 (activated B cells) and CD56 (Natural killer cells) between different prakriti groups. CD25 and CD56 expression was significantly higher in kapha prakriti samples than other prakriti groups. Similarly, slightly higher levels of CD14 were observed in pitta prakriti samples. Conclusion: Significant difference in the expression of CD14, CD25 and CD56 markers between three different prakriti is demonstrated. The increased level of CD25 and CD56 in kapha prakriti may indicate ability to elicit better immune response, which is in conformity with textual references in Ayurveda

Giant electrostriction-like response from defective non-ferroelectric epitaxial BaTiO3 integrated on Si (100)

Abstract Lead-free, silicon compatible materials showing large electromechanical responses comparable to, or better than conventional relaxor ferroelectrics, are desirable for various nanoelectromechanical devices and applications. Defect-engineered electrostriction has recently been gaining popularity to obtain enhanced electromechanical responses at sub 100 Hz frequencies. Here, we report record values of electrostrictive strain coefficients (M 31 ) at frequencies as large as 5 kHz (1.04×10−14 m2/V2 at 1 kHz, and 3.87×10−15 m2/V2 at 5 kHz) using A-site and oxygen-deficient barium titanate thin-films, epitaxially integrated onto Si. The effect is robust and retained upon cycling upto 6 million times. Our perovskite films are non-ferroelectric, exhibit a different symmetry compared to stoichiometric BaTiO3 and are characterized by twin boundaries and nano polar-like regions. We show that the dielectric relaxation arising from the defect-induced features correlates well with the observed giant electrostriction-like response. These films show large coefficient of thermal expansion (2.36 × 10−5/K), which along with the giant M 31 implies a considerable increase in the lattice anharmonicity induced by the defects. Our work provides a crucial step forward towards formulating guidelines to engineer large electromechanical responses even at higher frequencies in lead-free thin films