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The synchronous research and analysis of total and active soil microbial communities can provide insight into how these communities are impacted by continuous cropping years and pathogen infection. The diversity of total and active bacteria in rhizospheric soil of 2-year-old and 3-year-old healthy and diseased Panax notoginseng can comprehensively reveal the bacterial response characteristics in continuous cropping practice. The results showed that 4916 operational taxonomic units (OTUs) were found in the rhizospheric soil bacterial community of P. notoginseng at the DNA level, but only 2773 OTUs were found at the RNA level. The rhizospheric environment had significant effects on the active and bacterial communities, as indicated by the number of OTUs, Shannon, Chao1, Faith's phylogenetic diversity (Faith's PD), and Simpson's diversity indexes. The DNA level can better show the difference in diversity level before and after infection with root rot. The bacterial Chao1 and Faith's PD diversity indexes of 2-year-old root rot-diseased P. notoginseng rhizospheric soil (D2) were higher than that of healthy plants, while the bacterial Shannon diversity index of 3-year-old root rot-diseased P. notoginseng rhizospheric soil (D3) was the lowest in the total bacteria. Principal coordinate analysis (PCoA) illustrated that the total bacterial species composition changed markedly after root rot disease. There were significant differences in the composition of active bacterial species between the 2-year and 3-year rhizospheres. In conclusion, the total and active edaphic rhizospheric bacterial communities could provide important opportunities to understand the responses of bacteria to continuous cropping of P. notoginseng. Differential responses of total and active edaphic rhizosphere bacterial communities to continuous cropping of Panax notoginseng [ABSTRACT FROM AUTHOR]

Abstract Background Most cancers acquire numerous genetic changes in proto-oncogenes as well as tumor-suppressor genes. Cancer's early diagnosis remains a challenge. Recently, nonlinear polarization has revealed the potential as a promising tool for early cancer diagnosis. Laser-induced nonlinear polarization can offer a novel fingerprint signature. Methods In this study, nonlinear polarization was adopted for the characterization of both DNA and RNA samples from healthy volunteers. Total DNA and RNA were illuminated with a 656-nm LED source, and the resonance frequencies (scattered and re-emitted signals) were captured and recorded using a hyperspectral camera. Results Changes in signal frequency as well as phase shift offered a potent means to differentiate DNA (control) from RNA (control). DNA (control) demonstrated characteristic resonance frequencies that differ from total RNA (control) at the 2nd, 3rd, 4th, and 5th harmonics. While DNA demonstrated a phase shift dominating at 0.88 GHz, RNA dominates at 0.106 GHz. The resonance spectral signature of RNA samples from people with hepatocellular carcinoma (HCC) was compared to that of RNA (control). RNA (HCC) demonstrated distinctive frequency signals at 0.014, 0.021, 0.032, and 0.072 GHz. These characteristics feature could facilitate early HCC diagnosis. While RNA (control) dominates at 0.014 and 0.072 MHz, RNA (HCC) dominates at 0.021 and 0.032. Conclusion As far as we are aware, this is the initial investigation into the use of simple nonlinear polarization to generate spectral fingerprinting signatures of total DNA and RNA. Furthermore, RNA mutations due to HCC were identified via characteristic nonlinear spectral signature.

Background: Most cancers acquire numerous genetic changes in proto-oncogenes as well as tumor-suppressor genes. Cancer's early diagnosis remains a challenge. Recently, nonlinear polarization has revealed the potential as a promising tool for early cancer diagnosis. Laser-induced nonlinear polarization can offer a novel fingerprint signature. Methods: In this study, nonlinear polarization was adopted for the characterization of both DNA and RNA samples from healthy volunteers. Total DNA and RNA were illuminated with a 656-nm LED source, and the resonance frequencies (scattered and re-emitted signals) were captured and recorded using a hyperspectral camera. Results: Changes in signal frequency as well as phase shift offered a potent means to differentiate DNA (control) from RNA (control). DNA (control) demonstrated characteristic resonance frequencies that differ from total RNA (control) at the 2nd, 3rd, 4th, and 5th harmonics. While DNA demonstrated a phase shift dominating at 0.88 GHz, RNA dominates at 0.106 GHz. The resonance spectral signature of RNA samples from people with hepatocellular carcinoma (HCC) was compared to that of RNA (control). RNA (HCC) demonstrated distinctive frequency signals at 0.014, 0.021, 0.032, and 0.072 GHz. These characteristics feature could facilitate early HCC diagnosis. While RNA (control) dominates at 0.014 and 0.072 MHz, RNA (HCC) dominates at 0.021 and 0.032. Conclusion: As far as we are aware, this is the initial investigation into the use of simple nonlinear polarization to generate spectral fingerprinting signatures of total DNA and RNA. Furthermore, RNA mutations due to HCC were identified via characteristic nonlinear spectral signature. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a leading cancer whose incidence is increasing globally. In spite of advances in medical and surgical therapies, its prognosis remains deprived. Developing methods for early diagnosis is essential for better treatment outcomes. In this pilot study, laser-induced linear polarization was adopted for the characterization of total RNA samples obtained from hepatocellular carcinoma (HCC) patients against healthy volunteers. Total RNA samples were illuminated with non-ionizing laser source of 632.8 nm; the scattered light signatures were captured and processed using hyperspectral camera. Scattered light signature was found to depend on polarizabiltiy, size, shape, and chemical structure of scattering macromolecules. DNA (control) demonstrated the highest scattered intensity; with intense fringes compared with RNA (control). Meanwhile RNA (HCC) demonstrated the lowest scattered intensity as well as the lowest fringe spacing compared with RNA (control). For instance, at 380 fringe spacing DNA (Control), RNA (Control), RNA (HCC) demonstrated scatted light intensity of 0.049, 0.039, and 0.034, respectively. Novel column mean calculation was conducted for hyperspectral images at 632.8 nm to assess scattered signal intensity and fringe spacing. The scattered RNA signal could secure characteristic finger-print fringes; that are candidate for facile HCC early diagnosis. To the best of our knowledge, this is the first time to report an optical methodology based on linear polarization for HCC facile diagnosis as a pilot study. [ABSTRACT FROM AUTHOR]

