Nucleic Acid Content Research Articles

Identifying Neutrophil Extracellular Traps (NETs) in Blood Samples Using Peripheral Smear Autoanalyzers.

Neutrophil Extracellular Traps (NETs) are large neutrophil-derived structures composed of decondensed chromatin, cytosolic, and granule proteins. NETs play an important role in fighting infection, inflammation, thrombosis, and tumor progression processes, yet their fast and reliable identification has been challenging. Smudge cells (SCs) are a subcategory of white cells identified by CellaVision®, a hematology autoanalyzer routinely used in clinical practice that uses digital imaging to generate "manual" differentials of peripheral blood smears. We hypothesize that a proportion of cells identified in the SC category by CellaVision® Hematology Autoanalyzers are actually NETs. We demonstrate that NET-like SCs are not present in normal blood samples, nor are they an artifact of smear preparation. NET-like SCs stain positive for neutrophil markers such as myeloperoxidase, leukocyte alkaline phosphatase, and neutrophil elastase. On flow cytometry, cells from samples with high percent NET-like SCs that are positive for surface DNA are also positive for CD45, myeloperoxidase and markers of neutrophil activation and CD66b. Samples with NET-like SCs have a strong side fluorescent (SFL) signal on the white count and nucleated red cells (WNR) scattergram, representing cells with high nucleic acid content. When compared to patients with low percent SCs, those with a high percentage of SCs have a significantly higher incidence of documented bacterial and viral infections. The current methodology of NET identification is time-consuming, complicated, and cumbersome. In this study, we present data supporting identification of NETs by CellaVision®, allowing for easy, fast, cost-effective, and high throughput identification of NETs that is available in real time and may serve as a positive marker for a bacterial or viral infections.

The Activity of Calcium Glycerophosphate and Fluoride against Cariogenic Biofilms of Streptococcus mutans and Candida albicans Formed In Vitro.

This study evaluated the effects of calcium glycerophosphate (CaGP), with or without fluoride (F), on dual-species biofilms of Streptococcus mutans and Candida albicans. The biofilms were treated three times with 0.125, 0.25, and 0.5% CaGP solutions, with or without 500 ppm F (NaF). Additionally, 500 and 1100 ppm F-solutions and artificial saliva served as controls. After the final treatment, the microbial viability and biofilm structure, metabolic activity, total biomass production, and the composition of the extracellular matrix composition were analyzed. Regardless of the presence of F, 0.25 and 0.5% CaGP promoted a higher biomass production and metabolic activity increase than the controls (p < 0.05). F-free CaGP solutions reduced bacterial cell population significantly more than the 500 ppm F group or the negative control (p < 0.05). All the groups reduced the proteins, and 0.5% CaGP combined with F led to the highest reduction in the carbohydrate and nucleic acids content of the extracellular matrix (p < 0.05). It can be concluded that CaGP alone affected the number of bacterial cells and, when combined with F, reduced its production of biomass, metabolic activity, and the expression of the extracellular matrix components.

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

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

Extracellular Vesicles in Colorectal Cancer: From Tumor Growth and Metastasis to Biomarkers and Nanomedications.

Colorectal cancer (CRC) is a leading public health concern due to its incidence and high mortality rates, highlighting the requirement of an early diagnosis. Evaluation of circulating extracellular vesicles (EVs) might constitute a noninvasive and reliable approach for CRC detection and for patient follow-up because EVs display the molecular features of the cells they originate. EVs are released by almost all cell types and are mainly categorized as exosomes originating from exocytosis of intraluminal vesicles from multivesicular bodies, ectosomes resulting from outward budding of the plasma membrane and apoptotic bodies' ensuing cell shrinkage. These vesicles play a critical role in intercellular communications during physiological and pathological processes. They facilitate CRC progression and premetastatic niche formation, and they enable transfer of chemotherapy resistance to sensitive cells through the local or remote delivery of their lipid, nucleic acid and protein content. On another note, their stability in the bloodstream, their permeation in tissues and their sheltering of packaged material make engineered EVs suitable vectors for efficient delivery of tracers and therapeutic agents for tumor imaging or treatment. Here, we focus on the physiopathological role of EVs in CRCs, their value in the diagnosis and prognosis and ongoing investigations into therapeutic approaches.

