High Nucleic Acid Research Articles

Warming Disadvantages Phytoplankton and Benefits Bacteria During a Spring Bloom in the Mediterranean Thau Lagoon

To assess the response of a natural plankton community to the future scenario of a warming of +3°C predicted for coastal Mediterranean regions, an in situ mesocosm experiment was carried out over 19 days in a Mediterranean coastal lagoon in 2018. During this experiment, a phytoplankton bloom occurred and the abundances of several cytometric groups of phytoplankton (cyanobacteria, picophytoplankton and nanophytoplankton) and bacteria (low- and high-nucleic acid bacteria), as well as their growth and grazing mortality rates and their carbon biomass, were studied over the course of the experiment. Experimental warming led to a significant reduction of 47% of the phytoplankton biomass in average, based on Chl-a concentrations. This reduction was also observed for nanophytoplankton abundances during the whole experiment and for eukaryotic picophytoplankton only during the bloom period. This result coincided with a significant decrease in orthophosphate concentrations under warming during the prebloom and bloom periods simultaneous to an increase in bacterial abundances. At the same time, the higher growth rates of bacteria and the lower ones of phytoplankton observed at the beginning of the bloom could suggest that competition between phytoplankton and bacteria for orthophosphate might have contributed to the phytoplankton biomass decrease under warming. In addition, higher grazing mortality rates of phytoplankton groups under warming, notably nanophytoplankton at the end of the bloom and in postbloom (by 59 to 626%), as well as eukaryotic picophytoplankton over the course of the experiment (by 58 to 255%) could also have contributed to the lower phytoplankton biomass under warming. Based on these results, estimations of average phytoplankton carbon biomass production and transfer showed reductions of 42 and 45%, respectively, under warming, whereas those of bacteria were enhanced by 13 and 8%, respectively. These results indicated that warming induced a shift at the base of the microbial food web, going toward a more bacteria-based system. This suggested that under future warming scenarios, the microbial food web could become less productive and could negatively affect the functioning of the whole food web in coastal waters.

Purification of a Hydrophobic Elastin-Like Protein Toward Scale-Suitable Production of Biomaterials.

Elastin-like proteins (ELPs) are polypeptides with potential applications as renewable bio-based high-performance polymers, which undergo a stimulus-responsive reversible phase transition. The ELP investigated in this manuscript—ELP[V2Y-45]—promises fascinating mechanical properties in biomaterial applications. Purification process scalability and purification performance are important factors for the evaluation of potential industrial-scale production of ELPs. Salt-induced precipitation, inverse transition cycling (ITC), and immobilized metal ion affinity chromatography (IMAC) were assessed as purification protocols for a polyhistidine-tagged hydrophobic ELP showing low-temperature transition behavior. IMAC achieved a purity of 86% and the lowest nucleic acid contamination of all processes. Metal ion leakage did not propagate chemical modifications and could be successfully removed through size-exclusion chromatography. The simplest approach using a high-salt precipitation resulted in a 60% higher target molecule yield compared to both other approaches, with the drawback of a lower purity of 60% and higher nucleic acid contamination. An additional ITC purification led to the highest purity of 88% and high nucleic acid removal. However, expensive temperature-dependent centrifugation steps are required and aggregation effects even at low temperatures have to be considered for the investigated ELP. Therefore, ITC and IMAC are promising downstream processes for biomedical applications with scale-dependent economical costs to be considered, while salt-induced precipitation may be a fast and simple alternative for large-scale bio-based polymer production.

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.

Coupling Imaging and Omics in Plankton Surveys: State-of-the-Art, Challenges, and Future Directions