Fecal specimens have long been regarded as promising sources for gastrointestinal cancer screening and have, thus, been extensively investigated in biomarker research. MicroRNAs (miRNAs) are small, non-coding RNA molecules involved in regulating various biological processes. They are commonly dysregulated during tumor development and exhibit differential expression in feces. To assess the preanalytical feasibility of fecal miRNA analysis, we systematically compared the performance of commonly used total RNA extraction methods. Fecal samples from healthy subjects were utilized for this evaluation. Various methods, including miRNeasy, Universal, Trizol, RNeasy, and mirVana kits, were employed to isolate total RNA. MiRNA expression analyses were conducted using TaqMan or SYBR Green qRT-PCR for a subset of miRNAs, with externally spiked-in cel-miR-39 used for normalization. Most methods demonstrated similar performance in terms of the total RNA concentration and purity. Externally spiked cel-miR-39 and endogenous miRNAs (RNU6b, miR-16, and miR-21) exhibited comparable concentrations across the different RNA isolation methods, whereas the RNeasy mini kit consistently yielded lower values. Our findings suggest that various isolation methods produce reproducible and comparable miRNA expression results, supporting the potential comparability and translational applicability of miRNA-based biomarker research in the future. [ABSTRACT FROM AUTHOR]

The results of an analysis of the morphophysiological and biochemical indicators of somatic growth in Black Sea bivalve mollusks, the cockle Cerastoderma glaucum (Bruguiere, 1789) and the scallop Flexopecten glaber ponticus (Bucguoy, Dautzenberg et Dollfus, 1889), living in the vicinity of Sevastopol, are presented. The examples are of three size-age groups in the length range from 12 to 30 mm for both species; the features of tissue biosynthesis (somatic growth) of soft tissues are studied. According to the total RNA content and the RNA/DNA index in the C.glaucum, it is shown that, in the 17–22 and 23–28 mm groups (age 2–3 years), the level of synthesis of protein structures is 1.3–1.7 times higher than in the underyearlings. In individuals of F. glaber ponticus with linear sizes of 13–17 and 21–24 mm (underyearlings), the level of synthesis of protein structures is 1.2–1.5 times higher than in larger individuals (age 2 years). The species specificity of somatic growth is most significantly manifested in the middle and older size groups of the studied mollusks. The values of biochemical growth parameters in both species indicates an average level of tissue growth in comparison with other previously obtained data for the mass Black Sea bivalves. Linear regression models of size–weight values show that, for both species, the change in size explains 62–86% of the variation in body weight of the mollusk. An analysis of weight parameters (total mass/soft tissue mass) indicates a positive allometry of somatic tissue growth. [ABSTRACT FROM AUTHOR]

Tumor heterogeneity and its drivers impair tumor progression and cancer therapy. Single‐cell RNA sequencing is used to investigate the heterogeneity of tumor ecosystems. However, most methods of scRNA‐seq amplify the termini of polyadenylated transcripts, making it challenging to perform total RNA analysis and somatic mutation analysis.Therefore, a high‐throughput and high‐sensitivity method called snHH‐seq is developed, which combines random primers and a preindex strategy in the droplet microfluidic platform. This innovative method allows for the detection of total RNA in single nuclei from clinically frozen samples. A robust pipeline to facilitate the analysis of full‐length RNA‐seq data is also established. snHH‐seq is applied to more than 730 000 single nuclei from 32 patients with various tumor types. The pan‐cancer study enables it to comprehensively profile data on the tumor transcriptome, including expression levels, mutations, splicing patterns, clone dynamics, etc. New malignant cell subclusters and exploring their specific function across cancers are identified. Furthermore, the malignant status of epithelial cells is investigated among different cancer types with respect to mutation and splicing patterns. The ability to detect full‐length RNA at the single‐nucleus level provides a powerful tool for studying complex biological systems and has broad implications for understanding tumor pathology. [ABSTRACT FROM AUTHOR]

The current naso-oropharyngeal swab for SARS-CoV-2 detection faces several problems, such as waste issues and its use for quantitative studies. This study aimed to evaluate the total RNA and viral loads from different upper respiratory tract swabs types and whether SARS-CoV-2 quantification can use the current internal control for normalization. This cross-sectional study collected positive specimens with single oropharyngeal or nasopharyngeal swabs and naso-oropharyngeal swabs. The samples were extracted, tested with qualitative RT‒PCR, and then tested with quantitative RT‒PCR. The RNA eluate was measured for the total RNA concentration. The total RNA concentration, viral load, and RNaseP Ct values were collected and analysed statistically. The positive results came from 41 oropharyngeal swabs, 34 nasopharyngeal swabs, and 36 naso-oropharyngeal swabs. The total RNA increased significantly from oropharyngeal swabs to nasopharyngeal swabs to naso-oropharyngeal swabs. Significant differences in RNaseP Ct values between groups and their correlations with total RNA were found. In addition, the increase in the total RNA and the RNaseP Ct values were unrelated to the viral load. The physical features in the naso-oropharyngeal area and the swabbing procedures could affect the total RNA but not the viral load. However, since the virus particles could present inside and outside human cells, the increase in collected human cells may not always be followed by the viral load increase. Normalization using the RNaseP Ct value became unnecessary due to the factors mentioned above. Therefore, a careful approach is needed in viral load studies of swab specimens.