Preparation and antimicrobial mechanism of Maillard reaction products derived from ε-polylysine and chitooligosaccharides

Chitooligosaccharides can be combined with amino acids or polypeptide to form Maillard reaction products (MRPs) with the antibacterial characteristics through Maillard reaction. This research aims to clarify the structure, antimicrobial effect and mechanism against Shewanella putrefaciens (S. putrefaciens) of ε-polylysine and chitooligosaccharides Maillard reaction products (LC-MRPs). The results of intrinsic fluorescence (IF) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction, proton nuclear magnetic resonance (1H NMR) spectra and scanning electron microscope (SEM) indicated Maillard reaction occurred between ε-polylysine and chitooligosaccharides. The observation of confocal laser scanning microscopy (CLSM), SEM and growth curves of S. putrefaciens evidenced that LC-MRPs have the strongest antibacterial effects. The leakage of alkaline phosphatase (AKP) and lactate dehydrogenase (LDH) implied that LC-MRPs sabotaged bacterial barrier (cell wall and cell membrane). The changes in content of nucleic acids, reactive oxygen species (ROS) level, lipid peroxidation content (LPO), succinate dehydrogenase (SDH) activity and adenosine triphosphate (ATP) content showed LC-MRPs will affect bacterial genetic gene transcription, material and energy metabolism. Therefore, the LC-MRPs were effective antibacterial agents to inhibit S. putrefaciens, which will help to preserve food with S. putrefaciens as the main spoilage bacteria.

Metagenomic Next-Generation Sequencing for Diagnosis of Pulmonary Infections

Despite pneumonia being a leading cause of morbidity and mortality worldwide, diagnostics remains a challenge, hindering rapid organism identification and subsequent effective treatments. Current microbiological methods include culture, serology, and limited molecular panels. While helpful, these methods are unable to address the full range of potential pathogens (e.g., fastidious or noncultivable organisms or uncommon organisms not included in current panels). Metagenomic next-generation sequencing (mNGS) is a molecular technique that analyzes and compares the nucleic acid content in a patient sample to a reference database of organisms that may include bacteria, viruses, fungi, and/or parasites, depending on the mNGS technology used. By bypassing the limitations of culture and targeted molecular assays, mNGS offers the potential to identify countless organisms directly from a patient specimen to aid in the diagnosis of an infectious process. Although promising, mNGS does have considerable limitations related to cost, interpretation, standardization, clinical relevance, turnaround time (TAT), and widespread availability. Thus, these factors should be considered prior to implementing mNGS for clinical use. Moreover, additional studies are required to fully understand the clinical and epidemiological impact of mNGS for the diagnosis of infectious diseases, including respiratory infections.

Microbial Community Abundance and Metabolism Close to the Ice-Water Interface of the Blomstrandbreen Glacier (Kongsfjorden, Svalbard): A Sampling Survey Using an Unmanned Autonomous Vehicle

