Concentrations Of RNase Research Articles

Condensation of RNase L promotes its rapid activation in response to viral infection in mammalian cells.

Oligoadenylate synthetase 3 (OAS3) and ribonuclease L (RNase L) are components of a pathway that combats viral infection in mammals. Upon detection of viral double-stranded RNA (dsRNA), OAS3 synthesizes 2'-5'-oligo(A), which activates the RNase domain of RNase L by promoting the homodimerization and oligomerization of RNase L monomers. Activated RNase L rapidly degrades all cellular mRNAs, shutting off several cellular processes. We sought to understand the molecular mechanisms underlying the rapid activation of RNase L in response to viral infection. Through superresolution microscopy and live-cell imaging, we showed that OAS3 and RNase L concentrated into higher-order cytoplasmic complexes known as dsRNA-induced foci (dRIF) in response to dsRNA or infection with dengue virus, Zika virus, or West Nile virus. The concentration of OAS3 and RNase L at dRIF corresponded with the activation of RNase L-mediated RNA decay. We showed that dimerized/oligomerized RNase L concentrated in a liquid-like shell surrounding a core OAS3-dRIF structure and dynamically exchanged with the cytosol. These data establish that the condensation of dsRNA, OAS3, and RNase L into dRIF is a molecular switch that promotes the rapid activation of RNase L upon detection of dsRNA in mammalian cells.

Abstract LB333: Improved spatially resolved single-cell transcriptomic imaging in archival tissues with MERSCOPE

Abstract The advent of spatial transcriptomics has enabled a revolution in how complex tissues are studied. However, samples with lower quality RNA due to degradation, protein crosslinking, or high RNase content remain challenging for spatial transcriptomic measurement. In particular, formalin fixed, paraffin embedded (FFPE) tissues are the most widely used sample types in clinical and molecular diagnosis, yet they are notoriously difficult for single-cell transcriptomic analysis. To accurately profile the gene expression in FFPE samples in situ, a spatial transcriptomics technique with high detection efficiency and single molecule resolution is required. The Vizgen® MERSCOPE® Platform for spatial genomics is built on Multiplexed Error Robust in situ Hybridization (MERFISH) technology and directly profiles the transcriptome of intact tissues with high sensitivity in high-quality samples. Here we present an updated workflow to perform MERFISH in low and high-quality samples. We demonstrated its application in more than 5 FFPE sample types from mouse and human, including archival samples. For each tissue type, hundreds of thousands of cells were captured using the updated MERSCOPE Platform workflow, generating 100s million counts and their spatial information for profiled genes in each sample. The updated workflow involves streamlined sample preparation and chemistry optimization to improve sensitivity. MERSCOPE accurately profiled gene expression in situ and mapped cell types in archival human samples across a range of low and high RNA qualities. We compared the performance of MERSCOPE imaging using the updated protocol to the previous version and observed a significant increase in gene counts per 100 micron2 of tissue. We also demonstrated increased reproducibility between replicates with the streamlined workflow and chemistry. Furthermore, we demonstrated the updated workflow is compatible with simultaneous protein-based cell boundary staining. Finally, we constructed a spatially resolved single-cell atlas across low-quality archival breast and lung tumor types, mapped and cataloged different cell types within the tumor microenvironment, and systematically characterized the gene expression among cells. Spatially resolved transcriptomic profiling of low-quality samples at single-cell level provides enormous opportunities in cancer research. These improvements will enable new genomic inquiries into previously intractable tissues like FFPE, leading to new biological insights into cancer progression. Citation Format: Jiang He, Bin Wang, Justin He, Renchao Chen, Benjamin Patterson, Sudhir Tattikota, Timothy Wiggin, Lizi Maziashvili, Peter Reinhold, Manisha Ray, George Emanuel. Improved spatially resolved single-cell transcriptomic imaging in archival tissues with MERSCOPE [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB333.

Optimization of Enzymatic and Chemical Decellularization of Native Porcine Heart Valves for the Generation of Decellularized Xenografts.

