Nucleic Acid Content Research Articles

Histochemical Analysis of DNA and RNA Identified by Methyl Green - Pyronin ‘Y’ Method and Feulgen’s Reaction Staining in Liver of Channa Punctatus

The light microscopy was to identify cells, storage products, tissue deposits, and microbial pathogens. The Assessment of surrounding tissue with special stains may reveal aspects of interest for the tissue. We illustrate the expected staining characteristics of control Channa punctatus liver tissue with Methyl Green -Pyronin ‘Y’ and Feulgen’s Reaction stains. The aim of this study was to characterize the normal structure of the liver of Channa punctatus, a carnivorous freshwater fish found in Hasanparthy Lake, using gross anatomy, histology and histochemistry. Anatomically, the liver presents C-shaped and only two lobes smaller right and bigger left. The gallbladder is located in right lobe and shows elongated shaped. Histological analysis demonstrated that the hepatic parenchyma is made of two hepatocytes plates surrounded by sinusoids. The fish moreover pancreatic tissue was observed in visceral portion of liver, formed by exocrine pancreatic tissue and islet organ, constituting an extrahepatic pancreas. The nucleic acids content in different tissues in the freshwater fish of Channa punctatus. The fish were scarified and the tissues such as liver and Pancreas removed and processed for determination of nucleic acids. The results obtained in the present study may provide a contribution to the knowledge of the characteristics of nucleic acid as parameters of estimation of DNA and RNA levels in Liver of the fish.

Nucleic Acid-Rich Stress Granules Are Not Merely Crowded Condensates: A Quantitative Raman Imaging Study.

Liquid droplets, formed by intracellular liquid-liquid phase separation (LLPS), are called membraneless organelles. They provide transient enzymatic reaction fields for maintaining cellular homeostasis, although they might transform into aggregates, leading to neurodegenerative diseases. To understand the nature of intracellular droplets, it is crucial to quantify the liquid droplets inside a living cell as well as to elucidate the underlying biological mechanism. In this study, we performed near-infrared fluorescence and Raman imaging to quantify chemical components inside stress granules (SGs) formed by LLPS in living cells. The Raman images reveal that the nucleic acid concentration inside the SGs was more than 20% higher than the surrounding cytoplasm, whereas the lipid concentration was lower. Quantitative Raman intensity analysis using a water Raman band as an internal standard enables in situ concentration determination of nucleic acids in the SGs and other organelles. The intensity of the biomolecular C-H bands relative to the water band indicates that the crowding environment inside the SGs depends on the stress type; under oxidative stress, the inside of the SGs was nearly identical to the outside, whereas it was sparser in hyperosmotic stressed cells, suggesting that the high concentrations of nucleic acids play a pivotal role in maintaining the environments inside the SGs. These results demonstrate that intracellular droplets are not always highly condensed.

Recycling potential of Cupriavidus necator for life support in space: Production of SCPs from volatile fatty acid and urea mixture

The International Space Station currently requires four annual replenishments for food supply, a practice that won't be feasible for deep space missions due to the greater distances. Based on the design of closed ecological life support systems, two waste streams were identified: urea from the crew urine, volatile fatty acids (VFAs) from a first stage of anaerobic digestion of waste. The objective of this study was to assess the ability of bacterium Cupriavidus necator to produce single cell protein on urea and VFAs. Thus, the effect of carbon sources (glucose vs VFAs) and the dilution rate on the biomass composition was determined in continuous cultures. Complete transformation of the carbon source into protein-rich biomass was achieved up to 78 % cell dry weight (CDW). For both carbon sources, the protein content increased from 55.0 %CDW to 78 %CDW with a decrease in the dilution rate. Conversely, the nucleic acid and polyhydroxyalkanoate contents decreased with the dilution rate from 8.8 %CDW to 4.8 %CDW and 9.8 %CDW to 0.6 %CDW respectively. Working at a low dilution rate seems to be a good way to maximize protein content while minimizing unwanted nucleic acids and polyhydroxyalkanoates.