Polar marine environments host a complex assemblage of cold-adapted auto- and heterotrophic microorganisms that affect water biogeochemistry and ecosystem functions. However, due to logistical difficulties, remote regions like those in close proximity to glaciers have received little attention, resulting in a paucity of microbiological data. To fill these gaps and obtain novel insights into microbial structure and function in Arctic regions, a survey of microbial communities in an area close to the Blomstrandbreen glacier in Kongsfjorden (Svalbard Archipelago; Arctic Ocean) was carried out during an early summer period. An Unmanned Autonomous Vehicle designed to safely obtain seawater samples from offshore-glacier transects (PROTEUS, Portable RObotic Technology for Unmanned Surveys) was equipped with an automatic remotely-controlled water multi-sampler so that it could sample just beneath the glacier, where access from the sea is difficult and dangerous. The samples were analysed by image analysis for the abundance of total prokaryotes, viable and respiring cells, their morphological traits and biomass; by flow cytometry for autotrophic and prokaryotic cells (with high and low nucleic acid contents) as well as virus-like particle counts; by BIOLOG ECOPLATES for potential community metabolism; and by fluorimetry for potential enzymatic activity rates on organic polymers. Contextually, the main physical and chemical (temperature, salinity, pH, dissolved oxygen and nutrients) parameters were detected. Altogether, besides the PROTEUS vehicle’s suitability for collecting samples from otherwise inaccessible sites, the multivariate analysis of the overall dataset allowed the identification of three main sub-regions differently affected by the haline gradient (close to the glacier) or terrigenous inputs coming from the coast. A complex microbiological scenario was depicted by different patterns of microbial abundance and metabolism among the transects, suggesting that ice melting and Atlantic water inflow differently supported microbial growth.

Reliable cell preparation protocol for Raman imaging to effectively differentiate normal leukocytes and leukemic blasts

Leukemias are a remarkably diverse group of malignancies originating from abnormal progenitor cells in the bone marrow. Leukemia subtypes are classified according to the cell type that has undergone neoplastic transformation using demanding and time-consuming methods. Alternative is Raman imaging that can be used both for living and fixed cells. However, considering the diversity of leukemic cell types and normal leukocytes, and the availability of different sample preparation protocols, the main objective of this work was to verify them for leukemia and normal blood cell samples for Raman imaging.The effect of glutaraldehyde (GA) fixation in a concentration gradient (0.1 %, 0.5 %, and 2.5 % GA) on the molecular structure of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral blood mononuclear cells (PBMCs) was verified. Changes in the secondary structure of proteins within cells were indicated as the main effect of fixation, as shown by an increase in band intensity at 1041 cm−1, characteristic for in-plane δ(CH) deformation in phenylalanine (Phe).Different sensitivity of mononuclear and leukemic cells to fixation was observed. While the 0.1 % concentration of GA was too low to preserve the cell structure for an extended period of time, a GA concentration of 0.5 % seemed optimal for both normal and malignant cells. Chemical changes in PBMCs samples stored for 11 days were also investigated, which manifested in numerous modifications in the secondary structure of proteins and the content of nucleic acids. The impact of cell preculturing for 72 h after unbanking was verified, and there was no significant effect on the molecular structure of cells fixed with 0.5 % GA. In summary, the developed protocol for the preparation of samples for Raman imaging allows for the effective differentiation of fixed normal leukocytes from malignant T lymphoblasts.

Decellularized cryopreserved human pericardium: a validation study towards tissue bank practice

Pericardial patches are currently used as reconstructive material in cardiac surgery for surgical treatment of cardiac septal defects. Autologous pericardial patches, either treated with glutaraldehyde or not, can be used as an alternative to synthetic materials or xenograft in congenital septal defects repair. The availability of an allogenic decellularized pericardium could reduce complication during and after surgery and could be a valid alternative. Decellularization of allogenic tissues aims at reducing the immunogenic reaction that might trigger inflammation and tissue calcification over time. The ideal graft for congenital heart disease repair should be biocompatible, mechanically resistant, non-immunogenic, and should have the ability to growth with the patients. The aim of the present study is the evaluation of the efficacy of a new decellularization protocol of homologous pericardium, even after cryopreservation. The technique has proven to be suitable as a tissue bank procedure and highly successful in the removal of cells and nucleic acids content, but also in the preservation of collagen and biomechanical properties of the human pericardium.

Treatment of nitrogen and phosphorus in wastewater by heterotrophic N- and P-starved microalgal cell.

