Ribonucleic Acid Concentrations Research Articles

Development of a national proficiency test for SARS-CoV-2 detection by PCR in Colombia.

Proficiency testing (PT) is a tool for ensuring the validity of results of testing laboratories and is essential when laboratories are working with assays authorised for emergency use or implementing novel techniques for detecting emerging pathogens. In collaboration with the National Health Institute of Colombia and with international support, we developed a qualitative PT for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcription polymerase chain reaction (RT-PCR). A proficiency test item (PTI) based on reference material (research grade) produced by the National Institute of Standards and Technologies (NIST) was prepared and characterised using three positive samples with varying concentrations of SARS-CoV-2 ribonucleic acid (RNA) and two negative (control) samples. Tests were distributed to 121 laboratories across the national network of public health laboratories in Colombia. Positive samples had varying concentrations of SARS-CoV-2 RNA and were quantified by digital PCR (RT-ddPCR) assays for the E gene of SARS-CoV-2. We tested the ability of laboratories to detect low and high levels of viral RNA using samples with SARS-CoV-2 RNA concentrations of 1417 ± 216, 146 ± 28, and 14 ± 10 copies /uL (expanded uncertainty, k = 2, 95% confidence level) We also performed a semiquantitative analysis of instrumental responses (Ct values) reported by participating laboratories and homogeneity, stability, and characterisation studies of the produced materials to determine the adequacy of these materials and methods for use in the qualitative PT scheme. The PT evaluated reports for individual target genes from each laboratory; 98.3% of laboratories had satisfactory performance and the remaining 1.7% of laboratories had unsatisfactory performance for the detection of at least one of the reported genes. This PT scheme identified the potential metrological weaknesses of laboratories in the detection of SARS-CoV-2 by RT-PCR and may facilitate improvements in the quality of measurements from the perspective of public health surveillance.

Fat from Hermetia illucens Alters the Cecal Gut Microbiome and Lowers Hepatic Triglyceride Concentration in Comparison to Palm Oil in Obese Zucker Rats

BackgroundPalm oil (PO) is the most widely utilized plant oil for food production. Owing to the great ecologic problems associated with PO production, sustainably produced fats, such as insect fat, might be a suitable alternative. ObjectivesThe hypothesis was tested that fat from Hermetia illucens larvae (HF) compared with PO and soybean oil (SO) has no adverse effects on hepatic lipid metabolism, plasma metabolome, and cecal microbiome in obese Zucker rats. MethodsThirty male obese Zucker rats were randomly assigned to 3 groups (SO, PO, HF; n = 10 rats/group) and fed 3 different semisynthetic diets containing either SO, PO, or HF as the main fat source for 4 wk. The effects were evaluated by measurement of liver and plasma lipid concentrations, liver transcriptomics, targeted plasma metabolomics, and cecal microbiomics. ResultsSupplementation of HF reduced hepatic triglyceride concentration and messenger ribonucleic acid concentrations of selected genes involved in fatty acid and triglyceride synthesis in comparison to PO (P < 0.05). Pairwise comparison of the Simpson index and Jaccard index showed a higher cecal microbial α- and β-diversity in rats fed the HF diet than in rats fed the PO diet (P = 0.015 and P = 0.027), but no difference between rats fed the diets with SO or PO. Taxonomic analysis of the cecal microbial community revealed a lower abundance of Clostridium_sensu_stricto_1 and a higher abundance of Blautia, Mucispirillum, Anaerotruncus, Harryflintia, and Peptococcus in rats supplemented with HF than in rats supplemented with PO (P < 0.05). ConclusionsHF, compared with PO, has liver lipid-lowering effects in obese Zucker rats, which may be caused by a shift in the gut microbial community. Thus, HF might serve as a sustainably produced fat alternative to PO for food production.

Distinct Platelet Ribonucleic Acid Signatures in Patients with Pulmonary Hypertension.