One of the most important medical interventions for individuals with heart valvular disease is heart valve replacement, which is not without substantial challenges, particularly for pediatric patients. Due to their biological properties and biocompatibility, natural tissue-originated scaffolds derived from human or animal sources are one type of scaffold that is widely used in tissue engineering. However, they are known for their high potential for immunogenicity. Being free of cells and genetic material, decellularized xenografts, consequently, have low immunogenicity and, thus, are expected to be tolerated by the recipient's immune system. The scaffold ultrastructure and ECM composition can be affected by cell removal agents. Therefore, applying an appropriate method that preserves intact the structure of the ECM plays a critical role in the final result. So far, there has not been an effective decellularization technique that preserves the integrity of the heart valve's ultrastructure while securing the least amount of genetic material left. This study demonstrates a new protocol with untraceable cells and residual DNA, thereby maximally reducing any chance of immunogenicity. The mechanical and biochemical properties of the ECM resemble those of native heart valves. Results from this study strongly indicate that different critical factors, such as ionic detergent omission, the substitution of Triton X-100 with Tergitol, and using a lower concentration of trypsin and a higher concentration of DNase and RNase, play a significant role in maintaining intact the ultrastructure and function of the ECM.

Concentration of novel urinary tract infection biomarkers in neonates

Urinary tract infections (UTIs) are a common comorbidity in hospitalized neonates. The current UTI diagnostics have several limitations including invasive collection of urinary samples to ensure sterility, risk of contamination and lack of consensus definitions of UTI based on urine culture. Antimicrobial peptides (AMPs) have been recently utilized as novel biomarkers that can efficiently and accurately diagnose pediatric UTI. However, the concentration of AMPs in neonatal urine is not well-defined. Urine from neonates admitted to a single level IV neonatal intensive care unit was obtained to determine baseline concentration of two AMPs, Ribonuclease 7 (RNase 7) and Beta Defensin-1 (BD-1) and to define the relationship between AMP concentration and gestational age (GA). AMP levels were normalized to urine creatinine. RNase 7 and BD-1 were expressed in neonatal urine (n = 66) regardless of GA and as early as 22 weeks gestation. Urinary concentrations of both AMPs decreased as GA and birthweight increased. The overall median urinary RNase 7/UCr and BD-1/UCr values were 271 ng/mg, and 116 ng/mg, respectively. Median urinary concentrations of RNase 7/UCr for infants born at < 27, 27–32, 33–35 and ≥ 36 weeks were 569, 308, 254, and 124 ng/mg respectively. Similarly, the concentrations of BD-1/UCr at these GA were 166, 115, 108, and 14 ng/mg, respectively. Baseline neonatal urinary concentration of two AMPs (RNase 7 and BD-1) and the variation by GA were identified. This is an essential first step toward the potential utilization of AMPs in improving neonatal UTI diagnostics.

Mapping protein-RNA binding in plants with individual-nucleotide-resolution UV cross-linking and immunoprecipitation (plant iCLIP2).

Despite crucial roles of RNA-binding proteins (RBPs) in plant physiology and development, methods for determining their transcriptome-wide binding landscape are less developed than those used in other model organisms. Cross-linking and immunoprecipitation (CLIP) methods (based on UV-mediated generation of covalent bonds between RNAs and cognate RBPs in vivo, purification of the cross-linked complexes and identification of the co-purified RNAs by high-throughput sequencing) have been applied mainly in mammalian cells growing in monolayers or in translucent tissue. We have developed plant iCLIP2, an efficient protocol for performing individual-nucleotide-resolution CLIP (iCLIP) in plants, tailored to overcome the experimental hurdles posed by plant tissue. We optimized the UV dosage to efficiently cross-link RNA and proteins in plants and expressed epitope-tagged RBPs under the control of their native promoters in loss-of-function mutants. We select epitopes for which nanobodies are available, allowing stringent conditions for immunopurification of the RNA-protein complexes to be established. To overcome the inherently high RNase content of plant cells, RNase inhibitors are added and the limited RNA fragmentation step is modified. We combine the optimized isolation of RBP-bound RNAs with iCLIP2, a streamlined protocol that greatly enhances the efficiency of library preparation for high-throughput sequencing. Plant researchers with experience in molecular biology and handling of RNA can complete this iCLIP2 protocol in ~5 d. Finally, we describe a bioinformatics workflow to determine targets of Arabidopsis RBPs from iCLIP data, covering all steps from downloading sequencing reads to identifying cross-linking events ( https://github.com/malewins/Plant-iCLIPseq ), and present the R/Bioconductor package BindingSiteFinder to extract reproducible binding sites ( https://bioconductor.org/packages/release/bioc/html/BindingSiteFinder.html ).

