Nucleic Acid Content Research Articles

Refractive Index Determination of Individual Viruses and Small Extracellular Vesicles in Aqueous Media Using Nano-Flow Cytometry.

The refractive index (RI) is a fundamental physical property of materials. Although measurement of the RI of biological nanoparticles (BNPs) in aqueous media is of great importance to basic research and biomedical applications, it is hampered by their tiny size, large intrinsic heterogeneity, and weak scattering. Here, we report the development of a label-free technique that can determine the RI of individual viruses and small extracellular vesicles (sEVs) with high precision and an analysis rate up to 10 000 particles per minute. This was achieved via the combination of high-sensitivity light-scattering detection by nanoflow cytometry (nFCM) and the Mie theory calculation. With the measured RIs for T7 virions, T7 capsids, and sEVs, the concentrations of nucleic acid in viral particles and protein in the lumen of sEVs were estimated. Furthermore, building upon a simplified core-shell model, the RIs of sEVs ranging from 40 to 200 nm were obtained. By using these RIs, a statistically robust size distribution of sEVs was acquired in minutes with accuracy and resolution matched closely with those of cryo-TEM measurements. Our approach could become an important tool in the RI determination of single BNPs.

The Comparison between Tanzanian Indigenous (Ufipa Breed) and Commercial Broiler (Ross Chicken) Meat on the Physicochemical Characteristics, Collagen and Nucleic Acid Contents.

The objective of this study was to characterize the meat quality traits that affect the texture and savory taste of Ufipa indigenous chickens by comparing the proximate composition, physical characteristics, collagen, and nucleic acid contents with those of commercial broilers. It was found that Ufipa chicken breast and thigh meat had a higher protein content (p<0.05) than broiler chicken meat, whereas the fat content was lower (p<0.01). The moisture content of thigh meat was lower in Ufipa chicken meat than in broiler chicken meat (p<0.05). Regarding meat color, broiler chickens had considerably higher L* and b* than Ufipa chickens in both the breast and the thigh meat, except for a* (p<0.01). Regarding water holding capacity, Ufipa chicken breast exhibited higher drip loss but lower thawing and cooking losses than broiler chicken (p<0.01). In contrast, its thigh meat had a much lower drip and thawing losses but higher cooking losses (p<0.01). The shear force of Ufipa chickens’ breasts and thighs was higher than that of broiler chickens (p<0.05), while the amount of total collagen in the thigh meat was higher than that of broiler chickens (p<0.05). Additionally, the inosine-5’-monophosphate (IMP) of Ufipa chicken breast and thigh meat was higher than that of broiler meat (p<0.05). The principal component analysis of meat quality traits provides a correlation between the proximate and physical-chemical prosperties of both breeds with some contrast. In conclusion, the present study provides information on healthy food with good-tasting Ufipa indigenous chickens, which offer a promising market due to consumers’ preferences.

Coherent anti-Stokes Raman scattering cell imaging and segmentation with unsupervised data analysis.

Coherent Raman imaging has been extensively applied to live-cell imaging in the last 2 decades, allowing to probe the intracellular lipid, protein, nucleic acid, and water content with a high-acquisition rate and sensitivity. In this context, multiplex coherent anti-Stokes Raman scattering (MCARS) microspectroscopy using sub-nanosecond laser pulses is now recognized as a mature and straightforward technology for label-free bioimaging, offering the high spectral resolution of conventional Raman spectroscopy with reduced acquisition time. Here, we introduce the combination of the MCARS imaging technique with unsupervised data analysis based on multivariate curve resolution (MCR). The MCR process is implemented under the classical signal non-negativity constraint and, even more originally, under a new spatial constraint based on cell segmentation. We thus introduce a new methodology for hyperspectral cell imaging and segmentation, based on a simple, unsupervised workflow without any spectrum-to-spectrum phase retrieval computation. We first assess the robustness of our approach by considering cells of different types, namely, from the human HEK293 and murine C2C12 lines. To evaluate its applicability over a broader range, we then study HEK293 cells in different physiological states and experimental situations. Specifically, we compare an interphasic cell with a mitotic (prophase) one. We also present a comparison between a fixed cell and a living cell, in order to visualize the potential changes induced by the fixation protocol in cellular architecture. Next, with the aim of assessing more precisely the sensitivity of our approach, we study HEK293 living cells overexpressing tropomyosin-related kinase B (TrkB), a cancer-related membrane receptor, depending on the presence of its ligand, brain-derived neurotrophic factor (BDNF). Finally, the segmentation capability of the approach is evaluated in the case of a single cell and also by considering cell clusters of various sizes.