Rationale: Pulmonary hypertension encompasses progressive disorders leading to right ventricular dysfunction and early death. Late detection is an important cause of poor clinical outcomes. However, biomarkers that accurately predict the presence of pulmonary hypertension are currently lacking. Objectives: In this study, we provide evidence that blood platelets contain a distinctive ribonucleic acid (RNA) profile that may be exploited for the detection of pulmonary hypertension. Methods: Blood platelet RNA was isolated prospectively from 177 prevalent patients with different subtypes of pulmonary hypertension as well as 195 control subjects clinically not suspected of pulmonary hypertension. Sequencing libraries were created using SMARTer (Switching Mechanism at 5' end of RNA Template) copy desoxyribonucleic acid amplification and sequenced on the Illumina High Throughput Sequencing platform. RNA-sequencing reads were mapped to the human reference genome, and intron-spanning spliced RNA reads were selected. Differential spliced RNA panels were calculated by analysis of variance statistics. A particle swarm optimization-enhanced classification algorithm was built employing a development (n = 213 samples) and independent validation series (n = 159 samples). Results: We detected a total of 4,014 different RNAs in blood platelets from patients with pulmonary hypertension (n = 177) and asymptomatic control subjects (n = 195). Gene ontology analysis revealed enhanced RNA concentrations for genes related to RNA processing, translation, and mitochondrial function. A particle swarm optimization-selected RNA panel of 408 distinctive differentially spliced RNAs mediated detection of pulmonary hypertension with 93% sensitivity, 62% specificity, 77% accuracy, 0.89 (95% confidence interval, 0.83-0.93) area under the curve, and a negative predictive value of 91% in the independent validation series. The prediction score was independent of age, sex, smoking, pulmonary hypertension subtype, and the use of pulmonary hypertension-specific medication or anticoagulants. Conclusions: A platelet RNA panel may accurately discriminate patients with pulmonary hypertension from asymptomatic control subjects. In the light of current diagnostic delays, this study is the starting point for further development and evaluation of a platelet RNA-based blood test to ultimately improve early diagnosis and clinical outcomes in patients with pulmonary hypertension.

Design of robust [formula omitted] state estimator for delayed polytopic uncertain genetic regulatory networks: Dealing with finite-time boundedness

In this paper, a robust H∞ design approach is proposed to accomplish the state estimation task for a class of continuous-time gene regulatory networks (GRNs) in the finite-time framework. For the GRNs under investigation, the distributed inter-node time delays and intra-node time delays are, respectively, taken into account in the feedback regulation function and the translation process. Moreover, the parameter uncertainty with convex polytopic constraint is introduced to characterize the unavoidable modeling errors induced by parameter sensitivity, calibration accuracy, abrupt structural changes and failures, etc. The primary objective of this paper is to design a robust state estimator for the considered GRNs based on the measured concentrations of messenger ribonucleic acid and protein such that the finite-time boundedness and the H∞ performance are guaranteed. By resorting to the Lyapunov stability theory, the Jensen’s inequality and the Gronwall inequality, some sufficient conditions are established in terms of linear matrix inequalities. Subsequently, a set of parameter matrices with special structures is designed by means of the convex optimization techniques. Finally, a numerical example is provided to demonstrate the finite-time boundedness and the robust performance of the developed state estimation approach.

Degradation of SARS-CoV-2 specific ribonucleic acid in samples for nucleic acid amplification detection

The degradation of SARS-CoV-2 specific ribonucleic acid (RNA) was investigated by a numerical modeling approach based on nucleic acid amplification test (NAAT) results utilizing the SmartAmp technique. The precision of the measurement was verified by the relative standard deviation (RSD) of repeated measurements at each calibration point. The precision and detection limits were found to be 6% RSD (seven repeated measurements) and 94 copies/tube, respectively, at the lowest calibration point. RNA degradation curves obtained from NAAT data on four different temperatures were in good agreement with the first-order reaction model. By referring to rate constants derived from the results, the Arrhenius model was applied to predict RNA degradation behavior. If the initial RNA concentration was high enough, such as in samples taken from infected bodies, the NAAT results were expected to be positive during testing. On the other hand, if initial RNA concentrations were relatively low, such as RNA in residual viruses on environmental surfaces, special attention should be paid to avoid false-negative results. The results obtained in this study provide a practical guide for RNA sample management in the NAAT of non-human samples.

Tracking SARS-CoV-2 RNA through the Wastewater Treatment Process.

