Stranded RNA Molecules Research Articles

PHYLOGENETIC ANALYSIS OF TWELVE BULGARIAN SEQUENCES BASED ON PARTIAL OPEN READING FRAME 2 GENOME FRAGMENT OF HEPATITIS E VIRUS

Background: Hepatitis E virus (HEV) causes both acute and chronic liver inflammation. HEV is transmitted through the fecal-oral mechanism and infects both animals and humans. The virus belongs to the Hepeviridae family and its genome is a single stranded RNA molecule. Thanks to molecular sequencing methods different genotypes and subgenotypes have been established. The aim of the present study was to identify and characterize Bulgarian HEV sequences by applying Sanger sequencing technique for a genome fragment in Open Reading Frame 2 (ORF2 region). Material and methods: Twelve retrospective samples from patients with serologically confirmed HEV infection (anti-HEV IgM and IgG positive) were sequenced by Sanger sequencing. Sequencing data were analysed by BioEdit, MEGA11 and NCBI Genbank software tools. Results: The results revealed that all isolates assign to species Paslahepevirus balayani. Phylogenetic analysis showed that HEV isolates were characterized with considerable genetic diversity. The sequences were sub-clustered into the following subgenotypes: HEV-3e, 3m, 3f and 3c. Conclusion: We successfully applied the Sanger method for hepatitis E virus RNA sequencing. The established heterogeneity of subgenotypes requires further study in order to determine the circulation of all possible subgenotypes of HEV in the country.

The crosstalk between miRNAs and signaling pathways in human cancers: Potential therapeutic implications.

MicroRNAs (miRNAs) are increasingly recognized as central players in the regulation of eukaryotic physiological processes. These small double stranded RNA molecules have emerged as pivotal regulators in the intricate network of cellular signaling pathways, playing significant roles in the development and progression of human cancers. The central theme in miRNA-mediated regulation of signaling pathways involves their ability to target and modulate the expression of pathway components. Aberrant expression of miRNAs can either promote or suppress key signaling events, influencing critical cellular processes such as proliferation, apoptosis, angiogenesis, and metastasis. For example, oncogenic miRNAs often promote cancer progression by targeting tumor suppressors or negative regulators of signaling pathways, thereby enhancing pathway activity. Conversely, tumor-suppressive miRNAs frequently inhibit oncogenic signaling by targeting key components within these pathways. This complex regulatory crosstalk underscores the significance of miRNAs as central players in shaping the signaling landscape of cancer cells. Furthermore, the therapeutic implications of targeting miRNAs in cancer are substantial. miRNAs can be manipulated to restore normal signaling pathway activity, offering a potential avenue for precision medicine. The development of miRNA-based therapeutics, including synthetic miRNA mimics and miRNA inhibitors, has shown promise in preclinical and clinical studies. These strategies aim to either enhance the activity of tumor-suppressive miRNAs or inhibit the function of oncogenic miRNAs, thereby restoring balanced signaling and impeding cancer progression. In conclusion, the crosstalk between miRNAs and signaling pathways in human cancers is a dynamic and influential aspect of cancer biology. Understanding this interplay provides valuable insights into cancer development and progression. Harnessing the therapeutic potential of miRNAs as regulators of signaling pathways opens up exciting opportunities for the development of innovative cancer treatments with the potential to improve patient outcomes. In this chapter, we provide an overview of the crosstalk between miRNAs and signaling pathways in the context of cancer and highlight the potential therapeutic implications of targeting this regulatory interplay.