Analysis of Purity and Concentration Escherichia coli DNA by Boiling Method Isolation with Addition of Proteinase-K and RNase

Escherichia coli is a leading cause of Urinary Tract Infections (UTIs) in Indonesia, with approximately 180,000 cases reported annually. The more cases of UTIs, the more PCR diagnosis is needed with an accurate, fast, simple, and economical DNA isolation method. However, currently, there is no DNA purification stage from protein and RNA contaminants in the boiling DNA isolation method. This study aimed to investigate the impact of incorporating Proteinase-K and RNase into the boiling DNA isolation method on the purity and concentration of E. coli’s DNA during isolation. The boiling method involved heating to 95°C – 100°C bring to cell lysis and release of cellular components, including DNA. Urine samples were artificially contaminated with E. coli at different McFarland standards (0.25, 0.5, and 1). The boiling DNA isolation method was then performed and then analyzed for purity and concentration using a NanoDrop spectrophotometer. This study demonstrated a positive correlation between Proteinase-K and RNase concentrations used in the boiling DNA isolation method and the subsequent increase in DNA purity and concentration. An increase in DNA purity and concentration was obtained even though it was not statistically significant compared to that without Proteinase-K and RNase addition, with p-values of 0.245 for DNA purity and 0.353 for DNA concentration. Further research is recommended with higher Proteinase-K and RNase concentrations in the boiling DNA isolation method to achieve improved purity and concentration of E. coli DNA. Such enhancements could improve PCR amplification and help diagnose E. coli-related UTIs.

Antimicrobial skin peptides in premature infants: Comparison with term infants and impact of perinatal factors.

Preterm infants have an immature epidermis barrier function that may lead to an increased permeability to pathogens. On the surface of the human skin, antimicrobial peptides (AMPs) are important molecules of the innate immune system, have broad antimicrobial properties, and provide an essential role in integrity of the microbiome. Given the marked susceptibility of preterm infants to infection, we hypothesize a decreased expression of AMPs on the skin of preterm infants. In a prospective single-center study with 35 preterm and 20 term infants, we analyzed skin rinsing probes for the presence of the AMPs psoriasin (S100A7) and ribonuclease 7 (RNase 7) via enzyme-linked immunosorbent assay. Samples were taken from preterm infants < 34 0/7 weeks gestational age (mean ± SD gestational age, 28.8 ± 2.4 weeks) on days 0, 7, 14, and 28 after birth. Term infants (> 36 6/7 weeks) (controls) were washed on days 0 and 28. Psoriasin and RNase 7 were both expressed on skin of preterm and term infants and increased in concentration significantly over time. RNase 7 was more expressed in term infants on day 0 [preterm = 1.1 (0.7-2.9) vs. term = 2.0 (1.1-3.4) ng/ml, p = 0.017]. On day 28, premature infants showed higher values of psoriasin [preterm = 10.9 (5.6-14.2) vs. term = 6.3 (3.4-9.0) ng/ml, p < 0.001]. Notably, preterm infants with infectious or inflammatory context driven by histological proof of chorioamnionitis and early-onset or late-onset sepsis had higher concentrations of psoriasin as compared with non-affected preterm infants. After exclusion of infants with inflammatory hit, median concentrations of RNase 7 and psoriasin did not differ between preterm and full-term infants on days 0 and 28. Psoriasin and RNase 7 concentrations increase over time on the skin of newborn infants and seem to play a role in the first defense against infection. This is of particularly interest as the role of AMPs on a maturing skin microbiome and its possible new prevention strategies is unclear and needs to be determined.

Breast cancer nipple discharge exosomal microRNAs are stable under degradative conditions.