Municipal sewage carries degraded and intact viral particles and RNA (ribonucleic acid) of SARS-CoV-2 (severe acute respiratory coronavirus 2), shed by COVID-19 (coronavirus disease 2019) patients, to sewage and eventually to wastewater treatment plants. Proper wastewater treatment can prevent uncontrolled discharges of the virus into the environment. However, the role of different wastewater treatment stages in reducing viral RNA concentrations is, thus far, unknown. Here, we quantified SARS-CoV-2 RNA in raw sewage and during the main stages of the activated sludge process from two wastewater treatment plants in Israel, on three different days during the 2020 COVID-19 outbreak. To reduce the detection limit, samples were concentrated prior to quantification by real-time polymerase chain reaction by a factor of 2–43 using ultrafiltration. On average, ∼1 log RNA removal was attained by each of the primary and secondary treatment steps; however, >100 copies of SARS-CoV-2 RNA/mL remained in the secondary effluents. Following chlorination, SARS-CoV-2 RNA was detected only once, likely due to an insufficient chlorine dose. Our results emphasize the capabilities and limitations of the conventional wastewater treatment process in reducing the SARS-CoV-2 RNA concentration and present preliminary evidence for the importance of tertiary treatment and chlorination in reducing dissemination of the virus to the environment.

Ultrasensitive and Selective Detection of SARS-CoV-2 Using Thermotropic Liquid Crystals and Image-Based Machine Learning

Rapid, robust virus-detection techniques with ultrahigh sensitivity and selectivity are required for the outbreak of the pandemic coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Here, we report that the femtomolar concentrations of single-stranded ribonucleic acid (ssRNA) of SARS-CoV-2 trigger ordering transitions in liquid crystal (LC) films decorated with cationic surfactant and complementary 15-mer single-stranded deoxyribonucleic acid (ssDNA) probe. More importantly, the sensitivity of the LC to the SARS ssRNA, with a 3-bp mismatch compared to the SARS-CoV-2 ssRNA, is measured to decrease by seven orders of magnitude, suggesting that the LC ordering transitions depend strongly on the targeted oligonucleotide sequence. Finally, we design a LC-based diagnostic kit and a smartphone-based application (app) to enable automatic detection of SARS-CoV-2 ssRNA, which could be used for reliable self-test of SARS-CoV-2 at home without the need for complex equipment or procedures.

Ultrasensitive and Selective Detection of SARS-CoV-2 Using Thermotropic Liquid Crystals and Image-Based Machine Learning

Rapid, robust virus detection techniques with ultrahigh sensitivity and selectivity are required for the outbreak of the pandemic coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Here, we report that femtomolar concentrations of single-stranded ribonucleic acid (ssRNA) of SARS-CoV-2 trigger ordering transitions in liquid crystal (LC) films decorated with cationic surfactants and complementary 15-mer single-stranded deoxyribonucleic acid (ssDNA) probes. More importantly, the sensitivity of the LC to the severe acute respiratory syndrome (SARS) ssRNA, with a 3 base pair-mismatch compared to the SARS-CoV-2 ssRNA, was measured to decrease by seven orders of magnitude, suggesting that the LC ordering transitions depend strongly on the targeted oligonucleotide sequence. Finally, we designed a LC-based diagnostic kit and a smartphone-based application (App) to enable automatic detection of SARS-CoV-2 ssRNA, which can be used for reliable self-test of SARS-CoV-2 at home without the need for complex equipment or procedures.Funding: J.P. and X.W. thank the funding support by the startup funds of The Ohio State University (OSU) and X.W. thanks OSU Institute for Materials Research Kickstart Facility Grant. X.B. thanks the funding support by the startup funds of Davidson School of Chemical Engineering at Purdue University. S.S. and R.Q. thanks the funding support by Office of Naval Research (ONR Grant N00014-17-1-2928).Conflict of Interest: The Ohio State University has filed a patent application (Application Number 63066000) on the work described in this manuscript. The inventors listed on the patent application are X.W., X.B., Q.R., X.Y. and A.M.R. The authors declare no other competing interests.

Effect of digital hypothermia on lamellar inflammatory signaling in the euglycemic hyperinsulinemic clamp laminitis model.