Abstract Tumor heterogeneity and its drivers impair tumor progression and cancer therapy. Single‐cell RNA sequencing is used to investigate the heterogeneity of tumor ecosystems. However, most methods of scRNA‐seq amplify the termini of polyadenylated transcripts, making it challenging to perform total RNA analysis and somatic mutation analysis.Therefore, a high‐throughput and high‐sensitivity method called snHH‐seq is developed, which combines random primers and a preindex strategy in the droplet microfluidic platform. This innovative method allows for the detection of total RNA in single nuclei from clinically frozen samples. A robust pipeline to facilitate the analysis of full‐length RNA‐seq data is also established. snHH‐seq is applied to more than 730 000 single nuclei from 32 patients with various tumor types. The pan‐cancer study enables it to comprehensively profile data on the tumor transcriptome, including expression levels, mutations, splicing patterns, clone dynamics, etc. New malignant cell subclusters and exploring their specific function across cancers are identified. Furthermore, the malignant status of epithelial cells is investigated among different cancer types with respect to mutation and splicing patterns. The ability to detect full‐length RNA at the single‐nucleus level provides a powerful tool for studying complex biological systems and has broad implications for understanding tumor pathology.

High-purity total RNA extraction from animal embryos is essential for transcriptome analyses. lampreys, together with hagfish, are the only extant jawless vertebrates or cyclostomes and are thus key organisms for EvoDevo studies. However, extracting uncontaminated RNA from early-stage embryos remains challenging. RNA does not bind to the silica membrane in filter-based extractions, significantly reducing yields; and ethanol/isopropanol precipitation methods lead to contaminants, bringing down the optical density (OD) 260/280 ratio. The RNA extraction protocol was modified using precentrifugation and adding salts before isopropanol precipitation. This modification significantly increased RNA yield, removed contaminants and improved RNA integrity. Egg membrane sources were suspected to cause RNA purification problems because low-quality extraction does not occur in posthatching embryos.

Sap coconut (Cocos nucifera L. var. makapuno) in Vietnam is a mutant coconut variant; coconut water is in the state of lotus glue, and coconut rice is like cream. For high-quality transcriptome, sequencing, quality, purity and concentration of RNA are the key factors. However, coconut endosperm tissue has higher stiffness and fatness than the leaf tissue, which complicates the extraction process. Moreover, RNA is much more difficult to preserve than DNA. In this study, various RNA extraction methods were examined in Vietnamese waxy coconut endosperm tissue samples. Optimum extraction and preservation of the RNA using the simplest possible chemicals was the objective of this study. The modified CTAB method with LiCl and the TRI reagent method were tested and evaluated. The purity, concentration and quality of RNA after storage were improved. The findings indicated that the TRIsure extraction method with the addition of NaCl and β-mercaptoethanol yielded optimum RNA quality. The RNA concentration was 159 ng/µL, with a purity ratio of 1.94 ± 0.04 for A260/A280 and 1.58 ± 0.02 for A260/230. RNA samples remained stable for up to 3 weeks when stored in absolute ethanol at 8°C–10°C, which significantly reduced their degradation during transportation. This study facilitated the use of simple chemicals for high-quality RNA extraction from coconut endosperm and its preservation for applications in high throughput sequencing.

Single-cell sequencing of RNA (scRNA-seq) has advanced our understanding of cellular heterogeneity and signaling in developmental biology and disease. A large number of complementary assays have been developed to profile transcriptomes of individual cells, also in combination with other readouts, such as chromatin accessibility or antibody-based analysis of protein surface markers. As scRNA-seq technologies are advancing fast, it is challenging to establish robust workflows and up-to-date protocols that are best suited to address the large range of research questions. Here, we review scRNA-seq techniques from mRNA end-counting to total RNA in relation to their specific features and outline the necessary sample preparation steps and quality control measures. Based on our experience in dealing with the continuously growing portfolio from the perspective of a central single-cell facility, we aim to provide guidance on how workflows can be best automatized and share our experience in coping with the continuous expansion of scRNA-seq techniques. [ABSTRACT FROM AUTHOR]

Sepsis is a life-threatening condition mostly caused by a bacterial infection resulting in inflammatory reaction and organ dysfunction if not treated effectively. Rapid identification of the causing bacterial pathogen already in the early stage of bacteremia is therefore vital. Current technologies still rely on time-consuming procedures including bacterial culturing up to 72 h. Our approach is based on ultra-rapid and highly sensitive nanomechanical sensor arrays. In measurements we observe two clearly distinguishable distributions consisting of samples with bacteria and without bacteria respectively. Compressive surface stress indicates the presence of bacteria. For this proof-of-concept, we extracted total RNA from EDTA whole blood samples from patients with blood-culture-confirmed bacteremia, which is the reference standard in diagnostics. We determined the presence or absence of bacterial RNA in the sample through 16S-rRNA hybridization and species-specific probes using nanomechanical sensor arrays. Via both probes, we identified two clinically highly-relevant bacterial species i.e., Escherichia coli and Staphylococcus aureus down to an equivalent of 20 CFU per milliliter EDTA whole blood. The dynamic range of three orders of magnitude covers most clinical cases. We correctly identified all patient samples regarding the presence or absence of bacteria. We envision our technology as an important contribution to early and sensitive sepsis diagnosis directly from blood without requirement for cultivation. This would be a game changer in diagnostics, as no commercial PCR or POCT device currently exists who can do this. [ABSTRACT FROM AUTHOR]

A vast number of neurodegenerative disorders arise from neurotoxicity. In neurotoxicity, more than 250 RNA molecules are up and downregulated. The manuscript investigates the exposure of chlorpyrifos organophosphate pesticide (COP) effect on total RNA in murine brain tissue in 4 genotypes for in silico neurodegeneration development. The GSE58103 dataset from the Gene Expression Omnibus (GEO) database applies for data preprocessing, normalization, and quality control. Differential expression analysis (DEG) uses the limma package in R. Study compared expression profiles from murine fetal brain tissues across four genotypes: PON-1 knockout (KO), tgHuPON1Q192 (Q-tg), tgHuPON1R192 (R-tg), and wild-type (WT). We analyze 60 samples, 15 samples per genotype, to identify DEGs. The significance criteria are adjusted p -value <.05 and a |log2 fold change| > 1. The study identifies microRNA485 as the potential biomarker of COP toxicity using the GSE58103 dataset. Significant differences exist for microRNA485 between KO and WT groups by differential expression analysis. Moreover, graphical analysis shows sample relationships among genotype groups. MicroRNA485 represents a promising biomarker for developmental COP neurotoxicity by utilizing in silico analysis in scientific practice., Competing Interests: Declaration of conflicting interestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The author, a PhD student, Snežana Jovičić, declares no competing interest.