Effects of Thymoquinone (TQ) on the Molecular Structure and Total Antioxidant Capacity of Cerebellum Tissue of Healthy Rats

Objective: The neuroprotective effects of thymoquinone (TQ), the active compound of Nigella sativa, have been reported in accordance with the anti-inflammatory and antioxidant features of this drug. It has been suggested that the cerebellum plays a considerable role in neurodegenerative processes. In the current study, the possible effects of TQ on the structure and composition of cerebellum tissues and its total antioxidant capacity were studied dose-dependently. Materials and Methods: Fifteen adult Long Evans female rats were divided into groups as follows: G1: Control, G2: 10 mg/kg TQ treatment, G3: 20 mg/kg TQ treatment. TQ was injected into the rats intraperitoneally for two weeks. The control group only received corn oil used for the dissolving of TQ. Fourier-transform infrared spectroscopy (FTIR) studies and total protein, and antioxidant capacity measurements were carried out with cerebellum tissues which were removed following the decapitation of rats. Results and Conclusion: 10 mg/kg TQ treatment improved the saturated and unsaturated lipid and protein content in addition to decreasing nucleic acid content and lipid peroxidation and increasing the total antioxidant capacity of cerebellum tissues. However, 20 mg/kg TQ treatment did not have any significant effect.

Infrared spectrochemical findings on intermittent fasting-associated gross molecular modifications in rat myocardium

Cardiovascular diseases are among the primary life-threatening conditions affecting human society. Intermittent fasting is shown to be functional in the prevention of cardiovascular diseases, however, the information on fasting-associated modifications in myocardial biomolecules is limited. This study aimed to determine the impact of 18-h intermittent fasting administered for five weeks on 12 months-old rats using supervised linear discriminant analysis and support vector machine algorithms constructed on spectrochemical data obtained from myocardial tissues. These algorithms revealed gross biomolecular modifications, while quantitative analyses demonstrated higher amounts of saturated lipids (19%), triglycerides (11%), and lipids (56%), in addition to enhancement in membrane dynamics (18%). The concentrations of nucleic acids and glucose are increased by 52%, while the glycogen content is diminished by 61%. The protein carbonylation/oxidation is reduced by 38%, whereas a 35% increase in protein content was measured. Phosphorylated proteins have been calculated to be at higher concentrations in the 13–62% range. The study findings demonstrated significant molecular changes in the myocardium of rats subjected to intermittent fasting.

Rapid Nucleic Acid Extraction for Aquatic Animal DNA Virus Determination Using Chelex 100 Resin via Conventional PCR and Digital Droplet PCR Detection