A major challenge in characterizing plankton communities is the collection, identification and quantification of samples in a time-efficient way. The classical manual microscopy counts are gradually being replaced by high throughput imaging and nucleic acid sequencing. DNA sequencing allows deep taxonomic resolution (including cryptic species) as well as high detection power (detecting rare species), while RNA provides insights on function and potential activity. However, these methods are affected by database limitations, PCR bias, and copy number variability across taxa. Recent developments in high-throughput imaging applied in situ or on collected samples (high-throughput microscopy, Underwater Vision Profiler, FlowCam, ZooScan, etc) has enabled a rapid enumeration of morphologically-distinguished plankton populations, estimates of biovolume/biomass, and provides additional valuable phenotypic information. Although machine learning classifiers generate encouraging results to classify marine plankton images in a time efficient way, there is still a need for large training datasets of manually annotated images. Here we provide workflow examples that couple nucleic acid sequencing with high-throughput imaging for a more complete and robust analysis of microbial communities. We also describe the publicly available and collaborative web application EcoTaxa, which offers tools for the rapid validation of plankton by specialists with the help of automatic recognition algorithms. Finally, we describe how the field is moving with citizen science programs, unmanned autonomous platforms with in situ sensors, and sequencing and digitalization of historical plankton samples.

High sensitivity and fast response optical fiber nucleic acid sensor

A label-free optical fiber Mach-Zehnder interferometer (MZI) based biosensor was proposed for the specificity detection of single-stranded nucleic acid (NA). The MZI was made of a fiber sandwich structure by a core-offset fusion splicing means. The piranha solution, APTES (3-Aminopropyltriethoxylsilan) and GA (Glutaraldehyde) were used for the chemical modification of the optical fiber surface, and then the single-stranded probe NA with the known sequence was fixed on the optical fiber surface by covalent fixation method. The specificity of complementary target NA could be detected through the changing of the spectra of the MZI. Thanks to the high sensitivity of the MZI and the high fixation efficiency of the covalent fixation method, the optical fiber MZI based NA sensor showed a fast response of 30 s and a high sensitivity of 0.175 dB/nM with a minimum detection concentration of 1 nM.

Effects of various materials used to promote the direct interspecies electron transfer on anaerobic digestion of low-concentration swine manure

The activation of direct interspecies electron transfer (DIET) by the supplementation of conductive materials is one of the effective and available methods to enhance anaerobic digestion (AD). Microorganisms that colonize the surface of these materials form biofilms, the study of which could provide new insights into the character of the DIET process and its effect on AD. The present study focused on AD performance, microbial community, as well as morphological and topological features of biofilms on various materials used to promote DIET during AD of low-concentration swine manure. The best AD characteristics were observed in stainless steel mesh (SM)/digested cow manure (CM) and polyester felt (PF)/digested sewage sludge (SS) combinations used as material/inoculum, respectively. Thus, potential methane yields in CM-SM and SS-PF were up to 26.4% and 26.2% higher compared to the corresponding controls. Microbial analysis of biofilms revealed the dominance of putatively syntrophic bacteria of the MBA03 group of the Limnochordia class in CM inoculated reactors, and syntrophic proteolytic bacteria of the genus Coprothermobacter and acetogenic Clostridium sensu stricto 1, known for their ability to carry out DIET, in SS inoculated reactors. Biofilms on non-conductive materials contained pili-like structures, which were observed only in SS inoculated reactors. Polyester felt tended to biofoul better than carbon felt, resulting in up to 2.8, 3.2 and 1.8 higher nucleic acid, extracellular polymeric substances, and total biomass content, respectively, depending on the inoculum. These results provide new insights into the different types of DIET that can occur in low-loaded AD systems with attached growth.

Pulsed export of carbon in the north-western Mediterranean Sea

The short term (hourly scale) variability of heterotrophic prokaryote (HP) vertical distribution and respiratory activity, was investigated in the north-western (NW) Mediterranean Sea. HP vertical distribution was determined on board by flow cytometry analysis of seawater samples collected by series of CTD casts. Cell counts and viability were determined for all samples. HP respiratory rates were determined later in the laboratory from filtered seawater samples (23 dm3) from 300–1 150-m depth. The average cell viability was 94.8%±2.2% (n=240). There was no accumulation of dead cells, due to quick decay of damaged cells. In the epipelagic layer, three HP groups were distinguished, two (HNA1, HNA2) whose cells exhibited a high nucleic acid content and one (LNA) with low nucleic acid content cells. HNA2 was most populated at 50 m but not detected at 90 m and below, presumably aerobic anoxygenic photoheterotrophic bacteria (AAPs). The variability in HP abundance was mainly confined in the upper 80 m. A few secondary peaks of HP abundance were observed (80–150 m) in connection with abundance troughs in the surface layer. HP cells were continuously present in a wide layer around 500 m (mean 191×103 cells/cm3). Below this layer, HP abundance randomly exhibited peaks, coupled to respiratory rate peaks. The HP abundance and variability in the water column was suppressed during a strong wind event. The observed sporadic variability was tentatively interpreted through a pulsed carbon-export mechanism induced by the microorganism production of dissolved polysaccharides, followed by flocculation and rapid sinking. This mechanism would thus contribute to (i) preventing organic matter accumulation in the epipelagic layer, (ii) seeding the water column with live HP cells, and (iii) supplying the aphotic water column with fresh and labile organic matter. This important vertical flux mechanism needs further observations and modelling.