Grapevine germplasm collections are unique repositories of grape cultivars; therefore, it is necessary to minimize their infection with pathogens, including viruses, and develop various programs to maintain them in a virus-free state. In our study, we examined the virome of the largest Russian grapevine germplasm collection, the Anapa Ampelographic Collection, using high-throughput sequencing of total RNAs. As a result of bioinformatics analysis and validation of its results by reverse transcription PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR), we identified 20 viruses and 3 viroids in 47 libraries. All samples were infected with 2 to 12 viruses and viroids, including those that cause economically significant diseases: leafroll, fleck, and rugose wood complex. For the first time in Russia, we detected Grapevine virus B (GVB), Grapevine virus F (GVF), Grapevine asteroid mosaic-associated virus (GAMaV), Grapevine Red Globe virus (GRGV), Grapevine satellite virus (GV-Sat), Grapevine virga-like virus (GVLV), Grapevine-associated jivivirus 1 (GaJV-1) and Vitis cryptic virus (VCV). A new putative representative of the genus Umbravirus with the provisional name Grapevine umbra-like virus (GULV) was also identified in Russian grape samples. [ABSTRACT FROM AUTHOR]

In this study, some biochemical characteristics (total protein, total RNA, Catalase: CAT enzyme activity and malondialdehyde: MDA level) of Italian bee (A. m. ligustica) and Caucasian bee (A. m. caucasica), and Muğla and Anatolian bees (A. m. anatolica) from local honey bee races were investigated comparatively. Laboratory analyzes of biochemical characteristics were performed on worker bees aged 24 days old with 10 repetitions using appropriate methods informed in the literature. The amounts of total protein of bee races given above were 18.39±1.28, 20.71±0.63, 18.56±1.24 and 20.95±2.15 g/dL, respectively; the amounts of total RNA were 11.46±0.18, 12.10±0.26, 11.87±0.20 and 12.27±0.26 µg/µL, respectively; the CAT activities were 4.59±0.46, 5.12±0.67, 4.88±0.48 and 5.25±0.53 kU/g P, respectively; the levels of MDA were 0.52±0.04, 0.50±0.04, 0.48±0.02 ve 0.43±0.05 mmol/mg, respectively. Variance analysis showed that statistically significant differences among races in terms of the all characteristics examined. The results of CAT activity which is one of the indicators of antioxidant defense system, and levels of MDA which is an indicator of peroxidation of membrane lipids; and similarly total amount of protein also includes various proteins such as antioxidants and enzymes; it can be said that the Anatolian and Caucasian bee races (due to higher total protein, total RNA and CAT activities, and lower MDA level) are more resistant to various negative environmental factors (e.g. climate, flora, pesticide, etc.) than the Muğla and Italian bee races in the conditions of the Central Anatolia Region; there are significant differences between the bee races in terms of amounts of total RNA and this parameter can be also used in the characterization of bee races.

Objective: to study the peculiarities of the induction effect of total RNA (tRNA) from xenogenic bone marrow cells (BMCs) on regeneration processes in the recipient's native liver with extensive liver resection using an adoptive transfer model. Materials and methods. The study was carried out on an adoptive transfer model using male Wistar rats (n = 20) and guinea pigs (n = 17). The donors were rats (n = 10). 12 hours after extensive liver resection (70-75%), tRNA was isolated from BMCs and injected into intact (non-operated) recipients intraperitoneally at a dose of 30 μg/100 g of weight. The induction effect of the tRNA on operated rats was studied in 3 groups of recipients: Group 1 (control, n = 5) - administration of saline to guinea pigs; Group 2 (control, n = 10) - administration of tRNA from a donor rat to a recipient rat (allogeneic transfer); Group 3 (experiment, n = 12) - administration of tRNA from a donor rat to a recipient guinea pig (xenogeneic transfer). In histological preparations of recipient livers, after 48, 72 hours and 7 days, we studied the mitotic activity of hepatocytes and the features of the microscopic picture of the liver. The significance of differences in the compared groups was assessed using the parametric Student's t-test. Results. The ability of BMC tRNA to tissue-specifically activate regenerative and immune responses in the liver after extensive resection was found to depend on the donor and recipient species identity. Introduction of allogeneic donor tRNA in the recipient's liver resulted in predominant enhancement in hepatocyte mitotic activity (p < 0.05). The use of xenogeneic donor tRNA leads to enhanced activity of only immuno-inflammatory reactions in the recipient's liver, such as sinusoidal cell activation, lymphocytic infiltration into sinusoids, and portal tract infiltration by inflammatory cells. Conclusion. To induce regenerative processes in the liver, tRNA obtained from allogeneic BMCs should be used.

This study aimed to improve the heat shock method as a cost-effective and time-efficient for total RNA extraction. We compared the effectiveness of two total RNA extraction methods by using Real-Time PCR for nasopharynx swabs. Include: I; use of a commercial total RNA extraction kit as a standard. II; utilized a modified heat shock method (MHS). Time, centrifuge speed and duration, proteinase K, and RNA carrier were optimized. The optimized parameters included treating the sample with 5 μg/μL at 56°C for 5 minutes, heating at 95°C for 5 minutes followed by thermal shock in ice for 3 minutes, adding 4 μg/μL RNA carrier at room temperature for 3 minutes, and centrifuging at 7000 rpm for 10 minutes. This optimization demonstrated a sensitivity and specificity of 100% (CI: 95%) even in samples with low viral load. Our in-house method presents a rapid, and cost-effective alternative for total RNA extraction. [ABSTRACT FROM AUTHOR]

