Bushra Gull

MicroRNAs (miRNAs) are small RNA molecules implicated in controlling most cellular processes by either deadenylating/degrading mRNA transcripts or inhibiti

Proteomics research has increasingly focused on human cells, tissues, and fluids; however, comprehensive data on dental tissues remain limited. Dentine, a mineralized component of teeth, contains structural proteins and bioactive molecules that can modulate pulp cell activity and support regeneration when released. Understanding its protein composition is therefore essential. Previous studies have identified relatively few dentine proteins, and technical challenges have hindered reproducibility. Traditional extraction methods also rely on strong acids that lack clinical relevance. In this technical note, we introduce a workflow combining EDTA-based dentine extraction under clinically relevant conditions with peptide-level fractionation using high-pH reversed-phase chromatography. This approach was compared with unfractionated samples, SDS-PAGE protein-level fractionation, and strong cation exchange (SCX) peptide-level fractionation, all followed by LC–MS/MS. Data are available via ProteomeXchange (PXD070849). This workflow enabled the identification of 514 proteins compared with 238 (unfractionated), 428 (SDS-PAGE), and 193 (SCX). High-pH reversed-phase chromatography contributed 217 unique identifications, exceeding those from other techniques. Although used in other proteomic systems, this methodology has not previously been applied to dentine matrix extracts and represents a promising approach for improving protein discovery.

This study aimed to identify miRNA-based biomarkers in a multi-ethnic cohort of SARS-CoV-2-infected individuals to enhance preparedness for future variants of concern. A total of 31 healthy controls and 154 infected patients were enrolled, from which 13 matched controls and 38 infected nasal swab samples were analyzed using miRNA sequencing, followed by qRT-PCR validation. Among the 1788 miRNAs detected, 14 differentially expressed miRNAs and four novel miRNAs were identified, with novel-miR-264-5p showing a ≥ 2-fold change. Correlation with clinical markers highlighted several miRNAs as potential prognostic biomarkers. Seven miRNAs, including miR-146b-3p, miR-154-5p, miR-5010-3p, miR-127-3p, miR-335-3p, miR-30c-5p, and miR-202-5p, showed strong prognostic potential. Combined ROC analysis demonstrated that a panel of top-performing miRNAs significantly enhanced diagnostic accuracy (AUC 0.939–0.972; p < 0.0001). Moreover, integrating miRNA biomarkers with clinical parameters further improved performance (AUC = 0.982; p < 0.0001). miR-146b-3p, detected exclusively in infected patients, emerged as a highly specific biomarker. Several nasal miRNAs mirrored blood profiles, highlighting the utility of nasal swabs for non-invasive monitoring. Collectively, these findings suggest that miRNA-based biomarkers, alone or combined with clinical markers, offer a promising platform for COVID-19 prognosis and diagnosis, and lay groundwork for future miRNA-based antiviral strategies.

The intricate interplay between viruses and hosts involves microRNAs (miRNAs) to regulate gene expression by targeting cellular/viral messenger RNAs (mRNAs). Mouse mammary tumour virus (MMTV), the aetiological agent of breast cancer and leukaemia/lymphomas in mice, provides an ideal model to explore how viral and host miRNAs interact to modulate virus replication and tumorigenesis. We previously reported dysregulation of host miRNAs in MMTV-infected mammary glands and MMTV-induced tumours, suggesting a direct interaction between MMTV and miRNAs. To explore this further, we systematically examined all potential interactions between host miRNAs and the MMTV genome using advanced prediction tools. Leveraging miRNA sequencing data from MMTV-expressing cells, we identified dysregulated miRNAs capable of targeting MMTV. Docking analysis validated the interaction of three dysregulated miRNAs with the MMTV genome, followed by confirmation with RNA immunoprecipitation assays. We further identified host targets of these miRNAs using mRNA sequencing data from MMTV-expressing cells. These findings should enhance our understanding of how MMTV replicates and interacts with the host to induce cancer in mice, a model important for cancer research. Given MMTV’s potential zoonosis and association with human breast cancer/lymphomas, if confirmed, our work could further lead to novel miRNA-based antivirals/therapeutics to prevent possible MMTV transmission and associated cancers in humans.