Effect of concentration, temperature, and time on the stability of hepatitis C virus nucleic acid in serum.

Previously, there were few reports about the influence of different concentrations of sample nucleic acid on the stability of samples at various temperatures and times in various literatures. Therefore, it is necessary to analyze the influence of concentration factors on the stability of samples and test results at different storage times and temperatures. This study took the concentration of hepatitis C virus nucleic acid as the research object to further understand the stability of hepatitis C virus nucleic acid test samples under various storage conditions, to provide data reference for the treatment of hepatitis C virus nucleic acid and RNA test samples before clinical laboratory test, and provide guidance and help for the improvement of laboratory quality control.

The construction of an inflammation classification model for HDPCs applied in guiding pulpotomy based on single-cell Raman spectroscopy

The immune defense and the repair function of the pulp tissue serve as the biological foundation of pulpotomy. The precise evaluation of the pulp inflammation extent and determining its reversibility are essential for the success of pulpotomy. The objective of this study was to classify the molecular-level of dental pulp cell physiology and inflammatory state based on the biochemical changes obtained by single-cell Raman spectroscopy. Firstly, we differentiated the growth of HDPCs (human dental pulp cells) under physiological states by employing Raman spectroscopy with multivariate statistical analysis. Raman spectroscopy reflected the biochemical changes at different growth phases, including the lag phase, log phase, and stationary phase. Secondly, we evaluated the optimal concentration and duration of Porphyromonas gingivalis lipopolysaccharide (P.g.LPS) stimulation to establish a six-level inflammation classification model of HDPCs. Thirdly, we performed label-free characterization of biological component changes in cells of different inflammation grades by Raman spectroscopy. As a result, the differences of peaks in the region 600–1800 cm−1 demonstrated the biochemical molecular alterations in the different inflammation grades of HDPCs. As inflammation progresses in steps, protein peaks increased first and then decreased, while lipid and nucleic acid peaks gradually decreased compared to unstimulated cells. However, when the inflammatory stimulation reached grade V, the changes in the biological properties were characterized by a recovery in protein and lipid content, and a decrease in nucleic acid content. We then established the diagnostic model using the Raman spectra of HDPCs in physiological and inflammatory states, which had a prediction accuracy of 100 % and 97.4 %, respectively. Finally, we determined the reversibility threshold of HDPCs at different grades of inflammation. We observed that the inflammation of grade I and II cells had potential reversibility and could be attempted to be retained. In conclusion, Raman spectroscopy combined with multivariate statistical analysis has potential possibility to effectively distinguish the degree of inflammation in the dental pulp, thus providing new tools and perspectives on pulpotomy in clinical practice.

Chlorpyrifos modulates the mouse gut microbiota and metabolic activity

The organophosphate chlorpyrifos is a commonly used pesticide for fruits and vegetables despite its association with neurotoxicity in humans. While some studies suggest that organophosphates may impact the gut microbiota, no studies to date have investigated the direct effect of chlorpyrifos on the gut microbiota with doses that approximate environmentally relevant dietary concentrations (EPA chronic reference dose: 0.3 µg/kg/day in humans and EPA acute reference dose: 5 µg/kg/day in humans). Thus, we examined the influence of chlorpyrifos on the gut microbiota by assessment of bacterial physiology and metabolism using flow cytometry, 1H NMR-based metabolomics, and changes in the cecal microbiota community with 16S rRNA amplicon sequencing and analysis. Chlorpyrifos did not directly damage bacteria but rather perturbed bacterial metabolism. Chlorpyrifos exposure to bacteria increased the concentration of amino acids, carbohydrates, and nucleic acids. The relative abundances of Lactobacillus, Allobaculum, Roseburia, and Butyricicoccus increased after exposure to chlorpyrifos. Analyses of the 16S rRNA gene amplicon data predicted decreased amino acid biosynthesis and nucleic acid degradation and increased glycolysis which was supported by 1H NMR-based metabolomics. Collectively, these results demonstrate that environmentally relevant doses of chlorpyrifos can impact the metabolic activity of isolated gut microbes which may result in an imbalance in overall gut metabolic activity.