Objective: to study the cellular mechanisms of activation of regenerative processes in the liver when using total RNA (tRNA) of bone marrow cells (BMCs) based on an extended liver resection (ELR) model. Materials and methods. Male Wistar rats (n = 80) with ELR model (70%) were divided into 2 groups: group 1 (control group) had a single saline injection, while group 2 (experimental group) received a single tRNA injection at a 30 μg/100 g dose of animal weight. The biochemical parameters of liver function and weight were monitored over time. Also monitored were microstructural changes in hepatocytes 48 hours after ELR by examining mitotic activity, caspase-9 expression and morphometric parameters. Results. It was found that in group 2, in comparison to group 1, there was faster normalization of biochemical parameters (by 10–14 days), a higher mitotic index of hepatocytes (23.45‰ versus 5.37‰), and initially sharper decrease and then faster recovery of liver mass (by 10–12 days versus 18–20 days). Both groups showed almost total expression of caspase-9, including in mitotically splitting hepatocytes. Group 1 demonstrated decreased values of morphometric parameters of single and binuclear cells, decreased number of binucleated hepatocytes and increased total density of hepatocytes as compared to the intact liver. Intraperitoneal administration of tRNA increased morphometric parameters of mononuclear hepatocytes, did not affect their number, but increased the area of the nuclei of binuclear hepatocytes as compared to the control group. Conclusion. The proven capability of cell-bone marrow total RNA to simultaneously support apoptosis in liver cells after ELR and induce mitotic activity indicates that tRNA can switch activated apoptosis to cell proliferation at the early phase of the regenerative process. This effect may be due to the presence of regulatory RNA molecules in tRNA, including numerous non-coding RNAs.

Abstract Background Lactobacillus gasseri OLL2809 can highly induce interleukin (IL)-12 production in immune cells. Even though beneficial properties of this strain for both humans and animals have been reported, the mechanism by which the bacteria induces the production of IL-12 in immune cells remains elusive. In this study, we investigated the mechanism of induction of IL-12 using a mouse macrophage cell line J774.1. Results Inhibition of phagocytosis of L. gasseri OLL2809, and myeloid differentiation factor 88 and Toll-like receptors (TLRs) 7 and 9 signalling attenuated IL-12 production in J774.1 cells. Total RNA and genomic DNA of L. gasseri OLL2809, when transferred to the J774.1 cells, also induced IL-12 production. The difference in the IL-12-inducing activity of Lactobacilli is attributed to the susceptibility to phagocytosis, but not to a difference in the total RNA and genomic DNA of each strain. Conclusion We concluded that total RNA and genomic DNA of phagocytosed L. gasseri OLL2809 induce IL-12 production in J774.1 cell via TLRs 7 and 9, and the high IL-12-inducing activity of L. gasseri OLL2809 is due to its greater susceptibility to phagocytosis.

Sepsis is a life-threatening condition mostly caused by a bacterial infection resulting in inflammatory reaction and organ dysfunction if not treated effectively. Rapid identification of the causing bacterial pathogen already in the early stage of bacteremia is therefore vital. Current technologies still rely on time-consuming procedures including bacterial culturing up to 72 h. Our approach is based on ultra-rapid and highly sensitive nanomechanical sensor arrays. In measurements we observe two clearly distinguishable distributions consisting of samples with bacteria and without bacteria respectively. Compressive surface stress indicates the presence of bacteria. For this proof-of-concept, we extracted total RNA from EDTA whole blood samples from patients with blood-culture-confirmed bacteremia, which is the reference standard in diagnostics. We determined the presence or absence of bacterial RNA in the sample through 16S-rRNA hybridization and species-specific probes using nanomechanical sensor arrays. Via both probes, we identified two clinically highly-relevant bacterial species i.e., Escherichia coli and Staphylococcus aureus down to an equivalent of 20 CFU per milliliter EDTA whole blood. The dynamic range of three orders of magnitude covers most clinical cases. We correctly identified all patient samples regarding the presence or absence of bacteria. We envision our technology as an important contribution to early and sensitive sepsis diagnosis directly from blood without requirement for cultivation. This would be a game changer in diagnostics, as no commercial PCR or POCT device currently exists who can do this.

Background: Porcine milk is a complex fluid, containing a myriad of immunological, biochemical, and cellular components, made to satisfy the nutritional requirements of the neonate. Whole milk contains many different cell types, including mammary epithelial cells, neutrophils, macrophages, and lymphocytes, as well nanoparticles, such as milk exosomes. To-date, only a limited number of livestock transcriptomic studies have reported sequencing of milk. Moreover, those studies focused only on sequencing somatic cells as a proxy for the mammary gland with the goal of investigating differences in the lactation process. Recent studies have indicated that RNA originating from multiple cell types present in milk can withstand harsh environments, such as the digestive system, and transmit regulatory molecules from maternal to neonate. Transcriptomic profiling of porcine whole milk, which is reflective of the combined cell populations, could help elucidate these mechanisms. To this end, total RNA from colostrum and mature milk samples were sequenced from 65 sows at differing parities. A stringent bioinformatic pipeline was used to identify and characterize 70,841 transcripts. Results: The 70,841 identified transcripts included 42,733 previously annotated transcripts and 28,108 novel transcripts. Differential gene expression analysis was conducted using a generalized linear model coupled with the Lancaster method for P-value aggregation across transcripts. In total, 1667 differentially expressed genes (DEG) were identified for the milk type main effect, and 33 DEG were identified for the milk type x parity interaction. Several gene ontology (GO) terms related to immune response were significant for the milk type main effect, supporting the well-known fact that immunoglobulins and immune cells are transferred to the neonate via colostrum. Conclusions: This is the first study to perform global transcriptome analysis from whole milk samples in sows from different parities. Our results provide important information and insight into synthesis of milk proteins and innate immunity and potential targets for future improvement of swine lactation and piglet development. [ABSTRACT FROM AUTHOR]

RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3′ or 5′ termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.

RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3′ or 5′ termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples. [ABSTRACT FROM AUTHOR]

Aim: to conduct a comparative assessment of the effectiveness of liver regeneration occurring after induction of chronic fibrosing liver disease (CFLD) using bone marrow mononuclear cells (BMMCs) and total RNA (tRNA) extracted from BMMCs.Materials and methods. The study involved 140 Wistar rats. CFLD was modeled in 100 rats, of which 25 died. The surviving 75 rats (CFLD formed by the third month) were divided into 3 groups: Group 1 – control (administered with physiological saline); Group 2 – a single injection of tRNA from BMMCs at a dose of 30 μg/100g body weight; Group 3 – a single injection of BMMCs at a dose of (30–35) × 106 cells. The dynamics of regenerative processes in the liver was evaluated based on the animal mortality, dynamics of restoration of biochemical markers (ALAT, ASAT, alkaline phosphatase and total protein) and morphological picture of the liver on the seventh day and after three, six and nine months. The significance of differences in the compared values was determined through Student’s t-test for

Extensive liver resection (ELR), performed in a number of surgical operations, refers to a critical injury, which necessitates the improvement of methods of therapy of acute post-resection liver failure.Aim: to compare the effectiveness of stimulation of regenerative processes in the liver residue after ELR (60–70%) by intraperitoneal administration of lysed aspirate bone marrow cells (BMCs) and total RNA (tRNA) isolated from BMCs.Materials and methods. This work was performed on 175 rats-male Wistar breed 250–300 g, on 75 of which under the inhalation anesthesia it was reproduced the model of the ELR in three groups of experiments: group 1 – control (administration of isotonic solution after ELR), group 2 – in 3–5 hours after ELR the tRNA from BMCs was intraperitoneally injected at a dose of 30 μg/100 g, group 3 – in 3–5 hours after ELR BMCs was administered intraperitoneally at a dose of 30–35 × 106 cells per rat. Comparative studies of the restorative processes in the liver after the ELR in the three groups were carried out by dynamic control of the mitotic activity of hepatocytes in the liver residue, cytolytic enzymes, total bilirubin and total serum protein, as well as the liver residue (mass) weight.Results. The tRNA from BMCs and BMCs in the indicated doses prevent the risk of the development of lethal outcomes, and also contribute to an earlier (by 10–14 days) normalization of the functional indices of hepatic homeostasis. However, the tRNA from BMCs, compared with BMCs, has a stronger stimulating effect on the recovery processes: it promotes earlier intensification of mitotic activity of hepatocytes and provides a higher rate of recovery of liver mass.Conclusion. For the induction of recovery processes in the liver residue after ELR, the preference should be given to the tRNA from BMCs.

Grapevine germplasm collections are unique repositories of grape cultivars; therefore, it is necessary to minimize their infection with pathogens, including viruses, and develop various programs to maintain them in a virus-free state. In our study, we examined the virome of the largest Russian grapevine germplasm collection, the Anapa Ampelographic Collection, using high-throughput sequencing of total RNAs. As a result of bioinformatics analysis and validation of its results by reverse transcription PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR), we identified 20 viruses and 3 viroids in 47 libraries. All samples were infected with 2 to 12 viruses and viroids, including those that cause economically significant diseases: leafroll, fleck, and rugose wood complex. For the first time in Russia, we detected Grapevine virus B (GVB), Grapevine virus F (GVF), Grapevine asteroid mosaic-associated virus (GAMaV), Grapevine Red Globe virus (GRGV), Grapevine satellite virus (GV-Sat), Grapevine virga-like virus (GVLV), Grapevine-associated jivivirus 1 (GaJV-1) and Vitis cryptic virus (VCV). A new putative representative of the genus Umbravirus with the provisional name Grapevine umbra-like virus (GULV) was also identified in Russian grape samples.

Unlike for vertebrates, the impact of starvation on the gut microbiome of invertebrates is poorly studied. Deciphering shifts in metabolically active associated bacterial communities in vertebrates has led to determining the role of the associated microbiome in the sensation of hunger and discoveries of associated regulatory mechanisms. From an invertebrate perspective, such as the black soldier fly, such information could lead to enhanced processes for optimized biomass production and waste conversion. Bacteria associated with food substrates of black soldier fly are known to impact corresponding larval life-history traits (e.g., larval development); however, whether black soldier fly larval host state (i.e., starved) impacts the gut microbiome is not known. In this study, we measured microbial community structural and functional shifts due to black soldier fly larvae starvation. Data generated demonstrate such a physiological state (i.e., starvation) does in fact impact both aspects of the microbiome. At the phylum level, community diversity decreased significantly during black soldier fly larval starvation (p = 0.0025). Genus level DESeq2 analysis identified five genera with significantly different relative abundance (q < 0.05) across the 24 and 48 H post initiation of starvation: Actinomyces, Microbacterium, Enterococcus, Sphingobacterium, and Leucobacter. Finally, we inferred potential gene function and significantly predicted functional KEGG Orthology (KO) abundance. We demonstrated the metabolically active microbial community structure and function could be influenced by host-feeding status. Such perturbations, even when short in duration (e.g., 24 H) could stunt larval growth and waste conversion due to lacking a full complement of bacteria and associated functions.

Unlike for vertebrates, the impact of starvation on the gut microbiome of invertebrates is poorly studied. Deciphering shifts in metabolically active associated bacterial communities in vertebrates has led to determining the role of the associated microbiome in the sensation of hunger and discoveries of associated regulatory mechanisms. From an invertebrate perspective, such as the black soldier fly, such information could lead to enhanced processes for optimized biomass production and waste conversion. Bacteria associated with food substrates of black soldier fly are known to impact corresponding larval life-history traits (e.g., larval development); however, whether black soldier fly larval host state (i.e., starved) impacts the gut microbiome is not known. In this study, we measured microbial community structural and functional shifts due to black soldier fly larvae starvation. Data generated demonstrate such a physiological state (i.e., starvation) does in fact impact both aspects of the microbiome. At the phylum level, community diversity decreased significantly during black soldier fly larval starvation (p = 0.0025). Genus level DESeq2 analysis identified five genera with significantly different relative abundance (q < 0.05) across the 24 and 48 H post initiation of starvation: Actinomyces , Microbacterium , Enterococcus , Sphingobacterium , and Leucobacter. Finally, we inferred potential gene function and significantly predicted functional KEGG Orthology (KO) abundance. We demonstrated the metabolically active microbial community structure and function could be influenced by host-feeding status. Such perturbations, even when short in duration (e.g., 24 H) could stunt larval growth and waste conversion due to lacking a full complement of bacteria and associated functions. [ABSTRACT FROM AUTHOR]