Nitrogen (N) and phosphorus (P) are two major pollutants present in aquaculture wastewater, and their concentrations often do not meet discharge standards. In the present study, the N and P removal efficiency of nutrient-deficient cells (S group) was significantly higher than that of photoautotrophic cells (P group) and heterotrophic cells (H group). After incubation with wastewater, the N and P content of S group cells was significantly increased and reached a level similar to that of the P group and H group cells after 6 days of treatment. Additionally, in the S group cells, the content of total fatty acids (TFAs), which can be used to supply energy and organic carbon for N and P absorption, significantly decreased. In addition, the protein and nucleic acid contents of the S group cells also significantly increased, which revealed the biosynthetic flow of assimilated N and P. Comparative transcriptome analysis showed that compared with the P group and H group, the N metabolism, ribosome, RNA polymerase, and fatty acid degradation pathways were significantly upregulated in the S group cells, and the fatty acid biosynthesis pathway was significantly downregulated, which was in agreement with the biochemical results. In summary, our study showed that N- and P-starved heterotrophic cells are ideal for use in wastewater N and P removal processes. Keypoints • The N and P removal efficiencies of the S group were higher than P and H groups • Fatty acids were degraded to supply energy and carbon for N and P absorption • N metabolism and fatty acid degradation pathways were upregulated in the S group.

An innovative strategy to control Microcystis growth using tea polyphenols sustained-release particles: preparation, characterization, and inhibition mechanism.

Allelochemicals have been shown to inhibit cyanobacterial blooms for several years. In view of the disadvantages of "direct-added" mode, natural and pollution-free tea polyphenolic allelochemicals with good inhibitory effect on cyanobacteria were selected to prepare sustained-release particles by microcapsule technology. Results showed that the encapsulation efficiency of tea polyphenols sustained-release particles (TPSPs) was 50.6% and the particle size ranged from 700 to 970nm, which reached the nanoscale under optimum preparation condition. Physical and chemical properties of TPSPs were characterized to prove that tea polyphenols were well encapsulated and the particles had good thermal stability. The optimal dosage of TPSPs was determined to be 0.3g/L, at which the inhibition rate on Microcystis aeruginosa in logarithmic growth period could be maintained above 95%. Simultaneous decrease in algal density and chlorophyll-a content indicated that the photosynthesis of algal cells was affected leading to cell death. Significant changes of antioxidant enzyme activities suggested that Microcystis aeruginosa's antioxidant systems had been disrupted. Furthermore, TPSPs increased the concentration of O2- which led to lipid peroxidation of cell membrane and a subsequent increase in malondialdehyde (MDA) content. Meanwhile, the protein content, nucleic acid content, and electrical conductivity in culture medium rose significantly indicating the cell membrane was irreversibly damaged. This work can provide a basis for the utilization of environmentally friendly algal suppressants.

Production of Lactobacillus plantarum ghosts by conditional expression of a prophage-encoded holin.

Bacterial ghosts (BGs) are non-viable empty bacterial cell envelopes with intact cellular morphology and native surface structure. BGs made from pathogenic bacteria are used for biomedical and pharmaceutical applications. However, incomplete pathogenic cell inactivation during BG preparation raises safety concerns that could limit the intended use. Therefore, safer bacterial cell types are needed for BG production. Here, we produced BGs from the food-grade Gram-positive bacterium Lactobacillus plantarum TBRC 2-4 by conditional expression of a prophage-encoded holin (LpHo). LpHo expression was regulated using the pheromone-inducible pSIP system and LpHo was localized to the cell membrane. Upon LpHo induction, a significant growth retardation and a drastic decrease in cell viability were observed. LpHo-induced cells also showed membrane pores by scanning electron microscopy, membrane depolarization by flow cytometry, and release of nucleic acid contents in the cell culture supernatant, consistent with the role of LpHo as a pore-forming protein and L. plantarum ghost formation. The holin-induced L. plantarum BG platform could be developed as a safer alternative vehicle for the delivery of biomolecules.