MicroRNAs as Possible Biomarkers for Diagnosis and Therapy of Alzheimer’s Disease by Regulating the Abnormal Expression of Genes Related to Tau

Alzheimer’s disease (AD), the most common dementia with the symptom of deterioration of memory and cognitive functions, becomes one of the prevalent threats globally. Tau hyperphosphorylation is one of major risk factors of AD. At present, there is no effective treatment or quick diagnostic methods for the pre-clinical stage of this disease. MicroRNAs (miRNAs) are small (20-25bp) non-coding, double stranded RNA molecule. They mainly regulate gene expression during the post-transcription by binding to the 3-’UTR of the mRNA then stop translation. Unlike other biomarkers using for Alzheimer’s disease, miRNAs are stable and widely found in body fluids such as serum, tissues, and Cerebrospinal fluid (CSF) and one miRNA can regulate multiple genes. As a result, they are potentially used as diagnosis or therapeutic biomarkers for many diseases including Alzheimer’s disease. Currently, this field that using miRNAs as biomarkers for diagnosis and treatment has been rapidly developed. To examine the mechanism and function of miRNAs and potential in AD, this review summarizes current diagnostic and therapeutic techniques and comparing several microRNAs that especially regulating tau toxicity as practicable diagnostic biomarkers and treatment agents. It is likely to detect different stages of Alzheimer’s disease and reduce Tau hyperphosphorylation if this field of miRNA as biomarkers carries forward.

TRNA-derived fragments (tRFs) in cancer.

tRNA fragments (tRNA derived fragments or tRFs) are small single stranded RNA molecules derived from pre-tRNAs and mature tRNAs. tRFs have been known for a number of years, but previously they were believed to be not important products of tRNA degradation. tRFs can be unique, like tRF-1s, or redundant, like tRF-3s and tRF-5s. Scientific interest in tRFs has drastically increased in the last 5years. Many studies have found that tRFs are differentially expressed in many normal cellular processes as well as in transformed cancer cells. Dysregulation of tRFs expression have been reported in multiple major types of cancer including solid cancers and lymphoid malignancies. However the exact molecular role of these molecules is not entirely clear. A number of studies proposed that tRFs can work as microRNAs by targeting gene expression. Here we discuss recent studies showing differential expression of tRFs in many cancers as well as what is currently known about tRFs biological functions in cancer cells.

Gene Expression Analysis in MOTN-1 Cell Line after Treating with New Development Aptamer

Among age-related morality in humans, cancer stands at 2nd leading cause and this is why the ability to suppress carcinogenesis has got the cancer prevention and treatment researchers working hard on this. Leukemia (cancer of bone marrow and blood), is most commonly characterized by uncontrolled proliferation of some blood cells, in this case leukocytes. There are generally four types of leukemia: acute myelogenous, chronic myelogenous, acute lymphocytic and chronic myelogenous. There are quite a few different leukemia cell lines and among them, the MOTN-1 cell line was derived from acute lymphoblastic leukemia. Moreover, the MOTN-1 cells are commonly described a highly undifferentiated leukemia cells. In this study, we evaluated the effects of SIRT1 activation by aptamer (Aptamer are single strand DNA or RNA molecules, selected by an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment SELEX) on viability of MOTN-1 cancer cell line as an anticancer drug and analysed the gene expression profiles of MOTN-1 cells treated with aptamer and untreated cells by cDNA microarray analysis and RT-PCR to give us more details about the molecular mechanisms underlying aptamer- induced carcinogenesis, including leukaemia. The results demonstrate that aptamer is extremely efficient tools for anticancer activity on ALL by inhibited 91% of these cancer cells at 10 µM aptamer after 72 hours with IC50 = 3.5 μM. Besides that, we at first identified 13 genes with expression changed by aptamer treatment. For this set of genes, 11 of these were up-regulated and two were down-regulated by more than 3-fold, respectively. As a result, pharmacological activation of SIRT1 enhanced cell death suggesting a tumor suppressive function of aptamer and may be used in the future for cancer treatment.