[Objective) To explore the best approach for total RNA extraction. [Method ] three RNA extraction methods (Trizol, EasyPure Plant RNA Kit and TransZol Plant) were performed using the flower buds of fresh Iris tectorum Maxim. and frozen I. bulleyana Dykes. The integrity of RNA were tested by agarose gel electrophoresis, whereas the purity and concentration of total RNA were detected by micro UV spectrophotometry. The data was used to compare the mass and concentration of total RNA extracted by above different methods. [Result] The results showed that the total RNA of frozen I. bulleyana Dykes buds could only extracted by TransZol Plant method with good quality. Whereas, the RNA of fresh I. tectorum Maxim buds could be extracted by three methods for subsequent experiments, and the quality of RNA extracted by EasyPure Plant RNA kit was the best, and the yield of RNA extracted by TransZol Plant method was the highest, but Trizol method produced lowest yield. [Conclusion] Considering the plant sample materials, the cost, RNA purity and yield, the EasyPure Plant RNA Kit was suitable for high quality RNA yielding using fresh plant material, whereas the TransZol Plant was suitable for high RNA produe tion but lower quality using frozen fris flowers. This study provided the theory found for further research on Iris molecular biology, and also provided necessary reference for the RNA extraction of other Plant flowers. [ABSTRACT FROM AUTHOR]

The current naso-oropharyngeal swab for SARS-CoV-2 detection faces several problems, such as waste issues and its use for quantitative studies. This study aimed to evaluate the total RNA and viral loads from different upper respiratory tract swabs types and whether SARS-CoV-2 quantification can use the current internal control for normalization. This cross-sectional study collected positive specimens with single oropharyngeal or nasopharyngeal swabs and naso-oropharyngeal swabs. The samples were extracted, tested with qualitative RT‒PCR, and then tested with quantitative RT‒PCR. The RNA eluate was measured for the total RNA concentration. The total RNA concentration, viral load, and RNaseP Ct values were collected and analysed statistically. The positive results came from 41 oropharyngeal swabs, 34 nasopharyngeal swabs, and 36 naso-oropharyngeal swabs. The total RNA increased significantly from oropharyngeal swabs to nasopharyngeal swabs to naso-oropharyngeal swabs. Significant differences in RNaseP Ct values between groups and their correlations with total RNA were found. In addition, the increase in the total RNA and the RNaseP Ct values were unrelated to the viral load. The physical features in the naso-oropharyngeal area and the swabbing procedures could affect the total RNA but not the viral load. However, since the virus particles could present inside and outside human cells, the increase in collected human cells may not always be followed by the viral load increase. Normalization using the RNaseP Ct value became unnecessary due to the factors mentioned above. Therefore, a careful approach is needed in viral load studies of swab specimens., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Copyright of Forest Research is the property of Forest Research Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This paper presents a preliminary analysis of the morphological, physiological, and biochemical parameters of somatic growth in the Black Sea scallop, Flexopecten glaberponticus Bucguoy, Dautzenberg et Dollfus 1889, from a coastal site near Sevastopol, Crimea. The species is one of the few marine Black Sea mollusks listed in the Red Book of Sevastopol and Crimea with the status "Diminishing in numbers." The peculiarities of soft tissue somatic growth have been studied in three size groups differing in linear diameter: 13–17, 21–24, and 25–30 mm. An analysis of the total RNA in tissue homogenates has shown that the rate of protein synthesis in the first and second groups (under-yearlings) is 1.2–1.5 times higher compared to adults. The population reveals seven color morphs (phenotypes). Three phenotypes (beige, violet, and gray-and-brown) are characterized by high-level protein synthesis. Mollusks of a mixed "multi" phenotype have the lowest level of tissue biosynthesis, usually being 2.5 times lower than that for other morphs. The results obtained indicate that the growth processes are linked to the color of the mollusk shell. High direct correlations between the weights of the total wet mass and the tissue mass observed in all mollusk groups may be evidence of positive growth allometry. [ABSTRACT FROM AUTHOR]

Background: Lactobacillus gasseri OLL2809 can highly induce interleukin (IL)-12 production in immune cells. Even though beneficial properties of this strain for both humans and animals have been reported, the mechanism by which the bacteria induces the production of IL-12 in immune cells remains elusive. In this study, we investigated the mechanism of induction of IL-12 using a mouse macrophage cell line J774.1. Results: Inhibition of phagocytosis of L. gasseri OLL2809, and myeloid differentiation factor 88 and Toll-like receptors (TLRs) 7 and 9 signalling attenuated IL-12 production in J774.1 cells. Total RNA and genomic DNA of L. gasseri OLL2809, when transferred to the J774.1 cells, also induced IL-12 production. The difference in the IL-12-inducing activity of Lactobacilli is attributed to the susceptibility to phagocytosis, but not to a difference in the total RNA and genomic DNA of each strain. Conclusion: We concluded that total RNA and genomic DNA of phagocytosed L. gasseri OLL2809 induce IL-12 production in J774.1 cell via TLRs 7 and 9, and the high IL-12-inducing activity of L. gasseri OLL2809 is due to its greater susceptibility to phagocytosis. [ABSTRACT FROM AUTHOR]

We used high-throughput sequencing to identify viruses on tomato samples showing virus-like symptoms. Samples were collected from crops in the Iberian Peninsula. Either total RNA or double-stranded RNA (dsRNA) were used as starting material to build the cDNA libraries. In total, seven virus species were identified, with pepino mosaic virus being the most abundant one. The dsRNA input provided better coverage and read depth but missed one virus species compared with the total RNA input. By performing in silico analyses, we determined a minimum sequencing depth per sample of 0.2 and 1.5 million reads for dsRNA and rRNA-depleted total RNA inputs, respectively, to detect even the less abundant viruses. Primers and TaqMan probes targeting conserved regions in the viral genomes were designed and/or used for virus detection; all viruses were detected by qRT-PCR/RT-PCR in individual samples, with all except one sample showing mixed infections. Three virus species (Olive latent virus 1, Lettuce ring necrosis virus and Tomato fruit blotch virus) are herein reported for the first time in tomato crops in Spain.