We have previously shown that microRNAs (miRNAs) in nipple discharge are potential diagnostic biomarkers. In particular, exosomes are present in nipple discharge. Herein, we sought to elucidate the protective role of exosomes on miRNAs in nipple discharge and investigate the stability of miRNAs encapsulated in exosomes under degradative conditions. A novel TTMAAlPc-RNA complex method was used to measure the RNase concentration in colostrum and nipple discharge. Quantitative real-time polymerase chain reaction was performed to test the stability of exogenous synthetic miRNAs (cel-lin-4-5p and cel-miR-2-3p) and endogenous miRNAs (hsa-miR-4732-5p, hsa-miR-3646, hsa-miR-4484, and kshv-miR-K12-5-5p). RNase was present and functional in colostrum and nipple discharge. Endogenous miRNAs were more stably expressed compared to exogenous miRNAs at room temperature and 4°C. Triton X-100 (1%, 30 min) destroyed the exosomal membrane, causing RNA degradation in colostrum but not in nipple discharge. Therefore, we confirmed that exosomes in colostrum and nipple discharge could protect miRNAs from degradation by RNase. Exosomes in nipple discharge may be more resistant to Triton X-100 lysis compared to those in the colostrum. Exosomal miRNAs in nipple discharge in breast cancer are stable under degradative conditions. Differential Triton X-100 sensitivity of exosomes of nipple discharge and colostrum warrants further investigation.

Controlled Level of Contamination Coupled to Deep Sequencing (CoLoC-seq) Probes the Global Localisation Topology of Organelle Transcriptomes.

Information on RNA localisation is essential for understanding physiological and pathological processes, such as gene expression, cell reprogramming, host-pathogen interactions, and signalling pathways involving RNA transactions at the level of membrane-less or membrane-bounded organelles and extracellular vesicles. In many cases, it is important to assess the topology of RNA localisation, i.e., to distinguish the transcripts encapsulated within an organelle of interest from those merely attached to its surface. This allows establishing which RNAs can, in principle, engage in local molecular interactions and which are prevented from interacting by membranes or other physical barriers. The most widely used techniques interrogating RNA localisation topology are based on the treatment of isolated organelles with RNases with subsequent identification of the surviving transcripts by northern blotting, qRT-PCR, or RNA-seq. However, this approach produces incoherent results and many false positives. Here, we describe Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq), a more refined subcellular transcriptomics approach that overcomes these pitfalls. CoLoC-seq starts by the purification of organelles of interest. They are then either left intact or lysed and subjected to a gradient of RNase concentrations to produce unique RNA degradation dynamics profiles, which can be monitored by northern blotting or RNA-seq. Through straightforward mathematical modelling, CoLoC-seq distinguishes true membrane-enveloped transcripts from degradable and non-degradable contaminants of any abundance. The method has been implemented in the mitochondria of HEK293 cells, where it outperformed alternative subcellular transcriptomics approaches. It is applicable to other membrane-bounded organelles, e.g., plastids, single-membrane organelles of the vesicular system, extracellular vesicles, or viral particles. Key features • Tested on human mitochondria; potentially applicable to cell cultures, non-model organisms, extracellular vesicles, enveloped viruses, tissues; does not require genetic manipulations or highly pure organelles. • In the case of human cells, the required amount of starting material is ~2,500 cm2 of 80% confluent cells (or ~3 × 108 HEK293 cells). • CoLoC-seq implements a special RNA-seq strategy to selectively capture intact transcripts, which requires RNases generating 5'-hydroxyl and 2'/3'-phosphate termini (e.g., RNase A, RNase I). • Relies on nonlinear regression software with customisable exponential functions.

Sample Buffer Containing Guanidine-Hydrochloride Combines Biological Safety and RNA Preservation for SARS-CoV-2 Molecular Diagnostics.

