Ratio Of RNA Research Articles

Simultaneous Extraction of Bone Marrow RNA and DNA from Patients with Hematologic Diseases Using a Combined Magnetic Bead Method within 1 Hour.

TRIzolTM is widely used for RNA and DNA extraction. However, this method is laborious and time-consuming. The objective of this study was to validate a time-effective and labor-saving protocol. The TRIzol method was used to separate the aqueous phase, protein, and phenol layer of bone marrow samples from 12 patients with hematological diseases. Subsequently, RNA and DNA were extracted from the aqueous layer containing RNA and phenol layer containing DNA, respectively, using magnetic bead extraction kits. The quantity and purity of extracted RNA and DNA were examined using a NanoDrop spectrophotometer. Quantitative fluorescence PCR amplification of the ABL1 gene was performed to verify the effectiveness of the extracted RNA and DNA for downstream experiments. RNA and DNA from another 16 bone marrow samples were extracted to compare the performance of the two methods. Co-extraction of RNA and DNA was completed within 1 h. The data showed that RNA and DNA yield ranged from 13.1 to 204.5 ng/µL and 33.1 to 228.8 ng/µL, respectively. The A260/A280 ratios of RNA and DNA samples ranged from 1.82 to 2.01 and 1.73 to 1.91, respectively. RNA and DNA extracted using this scheme exhibited ideal performance in quantitative fluorescence PCR. The present protocol showed better quality and effectiveness in extracting RNA and DNA compared to the TRIzol method. This protocol for RNA and DNA co-extraction is fast, labor-saving, and high throughput. It can be adopted for routine molecular biology analyses, particularly for non-reproducible specimens.

Development of a multiple-biomarker approach using the green-lipped mussel Perna viridis for marine pollution monitoring: a case study in Victoria Harbour, Hong Kong

Pollutants often exist as mixtures in environmental settings, creating a challenge in selecting the most effective combination of biomarkers for routine monitoring. This study was conducted seasonally in Victoria Harbour, Hong Kong, to compare the responses of nine biomarkers in the green-lipped mussel Perna viridis with respect to its tissue levels of persistent organic pollutants and heavy metals. Multivariate statistical techniques were utilised to determine the single best predictor and optimal subset of biomarkers in P. viridis for each of the four scenarios: representing overall biomarker responses in the dry season, and wet season, as well as correlating tissue levels of mixed pollutants in the dry season, and wet season. Our findings recommend lysosomal destabilisation, and the nucleic acid ratio of RNA to DNA, as the core biomarkers in P. viridis for marine pollution monitoring. The non-specificity of these biomarkers allows effective identification of pollution hotspots and guides further detailed assessment.

A chromosome-level genome assembly of the forestry pest Coronaproctus castanopsis

As an important forestry pest, Coronaproctus castanopsis (Monophlebidae) has caused serious damage to the globally valuable Gutianshan ecosystem, China. In this study, we assembled the first chromosome-level genome of the female specimen of C. castanopsis by merging BGI reads, HiFi long reads and Hi-C data. The assembled genome size is 700.81 Mb, with a scaffold N50 size of 273.84 Mb and a contig N50 size of 12.37 Mb. Hi-C scaffolding assigned 98.32% (689.03 Mb) of C. Castanopsis genome to three chromosomes. The BUSCO analysis (n = 1,367) showed a completeness of 91.2%, comprising 89.2% of single-copy BUSCOs and 2.0% of multicopy BUSCOs. The mapping ratio of BGI, second-generation RNA, third-generation RNA and HiFi reads are 97.84%, 96.15%, 97.96%, and 99.33%, respectively. We also identified 64.97% (455.3 Mb) repetitive elements, 1,373 non-coding RNAs and 10,542 protein-coding genes. This study assembled a high-quality genome of C. castanopsis, which accumulated valuable molecular data for scale insects.

Cell-cell communication and initial population composition shape the structure of potato spindle tuber viroid quasispecies.