Simple SummaryConvenient, fast, and high-quality nucleic acid extraction methods are urgently needed in molecular diagnostic testing for viral pathogens in aquaculture. We developed a viral DNA extraction method from diseased tissues and cells using the Chelex 100 resin solution workflow. The only extraction reagents required are the Chelex 100 resin and phosphate-buffered saline. The whole extraction process only takes about 15 min from the tissue homogenate to obtain the DNA. The concentration of extracted DNA is at least 100 ng/µL. This methodology has clear benefits in terms of cost and time saving compared to the commercial kit extraction for aquatic animal DNA virus determination by PCR in the laboratory. In addition, the simplified method using Chelex 100 resin with a pH value of 10–11 presented excellent results in PCR application and could be a standard for the DNA extraction for DNA virus testing in the future.Molecular diagnostic testing for viral pathogens is crucial in aquaculture. The efficient and convenient preparation of pathogenic microbial nucleic acids is the basis of molecular diagnosis. Here, we developed a simplified deoxyribonucleic acid (DNA) extraction method from aquatic animal DNA viruses using the Chelex 100 resin. The nucleic acid was extracted from infected tissues and cell culture for the detection of three common aquatic viral pathogens (CEV, CyHV-2, and GSIV). We compared the extraction effects of a current commercial kit extraction method and the Chelex 100 resin extraction method according to nucleic acid concentration, conventional polymerase chain reaction (PCR), and digital droplet PCR (ddPCR). The results indicated that both extraction procedures could obtain high-quality nucleotide samples. Extracting DNA using the Chelex 100 resin led to better detective efficiency for ddPCR molecular diagnostic testing. The whole process took less than 20 min, and only Chelex 100 resin solution was added to the tissues or cells without multiple tubes being transferred several times. The extracted DNA concentration and the detection sensitivity were high. These results indicated that the Chelex 100 resin solution has the advantages of speed, efficiency, and economy compared to the commercial kit. In addition, the higher pH value (10–11) of the Chelex 100 resin solution markedly improved the detection sensitivity compared to a lower pH value (9–10). In conclusion, the comparison of the Chelex 100 Resin and commercial viral DNA extraction kits revealed the good performance of the Chelex 100 resin solution at pH 10–11 in DNA extraction for PCR amplification from aquatic animal viral samples of tissues and cells in molecular diagnostic testing. It is both rapid and cost-effective.

Effect of Sodium Fluoride (NaF) on Nucleic Acid (DNA) Concentration in Fresh Water Fishes Cirrhina Mrigala and Labeo Rohita

Effect of Sodium Fluoride (NaF) on Nucleic Acid (DNA) Concentration in Fresh Water Fishes Cirrhina Mrigala and Labeo Rohita

An Update on Non-invasive Approaches for Genetic Testing of the Preimplantation Embryo.

Preimplantation Genetic Testing (PGT) aims to reduce the chance of an affected pregnancy or improve success in an assisted reproduction cycle. Since the first established pregnancies in 1990, methodological approaches have greatly evolved, combined with significant advances in the embryological laboratory. The application of preimplantation testing has expanded, while the accuracy and reliability of monogenic and chromosomal analysis have improved. The procedure traditionally employs an invasive approach to assess the nucleic acid content of embryos. All biopsy procedures require high technical skill, and costly equipment, and may impact both the accuracy of genetic testing and embryo viability. To overcome these limitations, many researchers have focused on the analysis of cell-free DNA (cfDNA) at the preimplantation stage, sampled either from the blastocoel or embryo culture media, to determine the genetic status of the embryo non-invasively. Studies have assessed the origin of cfDNA and its application in non-invasive testing for monogenic disease and chromosomal aneuploidies. Herein, we discuss the state-of-the-art for modern non-invasive embryonic genetic material assessment in the context of PGT. The results are difficult to integrate due to numerous methodological differences between the studies, while further work is required to assess the suitability of cfDNA analysis for clinical application.

Clinical performance testing of the automated haematology analyzer XN-31 prototype using whole blood samples from patients with imported malaria in Japan

BackgroundThe automated haematology analyzer XN-31 prototype (XN-31p) is a new flow cytometry-based device developed to measure the number and the ratio of malaria-infected red blood cells (MI-RBC) with a complete blood count (CBC). The XN-31p can provide results in about one minute and also can simultaneously provide information on the malaria parasite (Plasmodium) species. In this study, clinical testing of the XN-31p was performed using blood samples from patients with imported malaria in Japan.MethodsBlood samples were collected from 80 patients who visited the hospital of the National Center for Global Health and Medicine, Tokyo, Japan, for malaria diagnosis from January 2017 to January 2019. The test results by the XN-31p were compared with those by other standard methods, such as microscopic observation, rapid diagnostic tests and the nested PCR.ResultsThirty-three patients were diagnosed by the nested PCR as being malaria positive (28 Plasmodium falciparum, 2 Plasmodium vivax, 1 Plasmodium knowlesi, 1 mixed infection of P. falciparum and Plasmodium malariae, and 1 mixed infection of P. falciparum and Plasmodium ovale), and the other 47 were negative. The XN-31p detected 32 patients as “MI-RBC positive”, which almost matched the results by the nested PCR and, in fact, completely matched with the microscopic observations. The ratio of RBCs infected with malaria parasites as determined by the XN-31p showed a high correlation coefficient of more than 0.99 with the parasitaemia counted under microscopic observation. The XN-31p can analyse the size and nucleic acid contents of each cell, and the results were visualized on a two-dimensional cytogram termed the “M scattergram”. Information on species and developmental stages of the parasites could also be predicted from the patterns visualized in the M scattergrams. The XN-31p showed a positive coincidence rate of 0.848 with the nested PCR in discriminating P. falciparum from the other species.ConclusionsThe XN-31p could rapidly provide instructive information on the ratio of MI-RBC and the infecting Plasmodium species. It was regarded to be of great help for the clinical diagnosis of malaria.