The COVID-19 pandemic has elicited the need to analyse and store large amounts of infectious samples for laboratory diagnostics. Therefore, there has been a demand for sample storage buffers that effectively inactivate infectious viral particles while simultaneously preserving the viral RNA. Here, we present a storage buffer containing guanidine-hydrochloride that fulfils both requirements. Its ability to preserve RNA stability was confirmed by RT-qPCR, and virus-inactivating properties were tested by tissue culture infectious dose assay. Our data revealed that RNA from samples diluted in this storage buffer was efficiently preserved. Spiking samples with RNase A resulted in RNAse concentrations up to 100 ng/mL being efficiently inhibited, whereas spiking samples with infectious SARS-CoV-2 particles demonstrated rapid virus inactivation. In addition, our buffer demonstrated good compatibility with several commercially available RNA extraction platforms. The presented guanidine-hydrochloride-based storage buffer efficiently inactivates infectious SARS-CoV-2 particles and supports viral RNA stability, leading to a reduced infection risk during sample analysis and an increased period for follow-up analysis, such as sequencing for virus variants. Because the presented buffer is uncomplicated to manufacture and compatible with a variety of commercially available test systems, its application can support and improve SARS-CoV-2 laboratory diagnostics worldwide.

RNase 7 Inhibits Uropathogenic Escherichia coli-Induced Inflammation in Bladder Cells under a High-Glucose Environment by Regulating the JAK/STAT Signaling Pathway.

Antimicrobial peptides (AMPs), which are natural antibiotics, protect against pathogens invading the urinary tract. RNase 7 with antimicrobial properties has rapid and powerful suppressive effects against Gram-positive and Gram-negative bacterial infections. However, its detailed antibacterial mechanisms have not been fully determined. Here, we investigate whether RNase 7 had an impact on bladder cells under uropathogenic Escherichia coli (UPEC) infection in a high-glucose environment using in vitro GFP-UPEC-infected bladder cell and PE-labeled TLR4, STAT1, and STAT3 models. We provide evidence of the suppressive effects of RNase 7 on UPEC infection and UPEC-induced inflammatory responses by regulating the JAK/STAT signaling pathway using JAK inhibitor and STAT inhibitor blocking experiments. Pretreatment with different concentrations of RNase 7 for 24 h concentration-dependently suppressed UPEC invasion in bladder cells (5 μg/mL reducing 45%; 25 μg/mL reducing 60%). The expressions of TLR4, STAT1, and STAT3 were also downregulated in a concentration-dependent manner after RNase 7 pretreatment (5 μg/mL reducing 35%, 54% and 35%; 25 μg/mL reducing 60%, 75% and 64%, respectively). RNase 7-induced decrease in UPEC infection in a high-glucose environment not only downregulated the expression of TLR4 protein and the JAK/STAT signaling pathway but also decreased UPEC-induced secretion of exogenous inflammatory IL-6 and IL-8 cytokines, although IL-8 levels increased in the 25 μg/mL RNase 7-treated group. Thus, inhibition of STAT affected pSTAT1, pSTAT3, and TLR4 expression, as well as proinflammatory IL-6 and IFN-γ expression. Notably, blocking JAK resulted in the rebound expression of related proteins, especially pSTAT1, TLR4, and IL-6. The present study showed the suppressive effects of RNase 7 on UPEC infection and induced inflammation in bladder epithelial cells in a high-glucose environment. RNase 7 may be an anti-inflammatory and anti-infective mediator in bladder cells by downregulating the JAK/STAT signaling pathway and may be beneficial in treating cystitis in DM patients. These results will help clarify the correlation between AMP production and UTI, identify the relationship between urinary tract infection and diabetes in UTI patients, and develop novel diagnostics or possible treatments targeting RNase 7.

Nano-Scaled Zeolitic Imidazole Framework-8 as an Efficient Carrier for the Intracellular Delivery of RNase A in Cancer Treatment.