Highly efficient and broad-spectrum antibacterial carbon dots combat antibiotic resistance

Bacterial infections have become a major global public health issue, particularly with the emergence of multidrug-resistant strains. Therefore, developing non-antibiotic antimicrobial agents is crucial for treating drug-resistant bacterial infections. Building on previous research into natural products as novel antibacterial agents, this study synthesized curcumin-derived carbon dots using curcumin and ethylenediamine as raw materials through a hydrothermal method. The resulting carbon dots not only improved the water solubility and stability of curcumin but also exhibited highly efficient broad-spectrum antibacterial activity. Detailed investigations into the antibacterial performance and mechanisms of the carbon dots were conducted through experiments such as minimum inhibitory concentration (MIC) determination, live/dead bacterial staining, morphological studies, nucleic acid concentration detection, and reactive oxygen species (ROS) detection. The results indicated that the carbon dots significantly damaged the structural integrity of bacteria and generated large amounts of ROS. They exhibited remarkable antibacterial effects against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, and effectively inhibited drug-resistant MRSA. Their antibacterial efficacy was notably superior to that of broad-spectrum antibiotics such as chloramphenicol and Sulfadiazine. This study highlights the potential application of curcumin-derived carbon dots in combating bacterial infections and provides valuable insights for developing novel antibacterial agents derived from natural products.

Comparison of human breast milk vs commercial formula-induced early trophic enteral nutrition during postoperative prolonged starvation in an animal model

The present study aimed to characterize the changes in macromolecular composition and structure in ileal tissue induced by postoperative prolonged starvation (PS), human breast milk feeding (HM) and commercial formula feeding (CF) for 48 and 72 h (h). Forty-two Wistar albino rats underwent an ileal transection and primary anastomosis and were then divided into six subgroups. Two groups of seven rats were food-deprived for 48 and 72 h with free access to water only in metabolic cages (48 h PS, 72 h PS). Then, two groups of seven rats received early enteral trophic nutrition (EEN) either using HM, and CF at 48 h post-operation (48 h HM, 48 h CF). The other two groups of seven rats received the same trophic enteral nutrition at 72 h post-operation (72 h HM, 72 h CF). An additional seven rats were fed normal rat chow (control), after which the ileal tissues were harvested and freeze-dried overnight. Then sample spectra were recorded by Fourier transform infrared (FTIR) spectroscopy. PS at 48 and 72 h resulted in an increase in the concentration of lipids and a decrease in the concentration of proteins. CF and HM trophic feeding induced a decrease in membrane fluidity and an increase in lipid order. Ileal tissues showed similar compositional and structural changes in lipids and proteins in the PS and CF groups after 48 and 72 h. A marked decrease in nucleic acid concentration was seen in CF at 48 h compared to HM. The human milk feeding groups did not induce any significant alterations and showed compositional and structural data similar to the controls. In conclusion, EEN application seems to be safer when introduced at 48 h rather than 72 h and time of this nutrition is crucial to maintain ileum structure and therefore immunity and well-being. HM-induced trophic nutrition is seen to protect the ileal tissue from significant alterations within lipid and protein compositions, whereas CF caused notable changes. HM is absolutely the best nutritional source for gut health in this animal model.

Comparison of commercially available DNA and RNA extraction kits for wildlife feces collected from the environment.

Wildlife fecal samples were collected across two Air Force installations to evaluate the effectiveness of commercially available DNA and RNA extraction kits. Four DNA kits, two DNA/RNA kits and one RNA only kit were used. Sample extracts were evaluated on nucleic acid concentration, TapeStation DNA or RNA Integrity Number values and presence of PCR inhibitors. For the DNA kits, PFP produced higher concentrations compared with PLM and RPM, while MWFM gave higher DNA Integrity Number values when compared with RPM. No PCR inhibition was detected. For the RNA kits, RPM gave higher concentrations compared with MWTV and no differences were seen in RNA Integrity Number values. PCR inhibition was observed in all RNA samples, with MWTV exhibiting higher inhibition compared with RPM.