Manually-operated, slider cassette for multiplexed molecular detection at the point of care

Effective control of epidemics, individualized medicine, and new drugs with virologic response-dependent dose and timing require, among other things, simple, inexpensive, multiplexed molecular detection platforms suitable for point of care and home use. Herein, we describe our progress towards developing such a platform that includes sample lysis, nucleic acid isolation, concentration, purification, and amplification. Our diagnostic device comprises a sliding component that houses the nucleic acid isolation membrane and a housing containing three amplification reaction chambers with dry stored reagents, blisters with buffers and wash solutions, and absorption pads to facilitate capillarity pull and waste storage. After sample introduction, the user slides the slider within the housing from one station to another to carry out various unit operations. The slider motion induces blisters to discharge their contents, effectuating washes, and eventual elution of captured nucleic acids into reaction chambers. The slider cassette mates with a processor that incubates isothermal amplification but can also be made to operate instrumentation-free. We demonstrate our cassette’s utility for the co-detection of the human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV). These three blood-borne pathogens co-infect many people worldwide with severe personal and public health consequences.

Engineered Riboswitch Nanocarriers as a Possible Disease-Modifying Treatment for Metabolic Disorders.

Both DNA- and RNA-based nanotechnologies are remarkably useful for the engineering of molecular devices in vitro and are applied in a vast collection of applications. Yet, the ability to integrate functional nucleic acid nanostructures in applications outside of the lab requires overcoming their inherent degradation sensitivity and subsequent loss of function. Viruses are minimalistic yet sophisticated supramolecular assemblies, capable of shielding their nucleic acid content in nuclease-rich environments. Inspired by this natural ability, we engineered RNA-virus-like particles (VLPs) nanocarriers (NCs). We showed that the VLPs can function as an exceptional protective shell against nuclease-mediated degradation. We then harnessed biological recognition elements and demonstrated how engineered riboswitch NCs can act as a possible disease-modifying treatment for genetic metabolic disorders. The functional riboswitch is capable of selectively and specifically binding metabolites and preventing their self-assembly process and its downstream effects. When applying the riboswitch nanocarriers to an in vivo yeast model of adenine accumulation and self-assembly, significant inhibition of the sensitivity to adenine feeding was observed. In addition, using an amyloid-specific dye, we proved the riboswitch nanocarriers' ability to reduce the level of intracellular amyloid-like metabolite cytotoxic structures. The potential of this RNA therapeutic technology does not apply only to metabolic disorders, as it can be easily fine-tuned to be applied to other conditions and diseases.

The Effect of Probiotic Lactobacillus acidophilus and Ethanolic Propolis Compound toward Nucleic Acid Deposition in the Extracellular Polymeric Substance of Root Canal Bacteria.

This study aimed to evaluate the effects of Apis trigona ethanolic propolis and probiotic bacteria Lactobacillus acidophilus on the nucleic acid concentration in the extracellular polymeric substances (EPS) derived from biofilm of root canal bacteria. Clinical bacteria of the root canal were cultured with ethanolic extract of propolis (EEP; 10 or 0.1%) and L. acidophilus. After the formation of biofilm was observed in the monolayer bacterial culture under several conditions, the enzymatic treatment and nucleic acid quantification were sequentially performed. Independent t-test and Mann-Whitney were performed following data normality to analyze the significant differences of the treatment effect on the nucleic acid concentration in EPS from the isolated biofilm. The results showed that the nucleic acid concentration in EPS biofilm were not increased by coculture with L. acidophilus as probiotics. However, the treatment with 10% EEP could significantly increase nucleic acid concentration. This study suggested that the biosurfactants from probiotic bacteria L. acidophilus might be a promising candidate for endodontic treatment, arguably better than EEP in inhibiting biofilm maturation and complexity.