Antimicrobial mechanism of semi-bionic extracts of three traditional medicinal plants-Rheum palmatum L., Scutellaria baicalensis Georgi, and Houttuynia cordata Thunb-That can be used as antibiotic alternatives.

The Chinese traditional medicinal plants Rheum palmatum L., Scutellaria baicalensis Georgi, and Houttuynia cordata Thunb in a ratio of 108:65:27 form a compound named Dahuang Qinyu San (DQS), which inhibits and kills Escherichia coli and Salmonella to a certain extent in fish and shrimp aquaculture environments. The active ingredients quercetin, emodin, baicalin, and aloe-emodin are obtained from the semi-biomimetic extract of DQS (SEDQS). However, the antibacterial mechanism of SEDQS against Salmonella is still unclear. This study used the microwell-plate method to determine the Minimum Inhibitory Concentration (MIC) of SEDQS against Salmonella enteritidis (S. enteritidis) isolated from geese. In addition, the effect of SEDQS on the growth curve, respiratory metabolic system, cell wall, soluble protein, and nucleic acid in bacterial liquid of S. enteritidis was detected by spectrophotometer and reagent kit. The effects of SEDQS on S. enteritidis DNA, binding gel blocking, virulence gene expression, and pathogenicity-related proteins were determined by gel electrophoresis, SDS-PAGE, and fluorescence quantitative PCR. The study found that a concentration of 1/4 MIC-2 MIC (2.27-18.2 mg/ml) SEDQS can significantly inhibit the normal growth of S. enteritidis, destroy the cell membrane structure of bacteria resulting in the leak of nucleic acid, protein, and other contents (P < 0.01). It also significantly inhibited the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH; P < 0.01) in a concentration-dependent manner. When the concentration of SEDQS was 1/2 MIC to 2 MIC (4.55-18.2 mg/ml), the expression levels of gyrB, fimA, filC, spvR, Hcp, and vgrG virulence genes (P < 0.01) all decreased by more than 31, 11, 18, 30, 34, and 21% respectively compared with the control group. SEDQS could significantly inhibit the expression of six virulence genes and play an important role in the pathogenicity of the S. enteritidis infected host. The SEDQS could exert antibacterial pharmacological effects by inhibiting the growth and metabolism of S. enteritidis and inhibiting the expression of major virulence factors. It has potential application value as an antibiotic alternative.

Molecular Mechanisms Underlying CRISPR/Cas-Based Assays for Nucleic Acid Detection

Applied to investigate specific sequences, nucleic acid detection assays can help identify novel bacterial and viral infections. Most up-to-date systems combine isothermal amplification with Cas-mediated detection. They surpass standard PCR methods in detection time and sensitivity, which is crucial for rapid diagnostics. The first part of this review covers the variety of isothermal amplification methods and describes their reaction mechanisms. Isothermal amplification enables fast multiplication of a target nucleic acid sequence without expensive laboratory equipment. However, researchers aim for more reliable results, which cannot be achieved solely by amplification because it is also a source of non-specific products. This motivated the development of Cas-based assays that use Cas9, Cas12, or Cas13 proteins to detect nucleic acids and their fragments in biological specimens with high specificity. Isothermal amplification yields a high enough concentration of target nucleic acids for the specific signal to be detected via Cas protein activity. The second part of the review discusses combinations of different Cas-mediated reactions and isothermal amplification methods and presents signal detection techniques adopted in each assay. Understanding the features of Cas-based assays could inform the choice of an optimal protocol to detect different nucleic acids.