A survey of miRNAs involved in biomineralization and shell repair in the freshwater gastropod Lymnaea stagnalis

MicroRNAs (miRNAs) are a deeply conserved class of small, single stranded RNA molecules that post-transcriptionally regulate mRNA levels via several targeted degradation pathways. They are involved in a wide variety of biological processes and have been used to infer the deep evolutionary relationships of major groups such as the Metazoa. Here we have surveyed several adult tissues of the freshwater pulmonate Lymnaea stagnalis (the Great Pond Snail) for miRNAs. In addition we perform a shell regeneration assay to identify miRNAs that may be involved in regulating mRNAs directly involved in the shell-forming process. From seven mature tissues we identify a total of 370 unique precursor miRNAs that give rise to 336 unique mature miRNAs. While the majority of these appear to be evolutionarily novel, most of the 70 most highly expressed (which account for 99.8% of all reads) share sequence similarity with a miRBase or mirGeneDB2.0 entry. We also identify 10 miRNAs that are differentially regulated in mantle tissue that is actively regenerating shell material, 5 of which appear to be evolutionarily novel and none of which share similarity with any miRNA previously reported to regulate biomineralization in molluscs. One significantly down-regulated miRNA is predicted to target Lst-Dermatopontin, a previously characterized shell matrix protein from another freshwater gastropod. This survey provides a foundation for future studies that would seek to characterize the functional role of these molecules in biomineralization or other processes of interest.

Emergence of ribozyme and tRNA-like structures from mineral-rich muddy pools on prebiotic earth

The RNA world hypothesis, although a viable one regarding the origin of life on earth, has so far failed to provide a compelling explanation for the synthesis of RNA enzymes from free nucleotides via abiotic processes. To tackle this long-standing problem, we develop a realistic model for the onset of the RNA world, using experimentally determined rates for polymerization reactions. We start with minimal assumptions about the initial state that only requires the presence of short oligomers or just free nucleotides and consider the effects of environmental cycling by dividing a day into a dry, semi-wet and wet phases that are distinguished by the nature of reactions they support. Long polymers, with maximum lengths sometimes exceeding 100 nucleotides, spontaneously emerge due to a combination of non-enzymatic, non-templated polymer extension and template-directed primer extension processes. The former helps in increasing the lengths of RNA strands, whereas the later helps in producing complementary copies of the strands. Strands also undergo hydrolysis in a structure-dependent manner that favour breaking of bonds connecting unpaired nucleotides. We identify the most favourable conditions needed for the emergence of ribozyme and tRNA-like structures and double stranded RNA molecules, classify all RNA strands on the basis of their secondary structures and determine their abundance in the population. Our results indicate that under suitable environmental conditions, non-enzymatic processes would have been sufficient to lead to the emergence of a variety of ribozyme-like molecules with complex secondary structures and potential catalytic functions.

Molecular characterization of RNase III protein of Asaia sp. for developing a robust RNAi-based paratransgensis tool to affect the sexual life-cycle of Plasmodium or Anopheles fitness

BackgroundAccording to scientific recommendations, paratransgenesis is one of the solutions for improving the effectiveness of the Global Malaria Eradication Programme. In paratransgenesis, symbiont microorganisms are used for distorting or blocking the parasite life-cycle, affecting the fitness and longevity of vectors or reducing the vectorial competence. It has been revealed recently that bacteria could be used as potent tools for double stranded RNA production and delivery to insects. Moreover, findings showed that RNase III mutant bacteria are more competent for this aim. Asaia spp. have been introduced as potent paratransgenesis candidates for combating malaria and, based on their specific features for this goal, could be considered as effective dsRNA production and delivery tools to Anopheles spp. Therefore, we decided to characterize the rnc gene and its related protein to provide the basic required information for creating an RNase III mutant Asaia bacterium.MethodsAsaia bacteria were isolated from field-collected Anopheles stephensi mosquitoes. The rnc gene and its surrounding sequences were characterized by rapid amplification of genomic ends. RNase III recombinant protein was expressed in E. coli BL21 and biological activity of the purified recombinant protein was assayed. Furthermore, Asaia RNaseIII amino acid sequence was analyzed by in silico approaches such as homology modeling and docking to determine its structural properties.ResultsIn this study, the structure of rnc gene and its related operon from Asaia sp. was determined. In addition, by performing superimposition and docking with specific substrate, the structural features of Asaia RNaseIII protein such as critical residues which are involved and essential for proper folding of active site, binding of magnesium ions and double stranded RNA molecule to protein and cleaving of dsRNA molecules, were determined.ConclusionsIn this study, the basic and essential data for creating an RNase III mutant Asaia sp. strain, which is the first step of developing an efficient RNAi-based paratransgenesis tool, were acquired. Asaia sp. have been found in different medically-important vectors and these data are potentially very helpful for researchers studying paratransgenesis and vector-borne diseases and are interested in applying the RNAi technology in the field.