Two Novel Polysaccharides From Clitocybe squamulosa: Their Isolation, Structures, and Bioactivities.

The crude polysaccharides from the fruiting bodies of Clitocybe squamulosa (CSFP) were isolated by hot-water extraction. Two novel polysaccharides, CSFP1-β and CSFP2-α, were further purified by DEAE-52 anion exchange and Sephacryl S-400 gel filtration chromatography, and the purities reached 98.44 and 97.83%, respectively. The structural characteristics and bioactivities of CSFP, CSFP1-β, and CSFP2-α were identified by the combination of chemical and instrumental analysis. Results showed that CSFP was formed by the aggregation of honeycomb spherical materials; CSFP1-β and CSFP2-α were interwoven by reticular and fibrous structures, respectively. Purified components of both CSFP1-β and CSFP2-α showed typical infrared absorption peaks of polysaccharides, and contents of nucleic acid and protein decreased significantly. Simultaneously, CSFP with a molecular weight (Mw) of 1.948 × 104 Da were composed mainly of glucose, mannose, galactose, and rhamnose. CSFP1-β was composed mainly of glucose, galactose, and mannose, while CSFP2-α was composed of glucose, and both their Mw distributions were uneven. Compared with CSFP, the antioxidant activities of CSFP1-β and CSFP2-α were significantly improved (p < 0.05), and they both showed good abilities to bind free cholesterol and bile acid salts in vitro. The binding abilities of the two compounds were found to be 68.62 and 64.43%, and 46.66 and 45.05 mg/g, respectively. CSFP, CSFP1-β, and CSFP2-α had good bacteriostatic effects with a linear increasing relationship to increasing concentration. In addition, CSFP promoted the growth of RAW264.7 cells and has potential immunomodulatory, anti-inflammatory, and anti-tumor activities.

Full integration of nucleic acid extraction and detection into a centrifugal microfluidic chip employing chitosan-modified microspheres

Microfluidic devices especially centrifugal ones have attracted great attention in the nucleic acid testing field, due to their automation, high efficiency, and simple operation. In which, nucleic acid extraction is the basic step, laying a foundation for the downstream amplification and detection procedures. Therefore, the integration of nucleic acid extraction on the chip is expected to achieve cost-efficiency, high-speed automation, diagnostic accuracy, and reaction robustness with the respect to real-time detection. In this work, we employ chitosan-modified magnetic microspheres for pH-induced nucleic acid extraction and integrate this approach into a centrifugal microfluidic chip. The microfluidic system comprises cell lysis, nucleic acids capture and release, isothermal amplification, and real-time fluorescence detection, which is manipulated by centrifugal force and magnetic control. The system exhibits comparable extraction and detection performance in respect of acceptable nucleic acid concentration and purity, high detection specificity and stability, as well as fast detection duration. These efforts to improve the integrated microfluidic detection chip could benefit the portable, efficient and simple nucleic acid diagnosis, especially under the resource-limited circumstance.

Untargeted metabolomics used to describe the chemical composition and antimicrobial effects of the essential oil from the leaves of Guatteria citriodora Ducke

Plant oils are sources of metabolites that have enormous potential for industrial applications. Herein, the chemical profile and in vitro antimicrobial activity of the essential oil (EO) from the leaves of Guatteria citriodora Ducke (Annonaceae) have been investigated for the first time. The composition of the hydrodistilled EO was analyzed using gas chromatography-mass spectrometry (GC-MS), which permitted the identification of oxygenated monoterpenes as the most highly representative class, and included citronellal (40.99%) and citronellol (14.6%) as the main compounds. The antimicrobial activity of G. citriodora EO (GcEO) was evaluated against pathogenic bacteria and phytopathogenic fungi. The experimental design was completely randomized (CRD), and used doses for each microorganism. Gram-positive strains were the most sensitive with a minimum inhibitory concentration (MIC) of 5.0 μL mL−1, while Gram-negative strains were 10.0 μL mL−1. The most potent antifungal activity was against Alternaria alternata (MIC of 1.25 μL mL−1). In addition, it fully inhibited A. alternata conidia germination at the minimum inhibitory concentration. The nucleic acid and soluble protein contents were significantly released from the conidia of A. alternata after treatment with GcEO. Using SEM (scanning electron microscopy), morphological alterations were observed in the conidia, which indicates that a lesion in the cytoplasmic membrane is one of its mechanisms of action. Overall, these results indicate that GcEO is an antimicrobial agent with potential applications in the agriculture, food, and pharmaceutical industries.