BackgroundZeolitic imidazole framework-8 (ZIF-8) as an emerging platform has exhibited great potential in the protein delivery owing to its tunable chemical functionality.Materials and methodsZIF-8 was employed as a carrier for the encapsulation and intracellular delivery of RNase A, aimed to achieve a rapid release of proteins in an acidic environment. The intracellular uptake of RNase A was studied by confocal laser scanning microscopy (CLSM), and the inhibition of cell proliferation after the delivery of RNase A was evaluated by MTT assay, Live/Dead staining, and TUNEL cell apoptosis analysis, using human lung adenocarcinoma cell line A549 as a model. The biocompatibility of RNase A@ZIF-8 nanoparticles was systematically detected through the hemolysis and cytotoxicity assay.ResultsThe RNase A@ZIF-8 nanoparticles constructed by biomimetic mineralization could not only facilitate the encapsulation of protein molecules (protein loading: 13.4%) but also maintain the enzymatic activity and stability of RNase A. The CLSM images showed that RNase A@ZIF-8 nanoparticles could efficiently improve the intracellular uptake of RNase A. Moreover, RNase A@ZIF-8 nanoparticles could obviously inhibit the cell proliferation through the induction of cell apoptosis, with 31.3% of cell death at an RNase A concentration of 10 μg/mL. Finally, RNase A@ZIF-8 nanoparticles were elucidated to possess excellent biocompatibility, with hemolysis of <5% using the same concentration of RNase A@ZIF-8.ConclusionZIF-8 could be used as an effective carrier to deliver the therapeutic protein RNase A into the cytosol, which will be beneficial for improving the efficacy of cancer treatment.

Homogeneous Electrochemiluminescence Biosensor for the Detection of RNase A Activity and Its Inhibitor.

Ribonuclease A (RNase A) is increasingly considered as a biomarker for tumor diagnosis, and it is of great significance to develop an ultrasensitive, cost-effective assay for RNase A detection. Electrochemiluminescence (ECL) technology has distinctive advantages in the development of biosensors for diverse targets. However, most of the ECL biosensors require the complex process of electrode modification, which is laborious and time consuming. In this work, an immobilization-free homogeneous ECL assay was developed for the highly sensitive detection of RNase A activity for the first time. On the basis of the fact that RNase A can specifically hydrolyze RNA at the site of ribonucleotide uracil (rU), a rU-containing chimeric DNA probe is designed and labeled with Ru(bpy)32+ (act as ECL indicator). The chimeric DNA probe hardly diffuses to the surface of negatively charged indium tin oxide (ITO) electrode due to the strong electrostatic repulsion between the negatively charged DNA and ITO electrode, resulting in a weak ECL signal detected. When the RNase A is present, the chimeric DNA probe is hydrolyzed into small fragments, which contains little negative charge and can diffuse easily to the ITO electrode surface due to the decreased electrostatic repulsion. In this case, an enhanced ECL signal can be detected. Under the optimal conditions, there is a linear relationship between the ECL signal and the concentration of RNase A in the range of 0.001-0.10 ng/mL, and the detection limit is 0.2 pg/mL. In addition, the proposed ECL sensing system is also applied to detect the RNase A inhibitor, taking As3+ as an example. The proposed homogeneous ECL sensing system provides a new approach for the highly sensitive and convenient detection of RNase A as well as other ribonucleases only by redesigning a responding chimeric DNA probe.

Immunoprecipitation and SDS-PAGE for Cross-Linking Immunoprecipitation (CLIP).

This first part of this protocol is designed to optimize purification of the RNABP by immunoprecipitation for cross-linking immunoprecipitation (CLIP) experiments. The key variables to assess are the quality and quantity of antibody needed to immunoprecipitate most but not quite all of the RNABP (the titration will decrease nonspecific binding), and the tolerance of the antibody:antigen interaction to stringent wash conditions. The results of these experiments can be checked first by western blot, and subsequently using the pilot CLIP protocol described in the second half of this protocol. RNase-treated cross-linked RNABP:RNA complexes from mixed lysates or cell pellets are immunoprecipitated using conditions optimized in the first half of the protocol. 3' Linkers are added, the RNA is radiolabeled, and the complexes are purified on SDS-PAGE. The pilot experiment will identify the optimal RNase concentration for the particular sample and will assess the quality and purity of the RNABP-RNA complexes following labeling of the RNA tags with 32P. This can be done without ligation of the 3' linker as described in the main protocol below. The pilot experiment assesses whether sufficient RNA-protein complexes can be detected by autoradiography and whether contaminating RNA ligands are present in immunoprecipitations compared with control samples. Once it is confirmed that the signal-to-noise ratio for detection of RNA-protein complexes after immunoprecipitation is sufficient, the optimal immunoprecipitation conditions should be incorporated into the general CLIP protocol including the steps of cross-linking, RNase digestion, linker ligation, and labeling of RNA "tags," and the results analyzed by autoradiography.