BackgroundContinuous digital hypothermia (CDH) prevents lamellar failure in the euglycemic hyperinsulinemic clamp (EHC) model of laminitis, but the protective mechanisms are unclear.Hypothesis/ObjectivesTo determine if CDH inhibits lamellar inflammatory signaling in the EHC model of laminitis.AnimalsEight Standardbred horses.MethodsProspective experimental study. Horses underwent an EHC, with 1 forelimb treated with CDH and the other kept at ambient temperature (AMB). Horses were euthanized 48 hours after initiation of the EHC and lamellar tissue was analyzed via polymerase chain reaction (pro‐inflammatory cytokine and chemokine genes—CXCL1, CXCL6, CXCL8, IL‐6, MCP‐1, MCP‐2, IL‐1β, IL‐11, cyclooxygenase 1 and 2, tumour necrosis factor‐alpha [TNF‐α], E‐selectin, and intercellular adhesion molecule‐1 [ICAM‐1]) and immunoblotting (phosphorylated and total signal transducer and activator of transcription 1 [STAT1] and STAT3).ResultsCompared to AMB, lamellar messenger ribonucleic acid (mRNA) concentrations of CXCL6 (P =.02), CXCL8 (P = .008), IL‐6 (P = .008), IL‐1β (P = .008), IL‐11 (P = .008), and cyclooxygenase‐2 (P = .008) were decreased in CDH. Cyclooxygenase‐1 (P = .008) was increased in CDH, while CXCL1 (P = .15), MCP‐1 (P = .05), MCP‐2 (P = .46), TNF‐α (P = .05), E‐selectin (P = .15), and ICAM‐1 (P = .15) mRNA were not significantly different. Compared to AMB, lamellar concentration of total STAT3 protein was decreased in CDH (P < .001), but there was no change in phosphorylated STAT3 (P‐STAT3 [S727] P = .19; P‐STAT3 [Y705] P = .05). There was no change in lamellar concentrations of total STAT1 (P = .75) or phosphorylated STAT1 (P‐STAT1 [S727], P = .25; P‐STAT1 [Y701], P = .64).Conclusions and Clinical ImportanceThese data add further support for the use of CDH as a first aid treatment for severe acute laminitis associated with hyperinsulinemia in horses.

An improved procedure for the isolation of Ribonucleic acid from methicillin-resistant Staphylococcus aureus

Extraction and purification of ribonucleic acid (RNA) from Gram-positive methicillin resistant Staphylococcus aureus (MRSA) is problematic, because the MRSA has a rigid cell wall that contains lipoteichoic acid and peptidoglycan, thus causing difficulty when utilizing the standard methods. For this reason, the aim of the current study was to improve and modify the method of extraction of RNA from MRSA, with good integrity, purity, low cost, and with saved time of extraction. A fast and an inexpensive method involving the use of acid phenol: chloroform (5: 1 [v/v]) at low pH (4.5), with lysostaphin and Triton X-100 for effective isolation of RNA from the MRSA is developed. As a result of this study, yields of this method presented high concentration of RNA 1175.26 ng/ µl/ 3 ml of bacterial culture broth, with high RNA integration number (RIN). In similar assays such as using; the RNeasy Mini kit, GeneJET RNA purification kit, TRIzol kit and hot phenol: chloroform (1: 1 [v/v]) extraction method, they yielded low concentrations of RNA (92-700 ng/ µl); with lower purity, quantity, and also little integrity, compared to using the current acid phenol chloroform (5: 1 [v/v]) extraction method. In conclusion, this new method for extraction of RNA from MRSA can be used to save time, cost, and provide high quality of RNA.

Long Non-Coding Ribonucleic Acid Small Nucleolar Host Gene 1 Regulates the Ischemic Stroke by Targeting Micro Ribonucleic Acid-376a-Mediated Cystathionine ? Synthase/Hydrogen Sulfide Axis Pathway