The mouse mammary tumor virus (MMTV) induces mammary tumors in mice. Recently, we have shown that MMTV perturbs expression of the host miR-17-92 cluster in MMTV-infected mammary glands and MMTV-induced mammary tumors. Known as oncomiR-1, this cluster is often dysregulated in a number of cancers, especially breast cancer. Therefore, we investigated whether there was a functional interaction between MMTV and the cluster. Our results reveal that MMTV expression led to dysregulation of the cluster in both mammary epithelial HC11 and HEK293T cells with the expression of miR-92a cluster member being affected the most. Conversely, over-expression of the whole or partial cluster significantly repressed MMTV expression. Interestingly, overexpression of miR-92a alone resulted in MMTV repression to the same extent as observed with the whole or partial cluster. Inhibition of miR-92a led to nearly a complete recovery of MMTV gene expression. Deletion/substitution of the miR-92a seed sequence within the cluster rescued MMTV expression. Dual luciferase assays identified MMTV genomic RNA as the target of miR-92a. These results show that the miR-17-92 cluster acts as part of the cell’s well-known miRNA-based anti-viral response to thwart incoming MMTV infection. Interestingly, miR-92a expression was the least amongst cluster members in MMTV-induced breast tumors, suggesting that the elevated level of MMTV expression in tumors is due perhaps to MMTV subverting miR-92a overexpression, restricting its ability to control MMTV replication during tumorigenesis. Thus, our study provides the first evidence demonstrating the biological significance of host miRNAs in controlling MMTV replication and influencing the process of tumorigenesis. Importance: MMTV is a non-acute, slow-transforming retrovirus that causes breast cancer and lymphomas in mice. Consequently, understanding how MMTV interacts with its host can reveal critical aspects of cellular anti-viral responses and the process of tumorigenesis. This study reveals that the host miR-17-92 cluster plays an anti-viral role during MMTV infection which seems to be counteracted by MMTV during tumorigenesis. This observation is significant since MMTV-induced tumors share similarity with human breast cancer and the MMTV/mouse model is the best mammalian animal model to study breast cancer initiation, progression, and therapy. Moreover, MMTV is increasingly (though controversial) being implicated in human breast cancer, leukemias/lymphomas, and biliary cirrhosis due to its potential zoonosis into humans. Thus, if proven true, understanding how miRNAs modulate MMTV replication will be critical in appreciating how hosts miRNAs affect virus replication/tumorigenesis, and towards the development of novel miRNA-based therapeutics (e.g., anti-miRs) to thwart MMTV replication in humans.

Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) is responsible for coronavirus disease of 2019 (COVID‐19) that infected more than 760 million people worldwide with over 6.8 million deaths to date. COVID‐19 is one of the most challenging diseases of our times due to the nature of its spread, its effect on multiple organs, and an inability to predict disease prognosis, ranging from being completely asymptomatic to death. Upon infection, SARS‐CoV‐2 alters the host immune response by changing host‐transcriptional machinery. MicroRNAs (miRNAs) are regarded as post‐transcriptional regulators of gene expression that can be perturbed by invading viruses. Several in vitro and in vivo studies have reported such dysregulation of host miRNA expression upon SARS‐CoV‐2 infection. Some of this could occur as an anti‐viral response of the host to the viral infection. Viruses themselves can counteract that response by mounting their own pro‐viral response that facilitates virus infection, an aspect which may cause pathogenesis. Thus, miRNAs could serve as possible disease biomarkers in infected people. In the current review, we have summarised and analysed the existing data about miRNA dysregulation in patients infected with SARS‐CoV‐2 to determine their concordance between studies, and identified those that could serve as potential biomarkers during infection, disease progression, and death, even in people with other co‐morbidities. Having such biomarkers can be vital in not only predicting COVID‐19 prognosis, but also the development of novel miRNA‐based anti‐virals and therapeutics which can become invaluable in case of the emergence of new viral variants with pandemic potential in the future.

Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes breast cancer in mice. The mouse mammary epithelial cells are the most permissive cells for MMTV, expressing the highest levels of virus upon infection and being the ones later transformed by the virus due to repeated rounds of infection/superinfection and integration, leading eventually to mammary tumors. The aim of this study was to identify genes and molecular pathways dysregulated by MMTV expression in mammary epithelial cells. Towards this end, mRNAseq was performed on normal mouse mammary epithelial cells stably expressing MMTV, and expression of host genes was analyzed compared with cells in its absence. The identified differentially expressed genes (DEGs) were grouped on the basis of gene ontology and relevant molecular pathways. Bioinformatics analysis identified 12 hub genes, of which 4 were up-regulated (Angp2, Ccl2, Icam, and Myc) and 8 were down-regulated (Acta2, Cd34, Col1a1, Col1a2, Cxcl12, Eln, Igf1, and Itgam) upon MMTV expression. Further screening of these DEGs showed their involvement in many diseases, especially in breast cancer progression when compared with available data. Gene Set Enrichment Analysis (GSEA) identified 31 molecular pathways dysregulated upon MMTV expression, amongst which the PI3-AKT-mTOR was observed to be the central pathway down-regulated by MMTV. Many of the DEGs and 6 of the 12 hub genes identified in this study showed expression profile similar to that observed in the PyMT mouse model of breast cancer, especially during tumor progression. Interestingly, a global down-regulation of gene expression was observed, where nearly 74% of the DEGs in HC11 cells were repressed by MMTV expression, an observation similar to what was observed in the PyMT mouse model during tumor progression, from hyperplasia to adenoma to early and late carcinomas. Comparison of our results with the Wnt1 mouse model revealed further insights into how MMTV expression could lead to activation of the Wnt1 pathway independent of insertional mutagenesis. Thus, the key pathways, DEGs, and hub genes identified in this study can provide important clues to elucidate the molecular mechanisms involved in MMTV replication, escape from cellular anti-viral response, and potential to cause cell transformation. These data also validate the use of the MMTV-infected HC11 cells as an important model to study early transcriptional changes that could lead to mammary cell transformation.

Background: The mouse mammary tumor virus (MMTV) Rem protein post translationally regulates nuclear export of unspliced RNAs by interacting with a Rem responsive element (RmRE) located at the 3’ end of the viral genome. Recently, we identified another cis -acting element at the 5’ end of the MMTV genome that facilitates elongation and stability of its gRNA. However, whether this element is Rem-responsive or has any functional interaction with host/viral factors to facilitate MMTV gene expression has not been elucidated. Thus, we investigated role of the 5’ element in transcriptional regulation of MMTV both in the presence and absence of RmRE. Results: Clones containing deletion of either the 5’ element and/or RmRE were tested in cells treated with Actinomycin D, a transcription inhibitor. Quantitative RT-PCRs on RNAs from nuclear and cytoplasmic fractions revealed defects in the efficiency of transcription, transcription elongation and RNA stability in the 5’ element mutants, while nuclear export of RNA was primarily affected in RmRE mutant. Luciferase-based reporter gene assays using wild type and mutant 5’ element revealed that the 5’ element was non-responsive to Rem, but responsive to both the HIV-1 Tat and HTLV-1 Tax as well as a factor from the MMTV genome as well as cellular factor(s). Reciprocally, the MMTV genome could activate the HIV-1 TAR hairpin similar to HTLV-1 Tax. Conclusions: MMTV not only harbors a 5’ cis -acting element similar to HIV-1-TAR/HTLV-1-TRE, but simultaneously contains a virally-encoded Tat/Tax-like factor capable of activating MMTV gene expression, making MMTV a bonafide complex betaretrovirus.

Northern blotting (NB), a gold standard for RNA detection, has lost its charm due to its hands-on nature, need for good quality RNA, and radioactivity. With the emergence of the field of microRNAs (miRNAs), the necessity for sensitive and quantitative NBs has again emerged. Here, we developed highly sensitive yet non-radiolabeled, fast, economical NB, and liquid hybridization (LH) assays without radioactivity or specialized reagents like locked nucleic acid (LNA)- or digoxigenin-labeled probes for mRNAs/small RNAs, especially miRNAs using biotinylated probes. An improvised means of hybridizing oligo probes along with efficient transfer, cross-linking, and signal enhancement techniques was employed. Important caveats of each assay were elaborated upon, especially issues related to probe biotinylation, use of exonuclease, and bioimagers not reported earlier. We demonstrate that, while the NBs were sensitive for mRNAs and small RNAs, our LH protocol could efficiently detect these and miRNAs using less than 10–100 times the total amount of RNA, a sensitivity comparable to radiolabeled probes. Compared to NBs, LH was a faster, more sensitive, and specific approach for mRNA/small RNA/miRNA detection. A comparison of present work with six seminal studies is presented along with detailed protocols for easy reproducibility. Overall, our study provides effective platforms to study large and small RNAs in a sensitive, efficient, and cost-effective manner.