Transesophageal ultrasound-guided bronchoscopic Acquire TBNB versus Vizishot2 TBNA needles for neoplastic lesions: A retrospective study

BackgroundLung cancer is often diagnosed at an advanced stage; however, it has shown improved therapeutic efficacy with the introduction of molecularly targeted drugs and immune checkpoint inhibitors, necessitating accurate molecular diagnosis for effective treatment planning. Traditional sampling techniques, including endobronchial ultrasound-guided transbronchial needle aspiration, frequently require multiple biopsies to obtain sufficient tissues for multiplex testing, highlighting the need for more efficient methods. Therefore, we explored the diagnostic utility of endoscopic ultrasound with bronchoscope-guided fine-needle biopsy (EUS-B-FNB) versus fine-needle aspiration (EUS-B-FNA) in patients with lung cancer, focusing on tissue sample collection for molecular testing. The introduction of the Franseen needle in EUS-B-FNB, characterized by three beveled edges, allows for more tissue collection in cylinder form. MethodsWe retrospectively analyzed the data of 97 patients who underwent EUS-B-FNB or EUS-B-FNA at Hakodate Goryoukaku Hospital and evaluated diagnostic yields, safety, and nucleic acid concentrations using collected specimens. ResultsThe diagnostic yields of EUS-B-FNB and EUS-B-FNA were comparable (92.2% vs. 92.3%), with no significant differences in complications. However, EUS-B-FNB provided significantly higher DNA and RNA concentrations (DNA; 41.05 vs. 10.20 ng/mL; P < 0.0001, RNA; 36.80 vs. 11.80 ng/mL; P = 0.0009), essential for comprehensive molecular testing. ConclusionThis study highlights the potential of EUS-B-FNB for enhancing the molecular diagnosis of lung cancer by ensuring adequate tissue sample collection for multiplex testing, paving the way for personalized medicine. This technique is comparable in safety and efficacy to traditional methods while offering a substantial improvement in the quality of molecular diagnostics.

Biocontrol of rusted root rot in Panax ginseng by a combination of extracts from Bacillus amyloliquefaciens YY8 crude protein and Enterobacteriaceae YY115 ethyl acetate

BackgroundRusted root rot is one of the most common root diseases in Panax ginseng, and Cylindrocarpon destructans is one of the main pathogenic fungus. The objective of this study was to screen and explore the extracts of biocontrol bacteria isolated from ginseng rhizosphere soil against Cylindrocarpon destructans.ResultsBacterial strains Bacillus amyloliquefaciens YY8 and Enterobacteriacea YY115 were isolated and found to exhibit in vitro antifungal activity against C. destructans. A combination of crude protein extract from B. amyloliquefaciens YY8 and ethyl acetate extract from Enterobacteriacea YY115 in a 6:4 ratio exhibited the strongest antifungal activity against C. destructans. Measurements of electrical conductivity, protein content, and nucleic acid content in suspension cultures of C. destructans treated with a mixture extracts indicated that the extracts disrupted the cell membranes of rusted root rot mycelia, resulting in the leakage of electrolytes, proteins, and nucleic acids from the cells, and ultimately inhibiting the growth of C. destructans. The combined extracts suppressed the infection of ginseng roots discs by C. destructans effectively.ConclusionThe extracts obtained from the two bacterial strains effectively inhibited C. destructans in P. ginseng. It can provide scientific basis for the development of new biological control pesticides, reduce the use of chemical pesticides, and promote the sustainable development of agriculture.

Preanalytical Impact of Incomplete K2EDTA Blood Tube Filling in Molecular Biology Testing.