Ultraviolet (UV) Cross-Linking of Live Cells, Lysate Preparation, and RNase Titration for Cross-Linking Immunoprecipitation (CLIP).

One of the great advantages of RNA CLIP (cross-linking immunoprecipitation) is that RNA-protein complexes can be "frozen" in situ in live cells by ultraviolet (UV) irradiation. This protocol describes UV cross-linking of mammalian tissue culture cells or whole tissues. For the latter, the tissue is typically triturated to allow UV penetration. However, depending on the thickness of the chosen tissue, this may not be necessary. It is preferable to handle the tissue as little as possible, to keep it in ice-cold buffers, and to cross-link as soon after the time of collection as is feasible to preserve native interactions at the time of cross-linking. This protocol also describes cell lysis following cross-linking, as well as treatment with RNase to partially hydrolyze the bound RNA. The first time this protocol is performed, a pilot experiment should be performed to determine the optimal RNase concentration for the particular sample. Once the RNase conditions are optimized this section of CLIP protocol can be repeated on experimental samples before proceeding through the rest of the protocol.

The Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly.

Supported by crystallography studies, secreted ribonuclease of Bacillus pumilus (binase) has long been considered to be monomeric in form. Recent evidence obtained using native polyacrylamide gel electrophoresis and size-exclusion chromatography suggests that binase is in fact dimeric. To eliminate ambiguity and contradictions in the data we have measured conformational changes, hypochromic effect, and hydrodynamic radius of binase. The immutability of binase secondary structure upon transition from low to high protein concentration was registered, suggesting the binase dimerization immediately after translocation through the cell membrane and leading to detection of binase dimers only in the culture fluid regardless of ribonuclease concentration. Our results made it necessary to take a fresh look at the binase stability and cytotoxicity towards virus-infected or tumor cells.

Method Optimization for Extracting High-Quality RNA From the Human Pancreas Tissue

Nucleic acid sequencing is frequently used to determine the molecular basis of diseases. Therefore, proper storage of biological specimens is essential to inhibit nucleic acid degradation. RNA isolated from the human pancreas is generally of poor quality because of its high concentration of endogenous RNase. In this study, we optimized the method for extracting high quality RNA from paired tumor and normal pancreatic tissues obtained from eight pancreatic cancer patients post-surgery. RNA integrity number (RIN) was checked to evaluate the integrity of RNA, we tried to extract the RNA with an RIN value of 8 or higher that allows for the latest genetic analysis. The effect of several parameters, including the method used for tissue lysis, RNAlater treatment, tissue weight at storage, and the time to storage after surgical resection, on the quantity and quality of RNA extracted was examined. Data showed that the highest quantity of RNA was isolated using a combination of manual and mechanical methods of tissue lysis. Additionally, sectioning the tissues into small pieces (<100 mg) and treating them with RNAlater solution prior to storage increased RNA stability. Following these guidelines, high quality RNA was obtained from 100% (8/8) of tumor tissues and 75% (6/8) of normal tissues. High-quality RNA was still stable under repeated freezing and thawing. The application of these results during sample handling and storage in clinical settings will facilitate the genetic diagnosis of diseases and their subsequent treatment.

Influence of RNase E deficiency on the production of stx2-bearing phages and Shiga toxin in an RNase E-inducible strain of enterohaemorrhagic Escherichia coli (EHEC) O157:H7.