RNA viruses and viroids replicate with high mutation rates, forming quasispecies, population of variants centered around dominant sequences. The mechanisms governing quasispecies remain unclear. Plasmodesmata regulate viroid movement and were hypothesized to impact viroid quasispecies. Here, we sequenced the progeny of potato spindle tuber viroid intermediate (PSTVd-I) strain from mature guard cells lacking plasmodesmal connections and from in vitro-cultivated mesophyll cell protoplasts from systemic leaves of early-infected tomato (Solanum lycopersicum) plants. Remarkably, more variants accumulated in guard cells compared to whole leaves. Similarly, after extended cell culture, we observed more variants in cultivated mesophyll protoplasts. Coinfection and single-cell sequencing experiments demonstrated that the same plant cell can be infected multiple times by the same or different PSTVd sequences. To study the impact of initial population composition on PSTVd-I quasispecies, we conducted coinfections with PSTVd-I and variants. Two inoculum ratios (10:1 or 1:10) established quasispecies with or without PSTVd-I as the master sequence. In the absence of the master sequence, the percentage of novel variants initially increased. Moreover, a 1:1 PSTVd-I/variant RNA ratio resulted in PSTVd-I dominating (>50%), while the variants reached 20%. After PSTVd-I-only infection, the variants reached around 10%, while after variant-only infection, the variants were significantly more than 10%. These results emphasize the role of cell-to-cell communication and initial population composition in shaping PSTVd quasispecies.

Effect of Replacement of Soybean Meal with Sesame Meal in The Diet of Thai-Chitralada Strain of Oreochromis niloticus (L)

Background: The present study was aimed to determine effect of replacement of soybean meal (SBM) with sesame meal (SSM) in the diet of Juvenile Thai chitralada tilapia. Methods: The study was undertaken with different inclusion levels of sesame meal (SSM) such as T1 (25% SSM), T2 (30% SSM), T3 (35% SSM) and control feed (C) for a period of 90 days. Result: Thai-Chitralada tilapia fed with T3 (35% SSM) diet attained maximum mean weight gain (28.09 g), highest specific growth rate (SGR) (2.69%), best feed conversion ratio (FCR) (1.04), maximum average daily growth (ADG) (0.31 g) and maximum protein efficiency ratio (PER) (8.87). The lowest growth rate (GR) was observed in the group fed with control diet. Diets with 35% SSM replacement upon SBM showed higher amino acid (AA) profile than other treatments and control diet. Analysed AA profile in T3 - 35% SSM was Arg - 2.88, Hist - 0.86, Isol - 0.92, Leu - 2.60, Lys - 3.78, Met - 0.39, Cys - 0.38, Phe - 1.77, Thr - 1.08, Val - 0.99, Tyro - 1.09, Trypt - 0.24. The higher DNA / RNA ratio of Thai-Chitralada tilapia was obtained in T3 - 0.459, followed by T1 - 0.44 and T2 - 0.323. The lower DNA / RNA ratio of Thai-Chitralada was obtained in control - 0.321. One way ANOVA of the data analysis and Duncan multiple range test clearly affirmed that different between and sampling periods, Thai-Tilapia fingerlings had significant difference (P less than 0.05) among the different experimental diets. From the present experiment, it could be concluded that, sesame seed meal can replace 35% SBM in the diets of Thai-Chitralada tilapia.

Cerebrospinal fluid pleocytosis is associated with HIV-1 neuroinvasion during acute infection.