The aim of this study was to investigate the possible preanalytical effect of incomplete filling of blood tubes on molecular biology assays. The study population consisted of 13 healthy volunteers from whom 11 mL of whole blood was collected and then distributed in different volumes (1.5, 3.0, and 6.0 mL, respectively) into three 6.0 mL spray-dried and evacuated K2EDTA blood tubes. Automated RNA extraction was performed using the Maxwell® CSC RNA Blood Kit. DNA was extracted with a MagCorePlusII, with concomitant measurement of glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) gene expression. The nucleic acid concentration was calculated using the NanoDrop 1000 spectrophotometer, and purity was assessed using A260/280 and A260/230 absorbance ratios. The RNA concentration was higher in the tubes filled with 1.5 and 3.0 mL of blood than in the reference 6 mL filled tube. The RNA 260/280 and RNA 260/230 ratios did not differ significantly between the differently filled blood tubes. The DNA concentration remained constant in the differently filled tubes. Compared to the 6.0 mL reference filled tube, the 1.5 mL and 3.0 mL filled blood tubes displayed a lower DNA 260/280 nm ratio. The DNA 260/230 ratio did not differ significantly in any of the variously filled tubes. Compared to the 6.0 mL reference filled blood tube, the 1.5 mL and 3.0 mL filled blood tubes showed a significant increase in the GAPDHcycle threshold. Our results suggest that underfilling of K2EDTA blood tubes may be a modest but analytically significant source of bias in molecular biology testing.

Efficient concentration of viral nucleic acid in wastewater through surfactant releasing and a two-step magnetic bead extraction and purification

Wastewater-based epidemiology (WBE) is a valuable complement to clinical monitoring, allowing for effective surveillance of viral infections in populations, and tracking the presence and the epidemiological dynamics of various infectious pathogens in communities. However, virus loads are usually low-abundant in wastewater, and current virus concentration methods for WBE are laborious and time-consuming with low recovery efficiency. To address these challenges, we have developed a magnetic bead-based semi-automated method involving extraction and purification to directly concentrate viral nucleic acids from sewage within 55 min. Prior to concentration, 0.5 % LDS was introduced to pretreat wastewater to inactivate viruses and release viral nucleic acids from both liquid and solid fractions to improve recovery. Under optimal conditions, the concentration method combined with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA added exogenously in wastewater as low as 4.9 copies/mL within 2.5 h, with an average recovery rate exceeding 80 %. Testing real sewages proved the applicability of the method to detect multiple viruses in different sewages. Additionally, variants of SARS-CoV-2 were successfully identified by multiplex amplicon sequencing in two samples. In conclusion, the new method could provide a much more efficient way for WBE of pathogenic viruses in various sewages.

The amount of DNA and RNA in primary cancer cells and their metastases

Considering the significant impact of oncopathology on global morbidity and mortality, as well as changes in its prevalence and typology, research into the molecular-genetic mechanisms of oncogenesis and metastasis, particularly the role of nucleic acids, becomes crucial. The aim of this study is to determine the content of DNA and RNA in cells of primary cancer and its metastases through microspectrophotometric analysis of malignant cells collected from 30 patients with primary and metastatic oncological processes. The following methods were employed: microspectrophotometric analysis using the automated complex “Protva-PM11-DVK-3m,” staining of histological specimens with halo cyanin-chrome alum, the discrete statistics method, frequency and cluster analysis, and the bibliographic method. Changes in the concentration of nucleic acids in the cells of primary and metastatic tumors were identified during the study. The changes did not always correlate with each other, indicating morphofunctional polymorphism in tumor cell populations. A trend towards a decrease in nuclear RNA concentration and an increase in cytoplasmic RNA concentration in metastases were observed, suggesting alterations in metabolic and synthetic processes in cells and a decrease in their differentiation degree.