Sensitive and selective DNA detecting electrochemical sensor via double cleaving CRISPR Cas12a and dual polymerization on hyperbranched rolling circle amplification

Electrochemical sensors are widely used for nucleic acid detection. However, they exhibit low sensitivity and specificity. To overcome these limitations, DNA amplification method is necessary. In this study, we introduced CRISPR (Clustered regularly interspaced short palindromic repeats) Cas12a-dependent hyperbranched rolling circle amplification (HRCA) into an electrochemical sensor platform. By resolving the existing false-positive issue of HRCA, CRISPR Cas12a determines the real positive amplification that able to enhance its sensitivity for extremely low concentrations of nucleic acids and specificity for single-point mutations. In detail, CRISPR Cas12a, which activates the nucleic acid amplification reaction, was used for both trans and cis cleavage for the first time. Finally, selectively amplified DNA was detected using a screen-printed electrode. Using the change in surface coverage by DNA, the electrochemical sensor detected a decrease in the redox signal. In summary, combining a novel DNA amplification method and electrochemical sensor platform, our proposed method compensates for the shortcomings of existing RCA and hyperbranched RCA, secures a high sensitivity of 10 aM, and overcomes false-positivity problems. Moreover, such creative applications of CRISPR Cas12a may lead to the expansion of its applications to other nucleic acid amplification methods.

Effect of Crude Oil Pollution on the Nucleic Acid Concentration of Portulaca Oleraceae

Aim of this study is to investigate the effect of crude oil pollution on the genome size, and nucleic acid concentration of Portulaca oleracea. Varying amounts of crude oil (0ml, 200ml, 400ml, 800ml and 1000ml) were used to pollute 10kg bags of loam soil, onto which Portulaca oleracea were transplanted, and the study lasted for 4 weeks. Data were collected for nucleic acid concentration. The application of crude oil on the soil consistently decreased the mean concentration of nucleic acid from 92.83 ng/µl, for the control treatment, to the lowest mean concentration of 9.77 ng/µl, for the 1000ml crude oil treatment. This shows an 89.41% decrement in concentration of nucleic acid. This is evident in decreasing staining with increase in crude oil treatment of the 10Kb bands observed in the pictogram of the gel electrophoresis. Crude oil treatment of P. oleracea does not affect the genome size of the plant, but it has an effect on the nucleic acid concentration of the plant, as is evident in the low staining effect of the gel electrophoresis bands as the crude oil treatment increases. Considering the resilience of P. oleracea and its ability to adapt to the high amounts of crude oil in the soil, it is advised that it be employed in the possible phyto-extraction of crude oil and/or phyto-remediation in crude oil polluted soils.

Dielectrophoresis-Assisted Self-Digitization Chip for High-Efficiency Single-Cell Analysis.

Single-cell analysis of cell phenotypic information such as surface protein expression and nucleic acid content is essential for understanding heterogeneity within cell populations. Here the design and use of a dielectrophoresis-assisted self-digitization (SD) microfluidics chip is described; it captures single cells in isolated microchambers with high efficiency for single-cell analysis. The self-digitization chip spontaneously partitions aqueous solution into microchambers through a combination of fluidic forces, interfacial tension, and channel geometry. Single cells are guided to and trapped at the entrances of microchambers by dielectrophoresis (DEP) due to local electric field maxima created by an externally applied AC voltage. Excess cells are flushed away, and trapped cells are released into the chambers and prepared for in situ analysis by turning off the external voltage, by running reaction buffer through the chip, and by sealing the chambers with a flow of an immiscible oil phase through the surrounding channels. The use of this device in single-cell analysis is demonstrated by performing single-cell nucleic acid quantitation based on loop-mediated isothermal amplification (LAMP). This platform provides a powerful new tool for single-cell research pertaining to drug discovery. For example, the single-cell genotyping of cancer-related mutant gene observed from the digital chip could be useful biomarker for targeted therapy.