Abstract 1943: Elucidating early tumor microenvironmental changes due to immunotherapy with label-free Raman spectroscopy and machine learning

Abstract Clinical benefit is observed in only a limited fraction of patients that undergo immunotherapy for a variety of cancer types. Therefore, the determination of robust biomarkers for accurate prediction of response to immunotherapeutic agents remains one of the grand challenges of 21st century. Our primary goal was to investigate if phenotypic differences due to treatment with ICI can be recognized with minimally perturbative molecular tools and guide personalization of immunotherapy. In this work, we provide first-in-class evidence that Raman spectroscopy (an optical method) and machine learning allows sensitive detection of early changes in biomolecular composition of tumors in response to immunotherapy with immune checkpoint inhibitors (ICI). By studying the widely investigated CT26 murine model of colorectal cancer treated with anti-CTLA-4 (n = 8) and anti-PD-L1 (n = 7) ICIs and controls (n = 10), we revealed new biological insights into the nature and degree of microenvironmental modifications induced by exposure to clinically relevant doses of ICI. Multivariate curve resolution-alternating least squares (MCR-ALS) decomposition of Raman spectral datasets revealed early changes in lipid, nucleic acid, and collagen content of the tumors following therapy. We trained supervised classification models - support vector machines and random forests on the spectral datasets and showed that the models provided excellent prediction accuracies for response to both ICIs and delineated spectral markers specific to each therapy, consistent with their differential mechanisms of action. On the basis of these findings, we sought to determine if the tumor microenvironment changes delineated by Raman spectroscopy correspond to proteomic alterations via quantitative mass spectrometry. Of the more than 6600 proteins identified, about 136 proteins were found to be significantly different in treated tumors relative to controls (p &amp;lt; 0.05 and log2 fold change of &amp;gt; 2). A subset of these differentially expressed proteins is known to regulate lipid metabolism and extracellular matrix composition, while others are known to control transcriptome dynamics or are associated with response to ICI therapy, thereby corroborating our Raman spectroscopic measurements. Our observation of biomolecular changes in the TME should catalyze detailed investigations for translating such markers and label-free Raman spectroscopy for clinical monitoring of immunotherapy response in cancer patients. Citation Format: Santosh K. Paidi, Joel R. Troncoso, Narasimhan Rajaram, Ishan Barman. Elucidating early tumor microenvironmental changes due to immunotherapy with label-free Raman spectroscopy and machine learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1943.

Characteristics, Biodiversity, and Cultivation Strategy of Low Nucleic Acid Content Bacteria.

Low nucleic acid content (LNA) bacteria are ubiquitous and estimated to constitute 20%–90% of the total bacterial community in marine and freshwater environment. LNA bacteria with unique physiological characteristics, including small cell size and small genomes, can pass through 0.45-μm filtration. The researchers came up with different terminologies for low nucleic acid content bacteria based on different research backgrounds, such as: filterable bacteria, oligotrophic bacteria, and low-DNA bacteria. LNA bacteria have an extremely high level of genetic diversity and play an important role in material circulation in oligotrophic environment. However, the majority of LNA bacteria in the environment remain uncultivated. Thus, an important challenge now is to isolate more LNA bacteria from oligotrophic environments and gain insights into their unique metabolic mechanisms and ecological functions. Here, we reviewed LNA bacteria in aquatic environments, focusing on their characteristics, community structure and diversity, functions, and cultivation strategies. Exciting future prospects for LNA bacteria are also discussed.