Development and validation of a rapid five-minute nucleic acid extraction method for respiratory viruses

BackgroundThe rapid transmission and high pathogenicity of respiratory viruses significantly impact the health of both children and adults. Extracting and detecting their nucleic acid is crucial for disease prevention and treatment strategies. However, current extraction methods are laborious and time-consuming and show significant variations in nucleic acid content and purity among different kits, affecting detection sensitivity and efficiency. Our aim is to develop a novel method that reduces extraction time, simplifies operational steps, and ensures high-quality acquisition of respiratory viral nucleic acid.MethodsWe extracted respiratory syncytial virus (RSV) nucleic acid using reagents with different components and analyzed cycle threshold (Ct) values via quantitative real-time polymerase chain reaction (qRT-PCR) to optimize and validate the novel lysis and washing solution. The performance of this method was compared against magnetic bead, spin column, and precipitation methods for extracting nucleic acid from various respiratory viruses. The clinical utility of this method was confirmed by comparing it to the standard magnetic bead method for extracting clinical specimens of influenza A virus (IAV).ResultsThe solution, composed of equal parts glycerin and ethanol (50% each), offers an innovative washing approach that achieved comparable efficacy to conventional methods in a single abbreviated cycle. When combined with our A Plus lysis solution, our novel five-minute nucleic acid extraction (FME) method for respiratory viruses yielded superior RNA concentrations and purity compared to traditional methods. FME, when used with a universal automatic nucleic acid extractor, demonstrated similar efficiency as various conventional methods in analyzing diverse concentrations of respiratory viruses. In detecting respiratory specimens from 525 patients suspected of IAV infection, the FME method showed an equivalent detection rate to the standard magnetic bead method, with a total coincidence rate of 95.43% and a kappa statistic of 0.901 (P < 0.001).ConclusionsThe FME developed in this study enables the rapid and efficient extraction of nucleic acid from respiratory samples, laying a crucial foundation for the implementation of expedited molecular diagnosis.

Unlocking the antibacterial activity and mechanisms of ε-poly-l-Lysine and perilla combination against Pseudomonas fluorescens: Insights from non-targeted metabolomic analyses

Perilla frutescens extract has been acknowledged for its robust antibacterial properties, while ε-Poly-Lysine (ε-PL) stands as a naturally occurring food preservative. However, their combined antibacterial activity and mechanisms has not been fully understood. Therefore, the aim of this study was to elucidate the combined antibacterial activity of the ε-PL/Perilla combination against Pseudomonas fluorescens. The findings unveiled a significant antibacterial efficacy of ε-PL and perilla against P. fluorescens, respectively. Upon treatment with ε-PL and perilla, a decline in membrane potential (MP) was observed. These alterations were further accompanied by reductions in nucleic acid content, metabolic activity, and respiratory chain dehydrogenase activity. And by morphological observation, it was found that the cells treated with perilla or ε-PL were severely broken. The metabolomic analysis following treatment with ε-PL and perilla unveiled substantial shifts in a multitude of metabolites. Multivariate statistical analysis showed that ε-PL and perilla hindered nucleotide metabolism and interfered with carbohydrate, amino acid, and lipid metabolism, impacting cell wall biosynthesis, cell membrane structure, and genetic material expression, leading to growth inhibition and bacterial demise. Collectively, these outcomes provide fresh insights into the intricate mechanisms underlying the inhibitory action of ε-PL and perilla against P. fluorescens.

Spectrochemical and explainable artificial intelligence approaches for molecular level identification of the status of critically ill patients with COVID-19

This study explores the molecular alterations and disease progression in COVID-19 patients using ATR-FTIR spectroscopy combined with spectrochemical and explainable artificial intelligence (XAI) approaches. Blood serum samples from intubated patients (IC), those receiving hospital services (SC), and recovered patients (PC) were analyzed to identify potential spectrochemical serum biomarkers. Spectrochemical parameters such as lipid, protein, nucleic acid concentrations, and IgG glycosylation were quantified, revealing significant alterations indicative of disease severity. Notably, increased lipid content, altered protein concentrations, and enhanced protein phosphorylation were observed in IC patients compared to SC and PC groups. The serum AGR (Albumin/Globulin Ratio) index demonstrated a distinct shift among patient groups, suggesting its potential as a rapid biochemical marker for COVID-19 severity. Additionally, alterations in IgG glycosylation and glucose concentrations were associated with disease severity. Spectral analysis highlighted specific bands indicative of nucleic acid concentrations, with notable changes observed in IC patients. XAI techniques further elucidated the importance of various spectral features in predicting disease severity across patient categories, emphasizing the heterogeneity of COVID-19's impact. Overall, this comprehensive approach provides insights into the molecular mechanisms underlying COVID-19 pathogenesis and offers a transparent and interpretable prediction algorithm to aid decision-making and patient management.