Ischemic stroke was a brain disease with high mortality and disability rate. Previous studies have shown that the expression of Micro Ribonucleic acid-376a in the serum of stroke patients was increased, suggesting that Micro Ribonucleic acid-376a was closely related to the occurrence and development of stroke. However, its mechanism has not been reported. The purpose of this study was to explore the regulatory mechanism of Long non-coding ribonucleic acid small nucleolar host gene 1 on Ischemic stroke through targeting the Micro Ribonucleic acid-376a-mediated cystathionine β synthase hydrogen sulfide axis pathway. The Ischemic stroke model of Human Cerebral Microvascular Endothelial Cell Line/D3 cells was established by the induce of Oxygen-glucose deprivation. The Micro Ribonucleic acid concentrations of small nucleolar host gene 1, Micro Ribonucleic acid-376a, and inflammatory cytokines were detected by Reverse transcription- polymerase chain reaction. Western blotting was used to detect the expression of cystathionine β synthase and apoptosis-related proteins. The apoptosis was analyzed by flow cytometry and the concentration of hydrogen sulfide was detected by the kit. The interaction between long non-coding ribonucleic acid, micro ribonucleic acid, and target genes was confirmed by the luciferase test. Our study showed that under the condition of Oxygen-glucose deprivation induction, when Micro Ribonucleic acid-376a was up-regulated, the Micro Ribonucleic acid concentration of small nucleolar host gene 1 and cystathionine β synthase in Human Cerebral Microvascular Endothelial Cell Line/D3 cells was down-regulated. Further results showed that the overexpression of Micro Ribonucleic acid-376a promoted apoptosis and inflammation, while overexpression of small nucleolar host gene 1 alleviated these processes. In terms of mechanism, Micro Ribonucleic acid-376a was the target of small nucleolar host gene 1, and cystathionine β synthase was the target of Micro Ribonucleic acid-376a. In addition, small nucleolar host gene 1 regulates the expression of cystathionine β synthase by inhibiting Micro Ribonucleic acid-376a, thus exerting its function. Long non-coding ribonucleic acid small nucleolar host gene 1 inhibits the apoptosis and inflammation of Ischemic stroke cells by inhibiting Micro Ribonucleic acid-376a and up-regulating cystathionine β synthase/hydrogen sulfide signal. These results reveal the potential mechanism of Ischemic stroke and provide a potential target for the treatment of Ischemic stroke.

Isolation of RNA and beta-NAD by phenylboronic acid functionalized, monodisperse-porous silica microspheres as sorbent in batch and microfluidic boronate affinity systems.

Monodisperse-porous silica microspheres 5.5 μm in size were obtained by a staged shape templated hydrolysis-condensation method, with a bimodal pore-size distribution. 3-aminophenylboronic acid (APBA) was covalently attached onto the silica microspheres with a capacity of 0.476 mmol APBA/g microspheres. The boronate affinity isolation behaviour of ribonucleic acid (RNA) containing cis-diol at 3'-end was investigated by using APBA attached-silica microspheres as the sorbent in batch fashion. A short-chain diol carrying agent, β-nicotinamide adenine dinucleotide (β-NAD) was used as a target molecule with stronger affinity for phenylboronic acid ligand. The maximum equilibrium adsorptions for RNA and β-NAD were determined as 60 and 159 mg/g sorbent, respectively. By using the synthesized sorbent, phosphate buffer at pH 7.0 containing sorbitol was successfuly used as a mild elution medium for obtaining quantitative desorptions with both RNA and β-NAD. RNA isolations from mammalian and bacterial cells were successfully performed while protecting the structural integrity of RNA via boronate affinity interaction in batch fashion. A microfluidic boronate affinity system including a microcolumn 300 μm in diameter was also constructed using APBA attached-silica microspheres as the stationary phase. The breakthrough curves of microfluidic system were obtained by studying with different feed concentrations of RNA and β-NAD. Quantitative desorptions and satisfactory isolation yields were obtained with RNA and β-NAD in the microfluidic system. The proposed system is useful for boronate affinity applications in genomics or proteomics in which valuable cis-diols at low concentrations are recovered from low-volume samples.

%MinMax: A versatile tool for calculating and comparing synonymous codon usage and its impact on protein folding.

