Overexpressed HSF1 cancer signature genes cluster in human chromosome 8q.

BACKGROUND: HSF1 (heat shock factor 1) is a transcription factor that is found to facilitate malignant cancer development and proliferation. In cancer cells, HSF1 mediates a set of genes distinct from heat shock that contributes to malignancy. This set of genes is known as the HSF1 Cancer Signature genes or simply HSF1-CanSig genes. HSF1-CanSig genes function and operate differently than typical cancer-causing genes, yet it is involved in fundamental oncogenic processes. RESULTS: By utilizing expression data from 9241 cancer patients, we identified that human chromosome 8q21-24 is a location hotspot for the most frequently overexpressed HSF1-CanSig genes. Intriguingly, the strength of the HSF1 cancer program correlates with the number of overexpressed HSF1-CanSig genes in 8q, illuminating the essential role of HSF1 in mediating gene expression in different cancers. Chromosome 8q21-24 is found under selective pressure in preserving gene order as it exhibits strong synteny among human, mouse, rat, and bovine, although the biological significance remains unknown. Statistical modeling, hierarchical clustering, and gene ontology-based pathway analyses indicate crosstalk between HSF1-mediated responses and pre-mRNA 3' processing in cancers. CONCLUSIONS: Our results confirm the unique role of chromosome 8q mediated by the master regulator HSF1 in cancer cases. Additionally, this study highlights the connection between cellular processes triggered by HSF1 and pre-mRNA 3' processing in cancers.

A multispecies polyadenylation site model.

BACKGROUND: Polyadenylation is present in all three domains of life, making it the most conserved post-transcriptional process compared with splicing and 5'-capping. Even though most mammalian poly(A) sites contain a highly conserved hexanucleotide in the upstream region and a far less conserved U/GU-rich sequence in the downstream region, there are many exceptions. Furthermore, poly(A) sites in other species, such as plants and invertebrates, exhibit high deviation from this genomic structure, making the construction of a general poly(A) site recognition model challenging. We surveyed nine poly(A) site prediction methods published between 1999 and 2011. All methods exploit the skewed nucleotide profile across the poly(A) sites, and the highly conserved poly(A) signal as the primary features for recognition. These methods typically use a large number of features, which increases the dimensionality of the models to crippling degrees, and typically are not validated against many kinds of genomes. RESULTS: We propose a poly(A) site model that employs minimal features to capture the essence of poly(A) sites, and yet, produces better prediction accuracy across diverse species. Our model consists of three dior-trinucleotide profiles identified through principle component analysis, and the predicted nucleosome occupancy flanking the poly(A) sites. We validated our model using two machine learning methods: logistic regression and linear discriminant analysis. Results show that models achieve 85-92% sensitivity and 85-96% specificity in seven animals and plants. When we applied one model from one species to predict poly(A) sites from other species, the sensitivity scores correlate with phylogenetic distances. CONCLUSIONS: A four-feature model geared towards small motifs was sufficient to accurately learn and predict poly(A) sites across eukaryotes.

In-silico study of antisense oligonucleotide antibiotics.

Background: The rapid emergence of antibiotic-resistant bacteria directly contributes to a wave of untreatable infections. The lack of new drug development is an important driver of this crisis. Most antibiotics today are small molecules that block vital processes in bacteria. To optimize such effects, the three-dimensional structure of targeted bacterial proteins is imperative, although such a task is time-consuming and tedious, impeding the development of antibiotics. The development of RNA-based therapeutics has catalyzed a new platform of antibiotics-antisense oligonucleotides (ASOs). These molecules hybridize with their target mRNAs with high specificity, knocking down or interfering with protein translation. This study aims to develop a bioinformatics pipeline to identify potent ASO targets in essential bacterial genes. Methods: Three bacterial species (P. gingivalis, H. influenzae, and S. aureus) were used to demonstrate the utility of the pipeline. Open reading frames of bacterial essential genes were downloaded from the Database of Essential Genes (DEG). After filtering for specificity and accessibility, ASO candidates were ranked based on their self-hybridization score, predicted melting temperature, and the position on the gene in an operon. Enrichment analysis was conducted on genes associated with putative potent ASOs. Results: A total of 45,628 ASOs were generated from 348 unique essential genes in P. gingivalis. A total of 1,117 of them were considered putative. A total of 27,273 ASOs were generated from 191 unique essential genes in H. influenzae. A total of 847 of them were considered putative. A total of 175,606 ASOs were generated from 346 essential genes in S. aureus. A total of 7,061 of them were considered putative. Critical biological processes associated with these genes include translation, regulation of cell shape, cell division, and peptidoglycan biosynthetic process. Putative ASO targets generated for each bacterial species are publicly available here: https://github.com/EricSHo/AOA. The results demonstrate that our bioinformatics pipeline is useful in identifying unique and accessible ASO targets in bacterial species that post major public health issues.