Comparing Conventional and Modern Methods for The Phycocyanin Extraction from Spirullina sp

Spirulina platensis, a blue-green algae abundant in tropical regions, is rich in minerals, vitamins, fibers, and pigments, with low nucleic acid content. It has unique chromoproteins called phycobiliproteins, notably phycocyanin, used in various applications. This study aims to optimize phycocyanin extraction using different solvents (distilled water and sodium phosphate buffer pH 6.7) and methods (freeze-thaw and sonication). Spirulina platensis biomass was extracted in both solvents, then some of them was freeze for 24 and 48 hours followed by thawing overnight. The other was sonicated for 2.5 minutes, 50 Hz then soaked for 1, 2, and 3 hours. All of the samples were centrifuged at 6000 rpm for 10 minutes and the absorbance was measured using a UV-Vis spectrophotometer at wavelengths of 280, 620, and 650 nm. with freeze-thawing for 48 hours yielded the highest phycocyanin concentration (0.55%), with a yield of 11.07 and purity of 0.21. Sonication improved phycocyanin concentration, yield, and purity significantly, yielding 1.108, 25.85, and 0.26, respectively.

A Review of Probe-Based Enrichment Methods to Inform Plant Virus Diagnostics.

Modern diagnostic techniques based on DNA sequence similarity are currently the gold standard for the detection of existing and emerging pathogens. Whilst individual assays are inexpensive to use, assay development is costly and carries risks of not being sensitive or specific enough to capture an increasingly diverse range of targets. Sequencing can provide the entire nucleic acid content of a sample and may be used to identify all pathogens present in the sample when the depth of coverage is sufficient. Targeted enrichment techniques have been used to increase sequence coverage and improve the sensitivity of detection within virus samples, specifically, to capture sequences for a range of different viruses or increase the number of reads from low-titre virus infections. Vertebrate viruses have been well characterised using in-solution hybridisation capture to target diverse virus families. The use of probes for genotyping and strain identification has been limited in plants, and uncertainty around sensitivity is an impediment to the development of a large-scale virus panel to use within regulatory settings and diagnostic pipelines. This review aims to compare significant studies that have used targeted enrichment of viruses to identify approaches to probe design and potential for use in plant virus detection and characterisation.

Human erythrocytes' perplexing behaviour: erythrocytic microRNAs.

Erythrocytes have the potential role in erythropoiesis and disease diagnosis. Thought to have lacked nucleic acid content, mammalian erythrocytes are nevertheless able to function for 120-140days, metabolize heme, maintain oxidative stress, and so on. Mysteriously, erythrocytes proved as largest repositories of microRNAs (miRNAs) some of which are selectively retained and function in mature erythrocytes. They have unique expression patterns and have been found to be linked to specific conditions such as sickle cell anaemia, high-altitude hypoxia, chronic mountain sickness, cardiovascular and metabolic conditions as well as host-parasite interactions. They also have been implicated in cell storage-related damage and the regulation of its survival. However, the mechanism by which miRNAs function in the cell remains unclear. Investigations into the molecular mechanism of miRNAs in erythrocytes via extracellular vesicles have provided important clues in research studies on Plasmodium infection. Erythrocytes are also the primary source of circulating miRNAs but, how they affect the plasma/serum miRNAs profiles are still poorly understood. Erythrocyte-derived exosomal miRNAs, can interact with various body cell types, and have easy access to all regions, making them potentially crucial in various pathophysiological conditions. Which can also improve our understanding to identify potential treatment options and discovery related to non-invasive diagnostic markers. This article emphasizes the importance of erythrocytic miRNAs while focusing on the enigmatic behaviour of erythrocytes. It also sheds light on how this knowledge may be applied in the future to enhance the state of erythrocyte translational research from the standpoint of erythrocytic miRNAs.