Investigation of the effect of sustainable magnetic treatment on the microbiological communities in drinking water

The drinking water scarcity is posing a threat to mankind, hence better water quality management methods are required. Magnetic water treatment, which has been reported to improve aesthetic water quality and reduce scaling problems, can be an important addition to the traditional disinfectant dependent treatment. Despite the extensive market application opportunities, the effect of magnetic fields on (microbial) drinking water communities and subsequently the biostability is still largely unexplored, although the first patent was registered already 1945.Here flow cytometry was applied to assess the effect of weak magnetic fields (≤10 G) with strong gradients (≈800 G/m) on drinking water microbial communities. Drinking water was collected from the tap and placed inside the magnetic field (treated) and 5 m away from the magnet to avoid any background interferences (control) using both a static set-up and a shaking set-up. Samples were collected during a seven-day period for flow cytometry examination. Additionally, the effects of magnetic fields on the growth of Pseudomonas aeruginosa in autoclaved tap water were examined. Based on the fluorescent intensity of the stained nucleic acid content, the microbial cells were grouped into low nucleic acid content (LNA) and high nucleic acid content (HNA). Our results show that the LNA was dominant under nutrient limited condition while the HNA dominates when nutrient is more available. Such behavior of LNA and HNA matches well with the long discussed r/K selection model where r-strategists adapted to eutrophic conditions and K-strategists adapted to oligotrophic conditions. The applied magnetic fields selectively promote the growth of LNA under nutrient rich environment, which indicates a beneficial effect on biostability enhancement. Inhibition on an HNA representative Pseudomonas aeruginosa has also been observed. Based on our laboratory observations, we conclude that magnetic field treatment can be a sustainable method for microbial community management with great potential.

Field Evaluation of a Duplex RT-RAA Assay for Rapid Detection of SARS-CoV-2 - Hebei Province, China, January 2021.

IntroductionRecently, a local cluster epidemic has occurred in Shijiazhuang City, Hebei Province. Failure to promptly identify patients with fever in rural areas was the major reason for this epidemic.MethodsWe presented the field evaluation of a new real-time reverse transcription recombinase-aided amplification (RT-RAA) kit incorporating an endogenous internal control in a single-tube format, completed at the Hebei CDC from January 17, 2021 to January 27, 2021.ResultsWe evaluated the diagnostic performance of RT-RAA assay using automatic extracted RNA of 808 clinical samples. Compared with reverse transcriptase real-time quantitative PCR (qRT-PCR), RT-RAA kit achieved 92.41% sensitivity, 98.78% specificity and a 96.29% coincidence rate, demonstrating an excellent agreement between the RT-RAA assay and qRT-PCR assay. Furthermore, 58 samples were extracted using a manual extraction method within 5 minutes, but only samples with high nucleic acid concentration (cycle threshold value not higher than 32) could be stably detected.DiscussionThe RT-RAA is more suitable to meet the needs of rapid, sensitive, and accurate detection in community-level medical institutions.

Potential of Aqueous Humor as a Liquid Biopsy for Uveal Melanoma.

Tumor biopsy can identify prognostic biomarkers for metastatic uveal melanoma (UM), however aqueous humor (AH) liquid biopsy may serve as an adjunct. This study investigated whether the AH of UM eyes has sufficient circulating tumor DNA (ctDNA) to perform genetic analysis. This is a case series of 37 AH samples, taken before or after radiation, and one tumor wash sample, from 12 choroidal and 8 ciliary body (CB) melanoma eyes. AH was analyzed for nucleic acid concentrations. AH DNA and one tumor wash sample underwent shallow whole-genome sequencing followed by Illumina sequencing to detect somatic copy number alterations (SCNAs). Four post-radiation AH underwent targeted sequencing of BAP1 and GNAQ genes. Post-radiation AH had significantly higher DNA and miRNA concentrations than paired pre-radiation samples. Highly recurrent UM SCNAs were identified in 0/11 post-radiation choroidal and 6/8 post-radiation CB AH. SCNAs were highly concordant in a CB post-radiation AH with its matched tumor (r = 0.978). BAP1 or GNAQ variants were detected in 3/4 post-radiation AH samples. AH is a source of ctDNA in UM eyes, particularly in post-radiation CB eyes. For the first time, UM SCNAs and mutations were identified in AH-derived ctDNA. Suggesting that AH can serve as a liquid biopsy for UM.