Electrocardiogram analysis of post-stroke elderly people using one-dimensional convolutional neural network model with gradient-weighted class activation mapping.

Stroke is the second leading cause of death globally after ischemic heart disease, also a risk factor of cardioembolic stroke. Thus, we postulate that heartbeats encapsulate vital signals related to stroke. With the rapid advancement of deep neural networks (DNNs), it emerges as a powerful tool to decipher intriguing heartbeat patterns associated with post-stroke patients. In this study, we propose the use of a one-dimensional convolutional network (1D-CNN) architecture to build a binary classifier that distinguishes electrocardiograms (ECGs) between the post-stroke and the stroke-free. We have built two 1D-CNNs that were used to identify distinct patterns from an openly accessible ECG dataset collected from elderly post-stroke patients. In addition to prediction accuracy, which is the primary focus of existing ECG deep neural network methods, we have utilized Gradient-weighted Class Activation Mapping (GRAD-CAM) to facilitate model interpretation by uncovering subtle ECG patterns captured by our model. Our stroke model has achieved ~90 % accuracy and 0.95 area under the Receiver Operating Characteristic curve. Findings suggest that the core PQRST complex alone is important but not sufficient to differentiate the post-stroke and the stroke-free. In conclusion, we have developed an accurate stroke model using the latest DNN method. Importantly, our work has illustrated an approach to enhance model interpretation, overcoming the black-box issue confronting DNNs, fostering higher user confidence and adoption of DNNs in medicine.

Brain delivering RNA-based therapeutic strategies by targeting mTOR pathway for axon regeneration after central nervous system injury.

Injuries to the central nervous system (CNS) such as stroke, brain, and spinal cord trauma often result in permanent disabilities because adult CNS neurons only exhibit limited axon regeneration. The brain has a surprising intrinsic capability of recovering itself after injury. However, the hostile extrinsic microenvironment significantly hinders axon regeneration. Recent advances have indicated that the inactivation of intrinsic regenerative pathways plays a pivotal role in the failure of most adult CNS neuronal regeneration. Particularly, substantial evidence has convincingly demonstrated that the mechanistic target of rapamycin (mTOR) signaling is one of the most crucial intrinsic regenerative pathways that drive axonal regeneration and sprouting in various CNS injuries. In this review, we will discuss the recent findings and highlight the critical roles of mTOR pathway in axon regeneration in different types of CNS injury. Importantly, we will demonstrate that the reactivation of this regenerative pathway can be achieved by blocking the key mTOR signaling components such as phosphatase and tensin homolog (PTEN). Given that multiple mTOR signaling components are endogenous inhibitory factors of this pathway, we will discuss the promising potential of RNA-based therapeutics which are particularly suitable for this purpose, and the fact that they have attracted substantial attention recently after the success of coronavirus disease 2019 vaccination. To specifically tackle the blood-brain barrier issue, we will review the current technology to deliver these RNA therapeutics into the brain with a focus on nanoparticle technology. We will propose the clinical application of these RNA-mediated therapies in combination with the brain-targeted drug delivery approach against mTOR signaling components as an effective and feasible therapeutic strategy aiming to enhance axonal regeneration for functional recovery after CNS injury.