The clinical significance of serum miRNA-224 expression in hepatocellular carcinoma.

Aim of the studyMicro-ribonucleic acids (miRNA) are small single stranded RNA molecules. They act as key regulators of several cellular processes such as proliferation, apoptosis, tumor differentiation, invasion and metastasis. Hepatocellular carcinoma (HCC) represents the most common primary liver cancer. miRNA-224 is an oncomiR that is highly upregulated in HCC tissues. The aim of the present study was to measure the relative expression of circulating miRNA-224 in the serum of patients with HCV-related liver cirrhosis and HCC and to assess its usefulness in the diagnosis of HCC.Material and methodsForty-eight patients were classified into two groups: 24 HCV-related HCC patients (HCC group), and 24 HCV-related liver cirrhosis patients (LC group). A third group included 24 healthy volunteers (control group). Clinical examination, imaging studies and routine laboratory investigations, including serum α-fetoprotein (AFP), were done. Quantification of serum miRNA-224 expression was performed using real time reverse transcription polymerase chain reaction (RT-PCR).ResultsThe relative expression of serum miRNA-224 was significantly higher in HCC patients compared to LC patients and healthy control subjects. Its level correlated positively with the serum concentration of AFP and with Barcelona Clinic Liver Cancer (BCLC) stage of HCC. By combining miRNA-224 relative expression with AFP, their diagnostic sensitivity, specificity and accuracy increased significantly (95.0%, 92.1% and 93.2%, respectively) compared with either of the two markers alone in discriminating HCC from liver cirrhosis.ConclusionsSerum miRNA-224 relative expression may aid in the diagnosis of HCC. Better diagnostic performance is obtained if miRNA-224 is combined with other tumor markers such as AFP.

MicroRNA-6744-5p promotes anoikis in breast cancer and directly targets NAT1 enzyme.

Objective: Anoikis is apoptosis that is induced when cells detach from the extracellular matrix and neighboring cells. As anoikis serves as a regulatory barrier, cancer cells often acquire resistance towards anoikis during tumorigenesis to become metastatic. MicroRNAs (miRNAs) are short strand RNA molecules that regulate genes post-transcriptionally by binding to mRNAs and reducing the expression of its target genes. This study aimed to elucidate the role of a novel miRNA, miR-6744-5p, in regulating anoikis in breast cancer and identify its target gene.Methods: An anoikis resistant variant of the luminal A type breast cancer MCF-7 cell line (MCF-7-AR) was generated by selecting and amplifying surviving cells after repeated exposure to growth in suspension. MiRNA microarray analysis identified a list of dysregulated miRNAs from which miR-6744-5p was chosen for overexpression and knockdown studies in MCF-7. Additionally, the miRNA was also overexpressed in a triple-negative breast cancer cell line, MDA-MB-231, to evaluate its ability to impair the metastatic potential of breast cancer cells.Results: This study showed that overexpression and knockdown of miR-6744-5p in MCF-7 increased and decreased anoikis sensitivity, respectively. Similarly, overexpression of miR-6744-5p in MDA-MB-231 increased anoikis and also decreased tumor cell invasion in vitro and in vivo. Furthermore, NAT1 enzyme was identified and validated as the direct target of miR-6744-5p.Conclusions: This study has proven the ability of miR-6744-5p to increase anoikis sensitivity in both luminal A and triple negative breast cancer cell lines, highlighting its therapeutic potential in treating breast cancer.