HIV-1 invades the brain within days post-transmission. This study quantitated cerebrospinal fluid (CSF) white blood cell count (WBC) and investigated whether it associated with plasma and CSF HIV-1 RNA during untreated acute HIV infection (AHI). Seventy participants underwent lumbar puncture during Fiebig stages I-V AHI. WBC and HIV-1 RNA with a lower limit of quantification (LLQ) of 80 copies/ml were measured in CSF. Sixty-nine (99%) participants were men, with a median age of 26. Their blood CD4 + and CD8 + T-cell counts were 335 [interquartile range (IQR) 247-553) and 540 (IQR 357-802) cells/μl, respectively. Forty-five (64%) were in Fiebig stages III-V whereas 25 (36%) were in Feibig stages I-II. Fifty-two (74%) experienced acute retroviral syndrome. Median plasma and CSF HIV-1 RNA were 6.10 (IQR 5.15-6.78) and 3.15 (IQR 1.90-4.11) log 10 copies/ml, respectively. Sixteen (23%) CSF samples had HIV-1 RNA below LLQ. Median CSF WBC was 2.5 (IQR 1-8) cells/μl. CSF pleocytosis (WBC >5) was observed in 33% and was only present in CSF samples with detectable HIV-1 RNA. The frequencies of CSF pleocytosis during Fiebig stages III-V and among CSF samples of higher viral load (>1000 copies/ml) were 42 and 45%, respectively. Pleocytosis independently associated with CSF HIV-1 RNA in multivariate analysis [adjusted coefficient: 0.79, 95% confidence interval (CI) 0.41-1.14), P < 0.001] and a lower plasma to CSF HIV-1 RNA ratio ( P < 0.001). CSF pleocytosis was present in one-third of participants with AHI. It associated with higher CSF HIV-1 RNA and a lower plasma to CSF HIV-1 RNA ratio, suggesting a potential association with HIV-1 neuroinvasion.

Screening Earth Analog Exoplanets on the Basis of a Predicted Nitrogen over Phosphorus Ratio

Since the first discovery in 1995, data for over 5300 exoplanets have been documented in the NASA archive, revealing a vast diversity. Identifying life-enabling analogs of the Earth among this rapidly expanding catalog is of major interest. The stability of liquid water at the planetary surface defining the concept of the habitable zone (HZ) around the host star, may be necessary for the emergence of life as we know it but not sufficient. The practically constant atomic ratio nitrogen:phosphorous = 16:1 in oceanic surface layers of our planet Earth was discovered by Redfield in 1934. It corresponds to phytoplanktonic biomass in suspension and appears optimal to fertilize phytoplankton development and therefore the food pyramid of marine life. Loladze and Elser have shown that it corresponds to a homeostatic protein:RNA ratio and is therefore “rooted in the stoichiometry of the foundational structures of life.” I show that according to the recent theory of the chemical differentiation of planets, this optimal ratio is also an intrinsic chemical property of our planet Earth uniquely determined in the solar system by its average orbital radius. On that basis, I propose a criterion of fertility within the HZ of a stellar system, which when applied to screen the public database allows us to sort out an extended list of up to 74 Earth analogs. The latter and its future extensions could provide priority targets for focused detection techniques.

Differential impact of exportin-1-mediated nuclear export of RNAs on the RNA content of extracellular vesicle subpopulations

Extracellular vesicles (EVs) are membrane-enclosed subcellular structures released by all cell types. EVs have important roles in both cellular homeostasis and intercellular communication. Recent progress in the field revealed substantial heterogeneity of EVs even within the size-based EV categories. Here we addressed the question whether the exportin-1 (XPO1)-mediated nuclear export of RNAs contributed to the EV heterogeneity. Size-based populations were separated from the conditioned media of three cell lines (U937, THP-1 and 5/4E8) in steady-state condition. The effects of activation and leptomycin B treatment (to inhibit the XPO1-mediated nuclear export of RNAs) were also tested in the case of the two monocytic cell lines. Agilent Pico and Small chips were used to characterize RNAs, fragment analysis was performed, and EV-associated miRNAs were tested by Taqman assays. As expected, we found the highest small RNA/total RNA ratio and the lowest rRNA/total RNA proportion in small EVs (~ 50–150 nm). Profiles of the small RNAs within different size-based EV categories significantly differed based on the activation status of the EV releasing cells. Leptomycin B had a differential inhibition on the tested small RNAs in EVs, even within the same EV size category. A similar heterogeneity of the EV miRNA content was observed upon cellular activation and nuclear export inhibition. Here we complement the already existing knowledge on EV heterogeneity by providing evidence that the RNA cargo varies depending on the EV size-based category, the releasing cell type, the functional status of the releasing cells and the exportin-1-mediated nuclear export of RNAs.