Mutational analysis of a Dcp2-binding element reveals general enhancement of decapping by 5'-end stem-loop structures.

mRNA decapping is a critical step in the control of mRNA stability and gene expression and is carried out by the Dcp2 protein. Dcp2 is an RNA-binding protein that must bind the RNA in order to recognize the cap for hydrolysis. We previously demonstrated that a 60 nucleotide (nt) element at the 5' end of the mRNA encoding Rrp41 is preferentially bound and decapped by Dcp2. Here, we demonstrate that enhanced decapping of this element is dependent on the structural integrity of its first 33 nt and not its primary sequence. The structure consists of a stem-loop positioned <10 nt from the 5' end of the mRNA. The generality of a stem-loop structure in enhanced Dcp2-mediated decapping was underscored by the identification of additional potential Dcp2 substrate mRNAs by a global analysis of human mRNAs containing a similar predicted stem-loop structure at their respective 5' end. These studies suggest a general role for 5' stem-loops in enhancing decapping activity and the utilization of this structure as a predictive tool for Dcp2 target substrates. These studies also demonstrate that Dcp2 alone in the absence of additional proteins can preferentially associate with and modulate mRNA decapping.

Identification of Blood Circular RNAs as Potential Biomarkers for Acute Ischemic Stroke.

Many hospitals lack facilities for accurate diagnosis of acute ischemic stroke (AIS). Circular RNA (circRNA) is highly expressed in the brain and is closely associated with stroke. In this study, we examined whether the blood-borne circRNAs could be promising candidates as adjunctive diagnostic biomarkers and their pathophysiological roles after stroke. We profiled the blood circRNA expression in mice subjected to experimental focal cerebral ischemia and validated the selected circRNAs in AIS patients. We demonstrated that 128, 198, and 789 circRNAs were significantly altered at 5 min, 3 h, and 24 h after ischemic stroke, respectively. Our bioinformatics analysis revealed that the circRNA-targeted genes were associated with the Hippo signaling pathway, extracellular matrix-receptor interaction, and fatty acid metabolism at 5 min, 3 h and 24 h after ischemic stroke, respectively. We verified that many of these circRNAs existed in the mouse brain. Furthermore, we found that most of the predicted circRNA-miRNA interactions apparently exhibited functional roles in terms of regulation of their target gene expression in the brain. We also verified that many of these mouse circRNAs were conserved in human. Finally, we found that circBBS2 and circPHKA2 were differentially expressed in the blood of AIS patients. These results demonstrate that blood circRNAs may serve as potential biomarkers for AIS diagnosis and reveal the pathophysiological responses in the brain after ischemic stroke.

A bipartite U1 site represses U1A expression by synergizing with PIE to inhibit nuclear polyadenylation.

U1A protein negatively autoregulates itself by polyadenylation inhibition of its own pre-mRNA by binding as two molecules to a 3'UTR-located Polyadenylation Inhibitory Element (PIE). The (U1A)2-PIE complex specifically blocks U1A mRNA biosynthesis by inhibiting polyA tail addition, leading to lower mRNA levels. U1 snRNP bound to a 5'ss-like sequence, which we call a U1 site, in the 3'UTRs of certain papillomaviruses leads to inhibition of viral late gene expression via a similar mechanism. Although such U1 sites can also be artificially used to potently silence reporter and endogenous genes, no naturally occurring U1 sites have been found in eukaryotic genes. Here we identify a conserved U1 site in the human U1A gene that is, unexpectedly, within a bipartite element where the other part represses the U1 site via a base-pairing mechanism. The bipartite element inhibits U1A expression via a synergistic action with the nearby PIE. Unexpectedly, synergy is not based on stabilizing binding of the inhibitory factors to the 3'UTR, but rather is a property of the larger ternary complex. Inhibition targets the biosynthetic step of polyA tail addition rather than altering mRNA stability. This is the first example of a functional U1 site in a cellular gene and of a single gene containing two dissimilar elements that inhibit nuclear polyadenylation. Parallels with other examples where U1 snRNP inhibits expression are discussed. We expect that other cellular genes will harbor functional U1 sites.

gb4gv: a genome browser for geminivirus.