Most amino acids can be encoded by more than one synonymous codon, but these are rarely used with equal frequency. In many coding sequences the usage patterns of rare versus common synonymous codons is nonrandom and under selection. Moreover, synonymous substitutions that alter these patterns can have a substantial impact on the folding efficiency of the encoded protein. This has ignited broad interest in exploring synonymous codon usage patterns. For many protein chemists, biophysicists and structural biologists, the primary motivation for codon analysis is identifying and preserving usage patterns most likely to impact high-yield production of functional proteins. Here we describe the core functions and new features of %MinMax, a codon usage calculator freely available as a web-based portal and downloadable script (http://www.codons.org). %MinMax evaluates the relative usage frequencies of the synonymous codons used to encode a protein sequence of interest and compares these results to a rigorous null model. Crucially, for analyzing codon usage in common host organisms %MinMax requires only the coding sequence as input; with a user-input codon frequency table, %MinMax can be used to evaluate synonymous codon usage patterns for any coding sequence from any fully sequenced genome. %MinMax makes no assumptions regarding the impact of transfer ribonucleic acid concentrations or other molecular-level interactions on translation rates, yet its output is sufficient to predict the effects of synonymous codon substitutions on cotranslational folding mechanisms. A simple calculation included within %MinMax can be used to harmonize codon usage frequencies for heterologous gene expression.

Identification of RNA Hydrolysis Products in NaOH-EDTA Extracts using 31P NMR Spectroscopy

Ribonucleic acid (RNA) is the most abundant form of organic phosphorus (P) in plant and microbial biomass and is therefore expected to be present in materials such as soils, sediments, composts, and manures. Phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy is increasingly used to characterize organic P in these materials, usually following extraction into a mixture of sodium hydroxide and ethylenediaminetetraacetic acid (NaOH-EDTA). Under these alkaline conditions, RNA is hydrolysed, providing a distinctive pattern or “fingerprint” in the 31P NMR spectrum. Complete assignment of the eight ribonucleotides produced was achieved using a spiking approach. The near coincidence in chemical shift of β-glycerophosphate and two of the ribonucleotide peaks complicates quantification of RNA concentrations when phospholipids are also present, but an approach based on quantifying signal in the most well-separated ribonucleotide peaks is suggested.

Genetic Polymorphism and Messenger Ribonucleic Acid Concentrations of TNFα and TGFβ Genes in Patients with Chronic Lower Limb Infections.

The number of chronic lower limb infections and their complications as venous and diabetic ulcers and chronic calf dermatitis is increasing worldwide. The clinical course and outcome in the immune responses to infection have been shown to be associated with genetic polymorphisms. The aim of study was to investigate frequencies of chosen single nucleotide polymorphisms (SNPs) in TNFα and TGFβ genes in patients with chronic lower limb infections and evaluate expression of messenger ribonucleic acid (mRNA) concentrations in chronic leg ulcers. Patients were divided into three groups: (group A) chronic venous leg ulcers, (group B) chronic post-traumatic non-healing wounds, and (group C) infected ischemic necrosis of the foot. Blood donors comprised the control group. Detection of polymorphisms was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and gene expression by real-time PCR methods. Patients in all groups showed higher frequency of TNFα gene polymorphism -308GG and lower frequency of -308GA genotypes than controls. The mutated homozygote AA was higher in groups A and B than in controls. The TGFβ74GG genotype was represented at highest values in group B. The GC genotype was found in all groups at a similar concentration lower than in controls. Genotypes TGFβ29TT and TC were represented at similar concentrations as controls. Analyses showed that the presence of the polymorphic allele -308A of TNFα gene was correlated with an increased concentration of gene expression in patients with chronic leg ulcers (group A). In the case of both TGFβ gene polymorphisms the presence of polymorphic allele C resulted in increased TGFβ gene expression. Comparison of genotypes in polymorphic sites in TNFα and TGFβ genes with their expression concentrations showed that the presence of polymorphic alleles could predispose to increased production of their proteins. Patients with prolonged non-healing wounds should have their genotypes studied, and in cases of mutation, long-term antibiotic and immune protein supply should be considered.