Optimization of RNA Extraction from Aedes aegypti and Aedes albopictus

RNA extraction is the critical initial stage in analyzing certain gene expressions, further analysis using Real Time PCR technology, and performing virus detection. However, the process of extracting RNA is often hampered by the risk of contamination, resulting in low concentrations of RNA and low purity of RNA. This is often an obstacle in extracting mosquito RNA especially detecting Dengue Virus (Den-V). Dengue virus (Den-V) can cause dangerous diseases in humans such as Dengue Fever (DHF) which is transmitted through the bites of Aedes aegypti and Aedes albopictus mosquitoes. This study aims to find out the effective steps for extracting RNA from Aedes aegypti and Aedes albopictus mosquitoes. The method being compared is a commercial RNA extraction kit with modification (addition of β-mercaptoethanol) and without modification. The results showed that the best DNA concentration and purity were obtained in mosquito samples from modified process. The purity ratio of RNA extracted without modification was 1.971 (0.021 ± 0.800) while with modification it was 2.003 (0.011 ± 0.112). Aedes aegypti had a better average concentration of 7.146 µg/ml for unmodified RNA and 7.613 µg/ml for modified RNA. This research is expected to be a reference for further studies on viruses in Aedes aegypti and Aedes albopictus.

The Mba1 homologue of Trypanosoma brucei is involved in the biogenesis of oxidative phosphorylation complexes.

Consistent with other eukaryotes, the Trypanosoma brucei mitochondrial genome encodes mainly hydrophobic core subunits of the oxidative phosphorylation system. These proteins must be co-translationally inserted into the inner mitochondrial membrane and are synthesized by the highly unique trypanosomal mitoribosomes, which have a much higher protein to RNA ratio than any other ribosome. Here, we show that the trypanosomal orthologue of the mitoribosome receptor Mba1 (TbMba1) is essential for normal growth of procyclic trypanosomes but redundant in the bloodstream form, which lacks an oxidative phosphorylation system. Proteomic analyses of TbMba1-depleted mitochondria from procyclic cells revealed reduced levels of many components of the oxidative phosphorylation system, most of which belong to the cytochrome c oxidase (Cox) complex, three subunits of which are mitochondrially encoded. However, the integrity of the mitoribosome and its interaction with the inner membrane were not affected. Pull-down experiments showed that TbMba1 forms a dynamic interaction network that includes the trypanosomal Mdm38/Letm1 orthologue and a trypanosome-specific factor that stabilizes the CoxI and CoxII mRNAs. In summary, our study suggests that the function of Mba1 in the biogenesis of membrane subunits of OXPHOS complexes is conserved among yeast, mammals and trypanosomes, which belong to two eukaryotic supergroups.

Environmental Nucleic Acid Pollution: Characterization of Wastewater Generating False Positives in Molecular Ecological Surveys

Environmental nucleic acids (eDNA and eRNA) metabarcoding analyses have attracted considerable research attention as they non-invasively and cost-effectively monitor ecosystems. For heretofore unknown reasons, however, they often detect false positives. Here, we focused on domestic wastewater as a possible source of false positives and characterized fish nucleic acids in the influent and effluent of a wastewater treatment plant (WWTP). The metabarcoding analysis revealed that DNA and RNA detected in the influent wastewater originated from over 120 fish species, and most of these were consumed fish. The nucleic acid content was significantly higher in the influent than in the effluent, and the copy numbers of DNA in the influent were significantly higher than those of RNA. These findings were corroborated by the current literature and indicated that eRNA could effectively mitigate false positives. The present study demonstrated that rivers may be extensively contaminated by fish nucleic acids, and WWTPs substantially alter the quantities and ratios of fish DNA and RNA. Therefore, analyses of the wastewater near sampling points and coverage of the sewage system enable us to select and use the appropriate nucleic acid type (DNA or RNA) for metabarcoding analysis and improve the accuracy of fish species detection and identification.

A glimpse into the trophic ecology of deep-water sharks in an important crustacean fishing ground.