BACKGROUND: Geminiviruses (family Geminiviridae) are prevalent plant viruses that imperil agriculture globally, causing serious damage to the livelihood of farmers, particularly in developing countries. The virus evolves rapidly, attributing to its single-stranded genome propensity, resulting in worldwide circulation of diverse and viable genomes. Genomics is a prominent approach taken by researchers in elucidating the infectious mechanism of the virus. Currently, the NCBI Viral Genome website is a popular repository of viral genomes that conveniently provides researchers a centralized data source of genomic information. However, unlike the genome of living organisms, viral genomes most often maintain peculiar characteristics that fit into no single genome architecture. By imposing a unified annotation scheme on the myriad of viral genomes may downplay their hallmark features. For example, the viron of begomoviruses prevailing in America encapsulates two similar-sized circular DNA components and both are required for systemic infection of plants. However, the bipartite components are kept separately in NCBI as individual genomes with no explicit association in linking them. Thus, our goal is to build a comprehensive Geminivirus genomics database, namely gb4gv, that not only preserves genomic characteristics of the virus, but also supplements biologically relevant annotations that help to interrogate this virus, for example, the targeted host, putative iterons, siRNA targets, etc. METHODS: We have employed manual and automatic methods to curate 508 genomes from four major genera of Geminiviridae, and 161 associated satellites obtained from NCBI RefSeq and PubMed databases. RESULTS: These data are available for free access without registration from our website. Besides genomic content, our website provides visualization capability inherited from UCSC Genome Browser. DISCUSSION: With the genomic information readily accessible, we hope that our database will inspire researchers in gaining a better understanding of the incredible degree of diversity of these viruses, and of the complex relationships within and between the different genera in the Geminiviridae. AVAILABILITY AND IMPLEMENTATION: The database can be found at: http://gb4gv.lafayette.edu.

U1 Adaptors Suppress the KRAS-MYC Oncogenic Axis in Human Pancreatic Cancer Xenografts.

Targeting KRAS and MYC has been a tremendous challenge in cancer drug development. Genetic studies in mouse models have validated the efficacy of silencing expression of both KRAS and MYC in mutant KRAS-driven tumors. We investigated the therapeutic potential of a new oligonucleotide-mediated gene silencing technology (U1 Adaptor) targeting KRAS and MYC in pancreatic cancer. Nanoparticles in complex with anti-KRAS U1 Adaptors (U1-KRAS) showed remarkable inhibition of KRAS in different human pancreatic cancer cell lines in vitro and in vivo As a nanoparticle-free approach is far easier to develop into a drug, we refined the formulation of U1 Adaptors by conjugating them to tumor-targeting peptides (iRGD and cRGD). Peptides coupled to fluorescently tagged U1 Adaptors showed selective tumor localization in vivo Efficacy experiments in pancreatic cancer xenograft models showed highly potent (>90%) antitumor activity of both iRGD and (cRGD)2-KRAS Adaptors. U1 Adaptors targeting MYC inhibited pancreatic cancer cell proliferation caused by apoptosis in vitro (40%-70%) and tumor regressions in vivo Comparison of iRGD-conjugated U1 KRAS and U1 MYC Adaptors in vivo revealed a significantly greater degree of cleaved caspase-3 staining and decreased Ki67 staining as compared with controls. There was no significant difference in efficacy between the U1 KRAS and U1 MYC Adaptor groups. Our results validate the value in targeting both KRAS and MYC in pancreatic cancer therapeutics and provide evidence that the U1 Adaptor technology can be successfully translated using a nanoparticle-free delivery system to target two undruggable genes in cancer. Mol Cancer Ther; 16(8); 1445-55. ©2017 AACR.

IDICAP: A Novel Tool for Integrating Drug Intervention Based on Cancer Panel.