High-Quality RNA Extraction and Evaluation of Reference Genes for qPCR Assay of &lt;i&gt;Pinus sylvestris&lt;/i&gt; L. Trunk Tissues

Scots pine (Pinus sylvestris L.) is a species of tree with heartwood (HW), which is forming during aging of sapwood (SW). Due to clear-cut border between SW and HW P. sylvestris should be used as a model woody plant for studying patterns of HW formation. Currently, molecular genetic methods are used to study the processes of trunk tissues formation in woody plants often. A feature of trunk tissues of coniferous trees is a high content of secondary metabolites, a low content of nucleic acids, and potential partial degradation of RNA. In this work we discuss the choice of most successful method for extraction a high-quality RNA for real-time PCR (RT-PCR) in P. sylvestris trunk tissues along the radial vector “conductive phloem/cambial zone – differentiating xylem – exterior part of SW (1–2 annual rings) – interior part of SW (1–2 annual rings afore transition zone (TZ)) – TZ (2 annual rings afore HW)” for reproducible RT-PCR data. The expression stability of six potential reference genes (Actin1, α-Tubulin, β-Tubulin, Ef1a, GAPDH, UBQ) was assessed in all describe tissues. Differences in expression levels of target genes are shown by data normalization using reference genes with different stability of expression.

State of Cyanobacteria &lt;i&gt;Arthrospira platensis&lt;/i&gt; and of Associated Microflora during Long-Term Storage in the State of Anhydrobiosis

Abstract—The biochemical composition of Arthrospira (Spirulina) platensis (Nordstedt) Gomont after long-term storage in the state of anhydrobiosis (4 years, 17 years) was determined by the standard methods. It was shown that protein content (55.3–61.2%) and total carbohydrates content (13.0–15.6%) in cyanobacterial cells were in agreement with the data known from the literature and with our results obtained at the onset of storage. The biomass had low content of free nucleotides (1.8–2.6%), lipids (1.3–11.0%), and especially pigments (0.5–1.3, 0.03–0.12, 1.4–2.0, 0.03–0.05% for chlorophyll a, carotenoids, C-phycocyanin, and allophycocyanin, respectively). The content of nucleic acids content was 3.1–24.0 and 0.11–0.16% for RNA and DNA, respectively. Microscopic examination of A. platensis (17 years of storage) showed high numbers of irreversibly damaged and dead cells (34.2 and 65.8%, respectively). To determine the quantitative and morphological parameters of associated microflora, a complex physicochemical treatment (methanol, ultrasound, and centrifugation) of the reactivated cyanobacterial suspension proved the most efficient. Three main groups were distinguished in the morphological structure of the microbiome (rod-shaped, rounded, and convoluted forms). The community was dominated by rod-shaped forms: large and small rods accounted for 60.5 and 14.4%, respectively. Mycelial forms (thin filaments), cocci, and convoluted forms were less common. On average, the volume of a bacterial cell was 0.27 ± 0.04 µm3. The contribution of bacteria to the biomass of A. platensis varied from 3.3 to 11.3% (8.3 ± 4.4% on average) of the dry weight of A. platensis. It has been suggested that the biochemical parameters and viability of cyanobacteria were affected by the accompanying microflora.

Isolation of Nuclei from Human Snap-frozen Liver Tissue for Single-nucleus RNA Sequencing.

Single-nucleus RNA sequencing (snRNA-seq) provides a powerful tool for studying cell type composition in heterogenous tissues. The liver is a vital organ composed of a diverse set of cell types; thus, single-cell technologies could greatly facilitate the deconvolution of liver tissue composition and various downstream omics analyses at the cell-type level. Applying single-cell technologies to fresh liver biopsies can, however, be very challenging, and snRNA-seq of snap-frozen liver biopsies requires some optimization given the high nucleic acid content of the solid liver tissue. Therefore, an optimized protocol for snRNA-seq specifically targeted for the use of frozen liver samples is needed to improve our understanding of human liver gene expression at the cell-type resolution. We present a protocol for performing nuclei isolation from snap-frozen liver tissues, as well as guidance on the application of snRNA-seq. We also provide guidance on optimizing the protocol to different tissue and sample types.