Evaluation of Ribonucleic Acid (RNA) amplification efficiency to determine optimal RNA concentration ranges from formalin-fixed paraffin-embedded rodent tissues for gene expression analysis

The goal of this study was to establish Ribonucleic Acid (RNA) amplification efficiencies and determine optimal concentration ranges for downstream real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses with RNA isolated from formalin-fixed paraffin-embedded (FFPE) and frozen rat tissues. Messenger RNA was isolated using magnetic particle processing technology. RNA concentrations and integrity were assessed from frozen and FFPE rat liver, lung, kidney, heart, mammary gland, pancreas, skin, and duodenum, and cycle thresholds (Cts) were obtained from each dilution of an RNA template. PCR amplification efficiencies and optimal template ranges were calculated for FFPE and frozen tissue samples. Calculated percent efficiency evaluations for FFPE and frozen tissues showed that FFPE samples, at the RNA concentrations tested, were of acceptable quality for downstream gene expression analyses.

The effect of tRNA levels on decoding times of mRNA codons.

The possible effect of transfer ribonucleic acid (tRNA) concentrations on codons decoding time is a fundamental biomedical research question; however, due to a large number of variables affecting this process and the non-direct relation between them, a conclusive answer to this question has eluded so far researchers in the field. In this study, we perform a novel analysis of the ribosome profiling data of four organisms which enables ranking the decoding times of different codons while filtering translational phenomena such as experimental biases, extreme ribosomal pauses and ribosome traffic jams. Based on this filtering, we show for the first time that there is a significant correlation between tRNA concentrations and the codons estimated decoding time both in prokaryotes and in eukaryotes in natural conditions (−0.38 to −0.66, all P values <0.006); in addition, we show that when considering tRNA concentrations, codons decoding times are not correlated with aminoacyl-tRNA levels. The reported results support the conjecture that translation efficiency is directly influenced by the tRNA levels in the cell. Thus, they should help to understand the evolution of synonymous aspects of coding sequences via the adaptation of their codons to the tRNA pool.

Metabolic regulation in meagre, Argyrosomus regius (Asso, 1801): Study of gene-diet interactions on lipid metabolism

Metabolic regulation in meagre, Argyrosomus regius (Asso, 1801): Study of gene-diet interactions on lipid metabolism

Enhancing the anaerobic digestion potential of dairy waste activated sludge by two step sono-alkalization pretreatment

High efficiency resource recovery from dairy waste activated sludge (WAS) has been a focus of attention. An investigation into the influence of two step sono-alkalization pretreatment (using different alkaline agents, pH and sonic reaction times) on sludge reduction potential in a semi-continuous anaerobic reactor was performed for the first time in literature. Firstly, effect of sludge pretreatment was evaluated by COD solubilization, suspended solids reduction and biogas production. At optimized condition (4172kJ/kg TS of supplied energy for NaOH – pH 10), COD solubilization, suspended solids reduction and biogas production was 59%, 46% and 80% higher than control. In order to clearly describe the hydrolysis of waste activated sludge during sono-alkalization pretreatment by a two step process, concentrations of ribonucleic acid (RNA) and bound extracellular polymeric substance (EPS) were also measured. Secondly, semi-continuous process performance was studied in a lab-scale semi-continuous anaerobic reactor (5L), with 4L working volume. With three operated SRTs, the SRT of 15 d was found to be most appropriate for economic operation of the reactor. Combining pretreatment with anaerobic digestion led to 58% and 62% of suspended solids and volatile solids reduction, respectively, with an improvement of 83% in biogas production. Thus, two step sono-alkalization pretreatment laid the basis in enhancing the anaerobic digestion potential of dairy WAS.

Waste activated sludge hydrolysis during ultrasonication: Two-step disintegration

In order to clearly describe the hydrolysis of waste activated sludge (WAS) during ultrasonication by a 2-step disintegration process, concentrations of ribonucleic acid (RNA) and bound extracellular polymeric substance (EPS) were measured. Apparently, different decreasing patterns of RNA and EPS concentrations during WAS hydrolysis made it possible to distinguish the floc disintegration (FD) and cell lysis (CL). Initially, FD and CL appear to take simultaneously, but the dominant hydrolytic process is shifted from FD to CL after 10min of ultrasonication. Additional kinetic analysis of WAS hydrolysis was also conducted. A five-fold greater hydrolysis rate constant of FD relative to that of CL was observed, reflecting the different strengths of floc and cells. Therefore, different rates of increased solubilization during WAS hydrolysis appear to account for the initial disintegration of the rather loose part (sludge floc) and the subsequent disintegration of the rigid part (microbial cells).