Cancer is a heterogeneous disease afflicting millions of people of all ages and their families worldwide. Tremendous resources have been and continue to be devoted to the development of cancer treatments that target the unique mutation profiles of patients, namely targeted cancer therapy. However, the sheer volume of drugs coupled with cancer heterogeneity becomes a challenge for physicians to prescribe effective therapies targeting patients' unique genetic mutations. Developing a web service that allows clinicians as well as patients to identify effective drug therapies, both approved and experimental, would be helpful for both parties. We have developed an innovative web service, IDICAP, which stands for Integrated Drug Intervention for CAncer Panel. It uses genes that have been linked to a cancer type to search for drug and clinical trial information from ClinicalTrials.gov and DrugBank. IDICAP selects and integrates information pertaining to clinical trials, disease conditions, drugs under trial, locations of trials, drugs that are known to target the queried gene, and any known single nucleotide polymorphism (SNP) effects. We tested IDICAP by gene panels that contribute to breast cancer, ovarian cancer, and cancer in general. Clinical trials and drugs listed by our tool showed improved precision compared to the results from ClinicalTrials.gov and Drug Gene Interaction Database (DGIdb). Furthermore, IDICAP provides patients and doctors with a list of clinical facilities in their proximity, a characteristic that lends credence to the Precision Medicine Initiative launched by the White House in the United States in 2015.

Homoharringtonine (omacetaxine mepesuccinate) as an adjunct for FLT3-ITD acute myeloid leukemia.

An in vitro drug-screening platform on patient samples was developed and validated to design personalized treatment for relapsed/refractory acute myeloid leukemia (AML). Unbiased clustering and correlation showed that homoharringtonine (HHT), also known as omacetaxine mepesuccinate, exhibited preferential antileukemia effect against AML carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD). It worked synergistically with FLT3 inhibitors to suppress leukemia growth in vitro and in xenograft mouse models. Mechanistically, the effect was mediated by protein synthesis inhibition and reduction of short-lived proteins, including total and phosphorylated forms of FLT3 and its downstream signaling proteins. A phase 2 clinical trial of sorafenib and HHT combination treatment in FLT3-ITD AML patients resulted in complete remission (true or with insufficient hematological recovery) in 20 of 24 patients (83.3%), reduction of ITD allelic burden, and median leukemia-free and overall survivals of 12 and 33 weeks. The regimen has successfully bridged five patients to allogeneic hematopoietic stem cell transplantation and was well tolerated in patients unfit for conventional chemotherapy, including elderly and heavily pretreated patients. This study validated the principle and clinical relevance of in vitro drug testing and identified an improved treatment for FLT3-ITD AML. The results provided the foundation for phase 2/3 clinical trials to ascertain the clinical efficacy of FLT3 inhibitors and HHT in combination.

Bioinformatic analysis reveals genome size reduction and the emergence of tyrosine phosphorylation site in the movement protein of New World bipartite begomoviruses.

Begomovirus (genus Begomovirus, family Geminiviridae) infection is devastating to a wide variety of agricultural crops including tomato, squash, and cassava. Thus, understanding the replication and adaptation of begomoviruses has important translational value in alleviating substantial economic loss, particularly in developing countries. The bipartite genome of begomoviruses prevalent in the New World and their counterparts in the Old World share a high degree of genome homology except for a partially overlapping reading frame encoding the pre-coat protein (PCP, or AV2). PCP contributes to the essential functions of intercellular movement and suppression of host RNA silencing, but it is only present in the Old World viruses. In this study, we analyzed a set of non-redundant bipartite begomovirus genomes originating from the Old World (N = 28) and the New World (N = 65). Our bioinformatic analysis suggests ∼ 120 nucleotides were deleted from PCP's proximal promoter region that may have contributed to its loss in the New World viruses. Consequently, genomes of the New World viruses are smaller than the Old World counterparts, possibly compensating for the loss of the intercellular movement functions of PCP. Additionally, we detected substantial purifying selection on a portion of the New World DNA-B movement protein (MP, or BC1). Further analysis of the New World MP gene revealed the emergence of a putative tyrosine phosphorylation site, which likely explains the increased purifying selection in that region. These findings provide important information about the strategies adopted by bipartite begomoviruses in adapting to new environment and suggest future in planta experiments.

U1 Adaptor Oligonucleotides Targeting BCL2 and GRM1 Suppress Growth of Human Melanoma Xenografts In Vivo.