Deep-water sharks are among the most vulnerable deep-water taxa because of their extremely conservative life-history strategies (i.e., late maturation, slow growth, and reproductive rates), yet little is known about their biology and ecology. Thus, this study aimed at investigating the trophic ecology of five deep-water shark species, the birdbeak dogfish (Deania calcea), the arrowhead (D. profundorum), the smooth lanternshark (Etmopterus pusillus), the blackmouth catshark (Galeus melastomus) and the knifetooth dogfish (Scymnodon ringens) sampled onboard a crustacean bottom-trawler off the south-west coast of Portugal. We combined carbon and nitrogen stable isotopes with RNA and DNA (RD) ratios to investigate the main groups of prey assimilated by these species and their nutritional condition, respectively. Stable isotopes revealed overall small interspecific variability in the contribution of different taxonomic groups to sharks' tissues, as well as in the origin of their prey. S. ringens presented higher δ15 N and δ13 C values than the other species, suggesting reliance on bathyal cephalopods, crustaceans and teleosts; the remaining species likely assimilated bathy-mesopelagic prey. The RD ratios indicated that most of the individuals had an overall adequate nutritional condition and had recently eaten. This information, combined with the fact that stable isotopes indicate that sharks assimilated prey from the local or nearby food webs (including commercially important shrimps), suggests a potential overlap between this fishing area and their foraging grounds, which requires further attention.

Risk Factors for CSF/Plasma HIV-1 RNA Discordance in HIV-Infected Patients.

Few large investigations have evaluated the association of cerebrospinal fluid/plasma (CSF/plasma) discordance with opportunistic neurological infections. We aimed to determine risk factors for CSF/plasma discordance to further assess whether CSF/plasma discordance is associated with antiretroviral therapy (ART) and opportunistic neurological infections. A retrospective study was conducted based on HIV RNA viral load and associated risk factors in plasma and CSF samples from 491 HIV-infected patients. HIV RNA levels higher in CSF compared with plasma was defined as CSF/plasma discordance. In this study, the rate of CSF/plasma discordance was 18.3%. We observed that headache, cryptococcal antigen, CSF cell count, Treponema pallidum particle assay positivity, and ART use were significantly associated with CSF/plasma discordance in the multivariate logistic regression model. The CSF RNA/plasma RNA ratio was significantly higher in HIV-infected patients with neurological infections than in HIV-infected cases without neurological infections (P < 0.001). CSF/plasma discordance was significantly different between HIV-infected patients without central nervous system (CNS) infection and those with CNS infection, tuberculous meningitis, cryptococcal meningitis, and neurosyphilis (P < 0.05). ART and CNS inflammation may influence CSF/plasma discordance.

Safe and efficient RNA and DNA introduction into cells using digital electroporation system

We systematically compared the delivery and expression efficiencies according to cell types (plant and animal cells) and genetic materials (RNA and DNA) to deliver RNA using a digital electroporation system. Despite the significantly lower RNA delivery in Chlamydomoans reinhartii than DNA delivery due to RNA secondary structure and cell wall, the expression/delivery ratio of RNA was significantly higher than that of DNA (up to 90%), confirming the generally known fact that RNA is more favorable for expression than DNA. On the other hand, in K562 cells, the difference in RNA and DNA delivery efficiency was negligible. Therefore, structural differences between DNA and RNA affect delivery efficiency differently depending on the cell type. RNA delivery efficiency of K562 cells was high, but expression efficiency was much lower than that of microalgae. According to the proposed strategy, compatibility between K562 cells and the nucleic acids used in this study is presumed to be one of the reasons for this low expression efficiency. Gene regulation by delivering small interfering RNA (siRNA) was demonstrated in K562 cells, confirming the feasibility of the digital electroporation system for RNA interference (RNAi) research as a safe and efficient delivery system.

Optimized production and fluorescent labeling of SARS-CoV-2 virus-like particles

SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemic that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100 nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the viral RNA ratio in infected cells. This allows SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and mature S. Furthermore, fluorescent or photoconvertible VLPs were generated by adding a fluorescent protein tag on N or M mixing with unlabeled viral proteins and characterized by western blots, atomic force microscopy coupled to fluorescence and immuno-spotting. Thanks to live fluorescence and super-resolution microscopies, we quantified VLPs size and concentration. SARS-CoV-2 VLPs present a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a concentration of 10e12 VLP/ml. In this condition, we were able to establish the incorporation of the Spike in the fluorescent VLPs. Finally, the Spike functionality was assessed by monitoring fluorescent MNES-VLPs docking and internalization in human pulmonary cells expressing or not the receptor hACE2. Results show a preferential maturation of S on N(GFP) labeled VLPs and an hACE2-dependent VLP internalization and a potential fusion in host cells. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.