Robust profiling of microRNAs and isomiRs in human plasma exosomes across 46 individuals

microRNAs (miRNAs) are small double stranded RNA molecules consisting of two complementary strands called the 5p and 3p arms. Following imprecise processing and/or addition of nucleotides at the ends, miRNA biogenesis can give rise to variants called isomiRs. Exosomes are small vesicles released by cells. They have attracted attention due to their potential use in biomarker development because of their content of biomolecules, including miRNAs and isomiRs. Exosomes are found in body fluids such as plasma. In this study we used next generation sequencing to investigate the distribution of 5p and 3p arms of both miRNAs and isomiRs in plasma exosomes from 46 individuals. Among the canonical miRNAs there was similar prevalence between 5p and 3p miRNAs. Most of the miRNAs had isomiRs, and in approximately half of the cases an isomiR was more abundant than the corresponding canonical miRNA. Most of the isomiRs were generated from 5p miRNAs. There were very small differences in the concentration of canonical miRNA and isomiR sequences between donors, suggesting tight control of isomiR generation and sorting into exosomes. IsomiRs are abundant in plasma exosomes and should be included in analysis when plasma exosomal miRNAs are investigated as potential biomarkers for disease development.

An Insight Into Dengue Virus Proteins As Potential drug/vaccine Targets

Dengue virus (DENV) is an arbovirus that belongs to family flaviviridae. Its genome is composed of a single stranded RNA molecule that encodes a single polyprotein. The polyprotein is processed by viral and cellular proteases to generate ten viral proteins. There are four antigenically distinct serotypes of DENV (DENV1, DENV2, DENV3 and DENV4), which are genetically related. Although protein variability is a major problem in dengue treatment, the functional and structural studies of individual proteins are equally important in treatment development. The data accumulated on dengue proteins are significant to provide detailed understanding of viral infection, replication, host-immune evasion and pathogenesis. In this review, we summarized the detailed current knowledge about DENV proteins.

Topical application of double stranded RNA molecules deriving from Sesbania mosaic virus (SeMV) CP and MP genes protects Sesbania plants against SeMV

RNA interference (RNAi) is a sequence-specific, gene silencing mechanism, induced by double-stranded RNA (dsRNA). It is a defense mechanism that protects eukaryotic cells from invasive nucleic acids such as viruses and transposons. In this study, we used a non-transgenic strategy in order to activate the antiviral RNAi mechanism against Sesbania mosaic virus (SeMV) in its natural host Sesbania grandiflora. DsRNA molecules from SeMV coat protein (CP) and movement protein (MP) (sobemovirus MP is considered a silencing suppressor) genes were produced by a two-step PCR approach followed by in vitro transcription and exogenously applied on sesbania plants along with SeMV. DsRNA for CP and MP conferred 53% and 64% resistance against SeMV, respectively based on the disease incidence data. The effect of dsRNA molecules against SeMV infection on sesbania plants was confirmed by direct antigen coating ELISA (DAC-ELISA). The strategy employed demonstrated the applicability of the RNA-based vaccination method, for the first time, to control sobemoviruses in a simple, highly specific and environmentally safe way.

MicroRNA-196a2 single nucleotide polymorphism rs11614913 in Egyptian patients with chronic lymphocytic leukemia

Background: MicroRNAs are recently identified class of small single stranded RNA molecules of 21-23 nucleotides that regulate gene expression at the posttranscriptional level. Chronic lymphocytic leukemia (CLL) is characterized by a typical defect in apoptosis resulting in accumulation of mature resting B cells within the peripheral blood, bone marrow and lymphoid organs. One of the relevant microRNAs to CLL is microRNA-196a2, as its potential targets of action include genes involved in apoptosis regulation. The single nucleotide polymorphism (SNP) in microRNA-196a2 rs11614913 C/T has been implicated as possible biomarker associated with multiple kinds of cancers. While multiple recent studies showed that genetic polymorphisms play an influential role in CLL susceptibility, the role of microRNA-196a2 SNP rs11614913in CLL remains elusive. Patients and methods: The present study was conducted on 40 newly diagnosed Egyptian CLL patients and 40 control subjects. Genotyping of microRNA-196a2 SNP rs11614913 was performed by real-time PCR. Results: The CC genotype was found in 55% of CLL patients versus 12.5% of the controls.On the other handthe TT and TC genotypes were significantly predominant in the control group compared to CLL patients with frequencies of 25% and 62.5% versus 10% and 35% respectively (p<0.001). The C allele carriers were found to have 3.38 fold increased risk for development of CLL (OR=3.38, CI=1.75–6.56) while the T allele seems to be protective against CLL (OR=0.29, CI=0.15–0.57). Conclusion: We could conclude that the CC genotype and the C allele of miR-1962a SNP rs3217927are implicated in the risk of CLL development in Egyptian patients while the T allele has a protective effect. However, additional well-designed large studies are required for the validation of these associations

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury.