In enterohaemorrhagic Escherichia coli (EHEC), stx1 or stx2 genes encode Shiga toxin (Stx1 or Stx2, respectively) and are carried by prophages. The production and release of both stx phages and toxin occur upon initiation of the phage lytic cycle. Phages can further disseminate stx genes by infecting naïve bacteria in the intestine. Here, the effect of RNase E deficiency on these two virulence traits was investigated. Cultures of the EHEC strains TEA028-rne containing low versus normal RNase E levels or the parental strain (TEA028) were treated with mitomycin C (MMC) to induce the phage lytic cycle. Phages and Stx2 titres were quantified by the double-agar assay and the receptor ELISA technique, respectively. RNase E deficiency in MMC-treated cells significantly reduced the yield of infectious stx2 phages. Delayed cell lysis and the appearance of encapsidated phage DNA copies suggest a slow onset of the lytic cycle. However, these observations do not entirely explain the decrease of phage yields. stx1 phages were not detected under normal or deficient RNase E levels. After an initial delay, high levels of toxin were finally produced in MMC-treated cultures. RNase E scarcity reduces stx2 phage production but not toxin. Normal concentrations of RNase E are likely required for correct phage morphogenesis. Our future work will address the mechanism of RNase E action on phage morphogenesis.

An Effective Wood DNA Extraction Protocol for Three Economic Important Timber Species of India

Extraction of DNA from fresh tissues is routine in studies of tropical forest species, but DNA extraction from wood is considered as difficult due to its highly degraded nature and adequate quality of genomic DNA extraction is essential for molecular studies. Very few studies have validated the potential for isolating DNA from dried wood (Heartwood and Sapwood). Wood genomic DNA extraction is difficult from mature timber (Teak (Tectona grandis f; verbanaceae), Black Rosewood (Dalbergia latifolia f; Fabaceae) Ben Teak (Lagerstroemia lanceolata f; Lytheraceae) tissues due to presence of high quantity of secondary metabolites polyphenols, tannins and terpenoids and protein inhibitors. Mostly in laboratories DNA extraction kits are available but by using kits, DNA yield is very low and it is quite expensive too. We have standardized and validated the DNA extraction through manual protocol which is applicable for Bark, Sapwood and Heartwood samples of tree species which contains huge amount of inflexible tissues and fibers. The quality of the DNA was tested by spectrophotometer, gel electrophoresis and PCR (ISSR and SSR) amplification. An avrage DNA yield for heartwood ranges from 0.186 - 0.166 μg/μL and sapwood was ranges from 0.26 - 0.244 μg/μL. Modification of CTAB method was by addition of polyvinylpyrrolidone (PVP) appx 0.25%, variation in Rnase concentration, proteinase treatment with different concentration and incubation time. In order to evaluate the standardized wood genomic DNA extraction protocol, we compared it with the mature leaf and core samples (heartwood and sapwood) of the same timber species. The outcome was also quantified and proved by means of polymerase chain reaction analysis by using ISSR and SSR microsatellite markers conducted with isolated pure DNAs. This modified protocol made increased yield and purity of wood total genomic DNA and facilitate the important application of forensic timber species effort.

Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery.

Small interfering RNA (siRNA) is a promising molecule for gene therapy, but its therapeutic administration remains problematic. Among the recently proposed vectors, cell-penetrating peptides show great promise in in vivo trials for siRNA delivery. Human protein DMBT1 (deleted in malignant brain tumor 1) is a pattern recognition molecule that interacts with polyanions and recognizes and aggregates bacteria. Taking advantage of these properties, we investigated whether specific synthetic DMBT1-derived peptides could be used to formulate nanoparticles for siRNA administration. Using an electrophoretic mobility shift assay and UV spectra, we identified two DMBT1 peptides that could encapsulate the siRNA with a self- and co-assembly mechanism. The complexes were stable for at least 2 hr in the presence of either fetal bovine serum (FBS) or RNase A, with peptide-dependent time span protection. ζ-potential, circular dichroism, dynamic light scattering, and transmission electron microscopy revealed negatively charged nanoparticles with an average diameter of 10–800 nm, depending on the reaction conditions, and a spherical or rice-shaped morphology, depending on the peptide and β-helix conformation. We successfully transfected human MCF7 cells with fluorescein isothiocyanate (FITC)-DMBT1-peptide-Cy3-siRNA complexes. Finally, DMBT1 peptides encapsulating an siRNA targeting a fluorescent reporter gene showed efficient gene silencing in MCF7-recombinant cells. These results lay the foundation for a new research line to exploit DMBT1-peptide nanocomplexes for therapeutic siRNA delivery.