U1 Adaptor is a recently discovered oligonucleotide-based gene-silencing technology with a unique mechanism of action that targets nuclear pre-mRNA processing. U1 Adaptors have two distinct functional domains, both of which must be present on the same oligonucleotide to exert their gene-silencing function. Here, we present the first in vivo use of U1 Adaptors by targeting two different human genes implicated in melanomagenesis, B-cell lymphoma 2 (BCL2) and metabotropic glutamate receptor 1 (GRM1), in a human melanoma cell xenograft mouse model system. Using a newly developed dendrimer delivery system, anti-BCL2 U1 Adaptors were very potent and suppressed tumor growth at doses as low as 34 µg/kg with twice weekly intravenous (iv) administration. Anti-GRM1 U1 Adaptors suppressed tumor xenograft growth with similar potency. Mechanism of action was demonstrated by showing target gene suppression in tumors and by observing that negative control U1 Adaptors with just one functional domain show no tumor suppression activity. The anti-BCL2 and anti-GRM1 treatments were equally effective against cell lines harboring either wild-type or a mutant V600E B-RAF allele, the most common mutation in melanoma. Treatment of normal immune-competent mice (C57BL6) indicated no organ toxicity or immune stimulation. These proof-of-concept studies represent an in-depth (over 800 mice in ~108 treatment groups) validation that U1 Adaptors are a highly potent gene-silencing therapeutic and open the way for their further development to treat other human diseases.Molecular Therapy - Nucleic Acids (2013) 2, e92; doi:10.1038/mtna.2013.24; published online 14 May 2013.

Long conserved fragments upstream of Mammalian polyadenylation sites.

Polyadenylation is a cotranscriptional nuclear RNA processing event involving endonucleolytic cleavage of the nascent, emerging pre-messenger RNA (pre-mRNA) from the RNA polymerase, immediately followed by the polymerization of adenine ribonucleotides, called the poly(A) tail, to the cleaved 3' end of the polyadenylation site (PAS). This apparently simple molecular processing step has been discovered to be connected to transcription and splicing therefore increasing its potential for regulation of gene expression. Here, through a bioinformatic analysis of cis-PAS-regulatory elements in mammals that includes taking advantage of multiple evolutionary time scales, we find unexpected selection pressure much further upstream, up to 200 nt, from the PAS than previously thought. Strikingly, close to 3,000 long (30-500 nt) noncoding conserved fragments (CFs) were discovered in the PAS flanking region of three remotely related mammalian species, human, mouse, and cow. When an even more remote transitional mammal, platypus, was included, still over a thousand CFs were found in the proximity of the PAS. Even though the biological function of these CFs remains unknown, their considerable sizes makes them unlikely to serve as protein recognition sites, which are typically ≤15 nt. By harnessing genome wide DNaseI hypersensitivity data, we have discovered that the presence of CFs correlates with chromatin accessibility. Our study is important in highlighting novel experimental targets, which may provide new understanding about the regulatory aspects of polyadenylation.

iTriplet, a rule-based nucleic acid sequence motif finder.

BACKGROUND: With the advent of high throughput sequencing techniques, large amounts of sequencing data are readily available for analysis. Natural biological signals are intrinsically highly variable making their complete identification a computationally challenging problem. Many attempts in using statistical or combinatorial approaches have been made with great success in the past. However, identifying highly degenerate and long (>20 nucleotides) motifs still remains an unmet challenge as high degeneracy will diminish statistical significance of biological signals and increasing motif size will cause combinatorial explosion. In this report, we present a novel rule-based method that is focused on finding degenerate and long motifs. Our proposed method, named iTriplet, avoids costly enumeration present in existing combinatorial methods and is amenable to parallel processing. RESULTS: We have conducted a comprehensive assessment on the performance and sensitivity-specificity of iTriplet in analyzing artificial and real biological sequences in various genomic regions. The results show that iTriplet is able to solve challenging cases. Furthermore we have confirmed the utility of iTriplet by showing it accurately predicts polyA-site-related motifs using a dual Luciferase reporter assay. CONCLUSION: iTriplet is a novel rule-based combinatorial or enumerative motif finding method that is able to process highly degenerate and long motifs that have resisted analysis by other methods. In addition, iTriplet is distinguished from other methods of the same family by its parallelizability, which allows it to leverage the power of today's readily available high-performance computing systems.