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments for toxicant-induced corneal injury are generally supportive as no specific therapeutics exist to treat these injuries. In the efforts to develop treatments and therapeutics to care for exposure, it can be important to understand the molecular and cellular mechanisms of these injuries. We propose that utilization of high throughput small inhibitory RNA (siRNA) screening can be an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. siRNA are double stranded RNA molecules that are 19-25 nucleotides long and utilize the post-transcriptional gene silencing pathway to degrade mRNA which have homology to the siRNA. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. The development and validation of in vitro exposure models and methods for the high throughput screening (HTS) of hydrogen fluoride- (HF) and chloropicrin- (CP) induced ocular injury are presented in this article. Although we selected these two toxicants, our methods are applicable to the study of other toxicants with minor modifications to the toxicant exposure protocol. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and IL-8 production were selected as endpoints in the screening protocol. Several challenges associated with the development of toxicant exposure and cell culture methods suitable for HTS studies are presented. The establishment of HTS models for these toxicants allows for further studies to better understand the mechanism of injury and to screen for potential therapeutics for chemical ocular injury.

Discovery and characterization of conserved and novel microRNAs from blunt snout bream (Megalobrama amblycephala) by deep sequencing

MicroRNAs (miRNAs) are short, single stranded RNA molecules with approximately 22 nts in length, which regulate the stability and translation of messenger RNAs in several organisms. To increase the repertoire of miRNAs characterized in M. amblycephala, we used the deep sequencing technology to sequence a small RNA library using pooled RNA sample isolated from the 4 different tissues of M. amblycephala. A total of 309 conserved miRNAs that originated from 131 miRNA families were detected. 15 novel candidates miRNA were identified. Randomly selected 6 miRNAs were analyzed by stem-loop qRT-PCR and differential expression patterns were observed in 6 different tissues of M. amblycephala. Furthermore, the potential targets were predicted. GO analysis showed that most of the targets were involved in a broad range of physiological functions including fish growth, development, metabolism, stress responses and so on. Overall, our results significantly increased the number of novel miRNAs in M. amblycephala, which should be useful for further investigation into the role of miRNAs in regulating diverse biological processes.

The role of miRNAs in the pathogenesis of pituitary adenomas

MicroRNAs (miRNAs) are short, single stranded RNA molecules which play regulatory roles through posttranscriptional regulation of their target genes. Based on our current knowledge, more than 30% of the human protein-coding genes are regulated by miRNAs, hence influencing basic cellular mechanisms including cell proliferation, differentiation and cell death. Differential miRNA expression pattern has been detected in many different types of tumors and, recently, several publications have referred to miRNAs as potential therapeutic targets. Through adjustment of miRNA levels by artificial miRNAs administration or miRNA inhibition, we can influence not only one target gene but also complex biological pathways. Pituitary adenoma is the second most frequent intracranial tumor. In spite of this, the molecular mechanism of the pituitary adenoma formation is not yet entirely revealed. Recently, more and more evidences have been found suggesting that miRNAs have an important role in pituitary adenoma pathogenesis. Here, we summarize the recent results related to this role and highlight the therapeutic potentials in pituitary adenomas. Orv Hetil. 2018; 159(7): 252-259.

A GPU-Accelerated Parameter Interpolation Thermodynamic Integration Free Energy Method.