A Simplified Method for RNA Isolation from Biofabricating Hydroxyapatite Scaffolds and Identification of Appropriate Reference Genes

PurposeTo validate a simplified RNA isolation method from biofabricating hydroxyapatite (HAp) scaffolds seeded with mesenchymal stem cells (MSCs) and to identify the appropriate reference gene.MethodsTen MSCs-HAp composites were used for RNA isolation by methods based on simplified homogenization steps and column-based purification procedures, while the remaining RNA (n = 13) was extracted by traditional single-step isolation methods. The differences between the two procedures regarding the operation time, RNA quantity and quality were evaluated. Quantitative real-time PCR (qRT-PCR) analysis was performed to identify the appropriate reference gene.ResultsThe simplified method showed significant superiority in operation time (P < 0.001), RNA concentration (P < 0.001), A260/280 ratio (P = 0.005) and A260/230 ratio (P < 0.001). The average integrity number and 28 s/18 s ratio of RNA yielded by the simplified method were 9.1 ± 0.2 and 1.3 ± 0.1, respectively. The qRT-PCR analysis results indicated that the cycle threshold (Ct) values of GAPDH were significantly higher than those of the remaining 2 reference genes (ACTB and RPL13A) in the RNA samples obtained by the simplified and traditional methods (P < 0.05). The standard deviations of the ΔCt value (the difference between the Ct value and the minimum) of ACTB were higher than those of GAPDH or RPL13A, regardless of the RNA isolation method.ConclusionThe simplified method could extract intact RNA from biofabricating MSCs-HAp scaffolds and was superior to the traditional single-step procedure in operation time, RNA quantity and quality. GAPDH was identified as the most appropriate reference gene in MSCs-HAp scaffold composites due to its high quantity and good stability.

Early Emergence of 5' Terminally Deleted Coxsackievirus-B3 RNA Forms Is Associated with Acute and Persistent Infections in Mouse Target Tissues.

Major EV-B populations characterized by 5′ terminal deletions (5′TD) have been shown to be associated with the development of myocarditis and type 1 diabetes in mice or humans. To date, the dynamics of EV-B 5′TD-RNA forms’ emergence during the course of infection and their impact on cellular functions remain unclear. Using a RACE-PCR approach in CVB3/28-infected mouse organs, we showed an early (3 days post infection, DPI) emergence of major 5′TD populations associated with minor full-length RNA forms. Viral replication activities with infectious particle production were associated with heart, liver, and pancreas acute inflammatory lesions, whereas clearance of viral RNA without organ lesions was observed in the brain, lung, intestines, and muscles from 3 to 7 DPI. At 28 DPI, low viral RNA levels, +/-RNA ratios < 5 associated with viral protein 1 expression revealed a persistent infection in the heart and pancreas. This persistent infection was characterized by molecular detection of only 5′TD RNA forms that were associated with dystrophin cleavage in the heart and insulin production impairment in beta-pancreatic cells. These results demonstrated that major EV-B 5′TD RNA forms can be early selected during systemic infection and that their maintenance may drive EV-induced acute and persistent infections with target cell dysfunctions.

Performance of the cobas® HBV RNA automated investigational assay for the detection and quantification of circulating HBV RNA in chronic HBV patients.