Meloidogyne spp. are plant-parasitic nematodes that form a very complex pseudo-organ, called gall, which contains the giant cells (GCs) to nourish them. During the last decade, several groups have been studying the molecular processes accompanying the formation of these structures, combining both transcriptomics and cellular biology. Among others, it was confirmed that a generalized gene repression is a hallmark of early developing GCs formed by Meloidogyne javanica in Arabidopsis and tomato. One of the main mechanisms behind this gene repression involve small RNAs (sRNAs) directed gene silencing. This is supported not only by the described action of several microRNAs differentially expressed in galls, but by the differential abundance of 24-nucleotide sRNAs in early developing Arabidopsis galls, particularly those rasiRNAs which are mostly involved in RNA-directed DNA methylation. Their accumulation strongly correlates to the repression of several retrotransposons at pericentromeric regions of Arabidopsis chromosomes in early galls. However, the contribution of this global gene repression to GCs/galls formation and maintenance is still not fully understood. Further detailed studies, as the correlation between gene expression profiles and the methylation state of the chromatin in galls are essential to raise testable working hypotheses. A high quality of isolated DNA and RNA is a requirement to obtain non-biased and comprehensive results. Frequently, the isolation of DNA and RNA is performed from different samples of the same type of biological material. However, subtle differences on epigenetic processes are frequent even among independent biological replicates of the same tissue and may not correlate to those changes on the mRNA population obtained from different biological replicates. Herein, we describe a method that allows the simultaneous extraction and purification of genomic DNA and total RNA from the same biological sample adapted to our biological system. The quality of both nucleic acids from Arabidopsis galls formed by M. javanica was high and adequate to construct RNA and DNA libraries for high throughput sequencing used for transcriptomic and epigenetic studies, such as the analysis of the methylation state of the genomic DNA in galls (MethylC-seq) and RNA sequencing (RNAseq). The protocol presents guidance on the described procedure, key notes and troubleshooting.

Copyright of Türkiye Klinikleri Journal of Veterinary Sciences is the property of Turkiye Klinikleri and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Meloidogyne spp. are plant-parasitic nematodes that form a very complex pseudo-organ, called gall, which contains the giant cells (GCs) to nourish them. During the last decade, several groups have been studying the molecular processes accompanying the formation of these structures, combining both transcriptomics and cellular biology. Among others, it was confirmed that a generalized gene repression is a hallmark of early developing GCs formed by Meloidogyne javanica in Arabidopsis and tomato. One of the main mechanisms behind this gene repression involve small RNAs (sRNAs) directed gene silencing. This is supported not only by the described action of several microRNAs differentially expressed in galls, but by the differential abundance of 24-nucleotide sRNAs in early developing Arabidopsis galls, particularly those rasiRNAs which are mostly involved in RNA-directed DNA methylation. Their accumulation strongly correlates to the repression of several retrotransposons at pericentromeric regions of Arabidopsis chromosomes in early galls. However, the contribution of this global gene repression to GCs/galls formation and maintenance is still not fully understood. Further detailed studies, as the correlation between gene expression profiles and the methylation state of the chromatin in galls are essential to raise testable working hypotheses. A high quality of isolated DNA and RNA is a requirement to obtain non-biased and comprehensive results. Frequently, the isolation of DNA and RNA is performed from different samples of the same type of biological material. However, subtle differences on epigenetic processes are frequent even among independent biological replicates of the same tissue and may not correlate to those changes on the mRNA population obtained from different biological replicates. Herein, we describe a method that allows the simultaneous extraction and purification of genomic DNA and total RNA from the same biological sample adapted to our biological system. The quality of both nucleic acids from Arabidopsis galls formed by M. javanica was high and adequate to construct RNA and DNA libraries for high throughput sequencing used for transcriptomic and epigenetic studies, such as the analysis of the methylation state of the genomic DNA in galls (MethylC-seq) and RNA sequencing (RNAseq). The protocol presents guidance on the described procedure, key notes and troubleshooting. [ABSTRACT FROM AUTHOR]

Total RNA isolated from peripheral blood lymphocytes of donor and patient with polycythemia, stimulates hematopoiesis in rats with toxic aplastic anemia due to benzene administration. Total RNA of lymphocytes from polycythemia patient has a more pronounced effect on the erythroid, myeloid and megakaryocytic hematopoiesis comparing to total RNA from donor lymphocytes. The greatest stimulatory effectof RNA observed after 21 days from the start of experiment. Total RNA of lymphocytes from polycythemia patient largely activates erythropoiesis promoting restoration of reticulocyte count in animals with aplastic anemia.

Ginger rhizomes contain high amount of water, polysaccharides, polyphenols and secondary metabolites which interfere with isolation of total RNA. High quality RNA with sufficient quantity is crucial for conducting gene expression studies. An effective protocol for isolation of RNA from ginger rhizomes is essential. The present study is focused on isolation of high quality RNA from ginger rhizome which can be further utilized for downstream applications like cDNA library preparation and differential gene expression studies. The protocol reported by Kumar et al. (2007) was modified and was compared with the original protocol. The modified protocol was found effective in getting higher quality (A260/A280-1.95 to 2.05) and quantity (51-58.6 µg/g) of isolated total RNA from fresh and frozen rhizomes. In the original protocol the quality obtained was (A260/A280-1.47 to 1.54) and quantity was 24.0-42.0 µg/g. [ABSTRACT FROM AUTHOR]