There has been a resurgence of interest in free energy methods motivated by the performance enhancements offered by molecular dynamics (MD) software written for specialized hardware, such as graphics processing units (GPUs). In this work, we exploit the properties of a parameter-interpolated thermodynamic integration (PI-TI) method to connect states by their molecular mechanical (MM) parameter values. This pathway is shown to be better behaved for Mg2+ → Ca2+ transformations than traditional linear alchemical pathways (with and without soft-core potentials). The PI-TI method has the practical advantage that no modification of the MD code is required to propagate the dynamics, and unlike with linear alchemical mixing, only one electrostatic evaluation is needed (e.g., single call to particle-mesh Ewald) leading to better performance. In the case of AMBER, this enables all the performance benefits of GPU-acceleration to be realized, in addition to unlocking the full spectrum of features available within the MD software, such as Hamiltonian replica exchange (HREM). The TI derivative evaluation can be accomplished efficiently in a post-processing step by reanalyzing the statistically independent trajectory frames in parallel for high throughput. We also show how one can evaluate the particle mesh Ewald contribution to the TI derivative evaluation without needing to perform two reciprocal space calculations. We apply the PI-TI method with HREM on GPUs in AMBER to predict p Ka values in double stranded RNA molecules and make comparison with experiments. Convergence to under 0.25 units for these systems required 100 ns or more of sampling per window and coupling of windows with HREM. We find that MM charges derived from ab initio QM/MM fragment calculations improve the agreement between calculation and experimental results.

Multi-Target Inhibition of Cancer Cell Growth by SiRNA Cocktails and 5-Fluorouracil Using Effective Piperidine-Terminated Phosphorus Dendrimers

Currently, RNAi based approaches for cancer treatment involving short double stranded RNA molecules (siRNA) are under vigorous scrutinization. Due to numerous biological obstacles, siRNA delivery into target cells requires protective escort. On the other hand, combining of siRNA-mediated gene silencing and action of conventional chemotherapeutics can propose additional enhancement of anticancer activity. In the present study, we investigated a siRNA cocktail able to downregulate anti-apoptotic genes (BCL-xL, BCL-2, MCL-1) and the chemotherapeutic agent 5-fluorouracil (5-FU) to evaluate multi-target cytotoxic effect on human cervical carcinoma cells (HeLa cell line). Novel phosphorus containing dendrimers of 3rd and 4th generations (namely AE2G3 and AE2G4) with voluminous piperidine terminal cationic groups were designed and tested as siRNA carriers. Dendrimers of both generations showed remarkable ability to bind pro-apoptotic siRNAs and provided 80–100% siRNA uptake by HeLa cells in the serum containing medium, while the widespread transfection agent Lipofectamine showed only ~40% uptake. SiRNA cocktail (in low concentrations 50 and 100 nM) delivered by AE2G3 dendrimer caused almost complete elimination of cancer cells. We have discovered considerable increase of 5-FU cytotoxic effect by addition of AE2G3/siRNA cocktail complexes in low doses. Thus, we demonstrated the effectiveness of combined multi-target siRNA anticancer approach and described new highly effective serum stable nanomaterial vehicle for gene-based drugs.

MicroRNA Biogenesis in Hypoxia.

MicroRNAs (miRNAs) are 17-22 nucleotide, non-coding, single stranded RNA molecules that play a key role in post-transcriptional gene regulation. Hypoxia is a reduction in the normal level of tissue oxygen (O2) tension, and is a feature of chronic vascular disease, pulmonary disease and many cancers. Tissue hypoxia can have widespread effects on cellular functions, as O2 availability is critical for many cellular processes. Cells respond to changes in O2 tension through multiple molecular and cellular mechanisms, including changes in gene expression through transcriptional and translational mechanisms. The transcription factor, hypoxia inducible factor-1, plays a dominant role in transcriptional gene regulation in hypoxia. Several hypoxically induced miRNAs have been shown to play important roles in the hypoxic adaptation of cancer cells. Global repression of enzymes critical for miRNA biogenesis seems to be a widespread phenomenon with several different mechanisms operating. This review describes the effects of hypoxia on specific miRNAs and more global effects on miRNA biogenesis, demonstrating that hypoxia is an important regulator of miRNA biogenesis and function.