The amount of HBV RNA in peripheral blood may reflect HBV covalently closed circular DNA (cccDNA) transcriptional activity within infected hepatocytes. Quantification of circulating HBV RNA (cirB-RNA) is thus a promising biomarker for monitoring antiviral treatment. We evaluated the performance of an automated, prototype quantitative HBV RNA assay for use on the Roche cobas® 6800/8800 systems. The sensitivity, specificity, linearity, and potential interference by HBV DNA of the cobas® HBV RNA assay were assessed using synthetic HBV armored RNA and clinical specimens. cobas® HBV RNA results were linear between 10 and 107 copies/mL in clinical samples of several HBV genotypes, and up to 109 copies/mL with synthetic RNA. Precision and reproducibility were excellent, with standard deviation below 0.15 log10 copies/mL and coefficients of variation below 5% throughout the linear range. The presence of HBV DNA had minimal (<0.3 log10 copies/mL) impact on HBV RNA quantification at DNA:RNA ratios of up to approximately one million. In a panel of 36 untreated patient samples, cirB-RNA concentrations were approximately 200-fold lower than HBV DNA. cirB-RNA was detected in all 13 HBeAg-positive patients (mean 6.0 log10 copies/mL), and in 20 of 23 HBeAg-negative patients (mean of quantifiable samples 2.2 log10 copies/mL). Finally, cirB-RNA was detected in 12 of 20 nucleoside analog-treated patients (mean of quantifiable samples 3.4 log10 copies/mL). The cobas® 6800/8800 investigational HBV RNA assay is a high throughput, sensitive and inclusive assay to evaluate the clinical relevance of cirB-RNA quantification in patients with chronic hepatitis B.

Effects of oral glutamine supplementation on jejunal morphology, development, and amino acid profiles in male low birth weight suckling piglets.

BackgroundIt has been shown that small intestine development in low birth weight (LBW) piglets is impaired. Glutamine (Gln) has been reported to improve piglet health and intestinal function in weaned piglets, but data is scarce in suckling piglets. This study was conducted to investigate the effects of oral Gln supplementation compared to Alanine (Ala) on jejunal development and function in 5 and 12 d old male LBW and normal birth weight (NBW) suckling piglets.ResultsGln had no effect on the jejunal morphology, development, tissue and digesta amino acid profiles and mRNA abundance of genes involved in amino acid transport, metabolism, glutathione synthesis in LBW piglets when compared to Ala supplementation and birth weight controls at 5 and 12 d. Only the concentration of Gln in jejunal tissue was higher in NBW piglets supplemented with Gln compared to Ala at 5 d (P < 0.05). A comparison of the birth weight groups showed no differences between LBW and NBW piglets at 5 and 12 d in any parameter. Jejunal crypt depth, villus height / width, tunica muscularis thickness, number of goblet and IgA positive cells, the ratio of jejunal RNA to DNA and the concentration of DNA, protein and RNA changed (P < 0.05) from 5 compared to 12 d. The concentrations of several free, and protein bound amino acids as well as amino metabolites differed between age groups in jejunal tissue but the digesta concentrations were affected to a lesser extent.ConclusionsOral Gln supplementation to suckling male piglets over the first 12 d of life was not associated with changes in jejunal parameters measured in this study. The absence of effects may indicate that Gln is absorbed as well as metabolized in the upper intestinal tract and thus could benefit intestinal development at a more proximal location.

Development of rapid and easy detection of Salmonella in food matrics using RPA-CRISPR/Cas12a method

Salmonella species are common foodborne pathogenic bacteria. At present, most detection methods for Salmonella are unsuitable for on-site applications because they require large instruments or complicated procedures. This study developed a novel method of the on-site detection for Salmonella in food by combining the CRISPR/Cas12a system with recombinant polymerase amplification (RPA). The optimal concentration ratio of Cas12a enzyme, CRISPR RNA (crRNA) and FQ-probe is 1 : 1: 2.5. The detection limit of the RPA–CRISPR/Cas12a method was 1 × 10−4 ng/μL for genomic DNA (gDNA), and 102 CFU/mL for bacterial liquid. The method exhibited no cross-reactivity to 4 common pathogenic bacteria. A simple boiling method was used to pretreat chicken and egg samples to meet on-site testing requirements. The detection limits of chicken and egg samples were 103 CFU/mL initially and could reach 102 CFU/mL and 101 CFU/mL, respectively, after 3 h enrichment at 37 °C. The sensitivity of the RPA–CRISPR/Cas12a method was comparable to that of the quantitative polymerase chain reaction (qPCR) technique. In addition, the operation of this method is simpler and faster than qPCR. Thus, the method is applicable to the on-site detection for Salmonella in food.