P263 Autoantibodies in ankylosing spondylitis: a systematic literature review

Abstract Background/Aims Ankylosing spondylitis (AS) is a chronic inflammatory arthritis leading to long term disability. It is frequently diagnosed late when irreversible damage has already occurred. Unlike most autoimmune rheumatological diseases, there are no known autoantibodies associated with AS; this can confound the diagnostic challenge as diagnosis depends not only on symptoms but on finding evidence of active inflammation at the time of assessment or damage from previously active disease. Our aim was to determine the extent to which autoantibodies may be pathognomonic of AS. Methods A systematic literature review was conducted to identify all articles reporting on autoantibodies in AS. The protocol was preregistered on PROSPERO. Articles were screened and reviewed independently by two reviewers. Disagreements were solved by consensus. Data were extracted and the MINORS (Methodological Index for Non-Randomised Studies) tool was used to assess and compare the quality of the studies. Data were pooled in a narrative synthesis. Results 743 papers were identified after removal of duplicates. Following review of abstracts, 31 full text articles were assessed for eligibility and 18 were excluded, leaving 13 studies to be included in the final systematic review. 12 articles looked at 14 biomarkers; one article looked at 2 high density nucleic acid protein arrays expressing 3,498 proteins. Samples sizes were small and the papers were found to be of modest quality. AS patients showed a wide-ranging autoantibody response across studies. One study found that 60% of autoantibodies detected were found only in the AS cohort versus the rheumatoid arthritis cohort and heathy controls. Antibodies against HLA-B27, pANCA, CD74, OmpC, collagens, PPM1A, noggin, sclerostin, Klebsiella pneumoniae and Breg cells were found more commonly in AS patients than in controls (both healthy controls and those with other autoimmune diseases). Antibodies to human tTG and anti DFS70 were not found to be associated with AS and there was conflicting evidence regarding the association of antibodies to the cell-wall mannan of Saccharomyces cerevisiae (ASCA) in AS from different papers. Conclusion Although many autoantibodies have been found among persons with AS, there is currently no evidence that any are specifically and independently associated with the disease. We therefore conclude that as yet no autoantibodies can be considered to be pathognomonic for AS. However, some autoantibodies may be clinically significant and combined with other biomarkers, such as HLA-B27, may prove fruitful in reducing the delay to diagnosis. Further work should seek to explore other avenues, in particular autoantibodies in closely related diseases, such as inflammatory bowel disease, to try to determine autoantibodies that may aid in the earlier diagnosis of this chronic disease. Disclosure E.J. Davies: None. G.T. Jones: None. R. Sengupta: Honoraria; Received grants, honoraria, and expenses for attendance at advisory board meetings or conferences and for giving lectures from AbbVie, Biogen, Celgene, Lilly, MSD, Novartis, Roche and UCB. Grants/research support; Received grants, honoraria, and expenses for attendance at advisory board meetings or conferences and for giving lectures from AbbVie, Biogen, Celgene, Lilly, MSD, Novartis, Roche and UCB.

Isothermal Background-Free Nucleic Acid Quantification by a One-Pot Cas13a Assay Using Droplet Microfluidics.

High sensitivity and specificity nucleic acid detection has been achieved by the Cas13a collateral effect in combination with a separate recombinase polymerase amplification (RPA). However, these emerging methods cannot provide accurate quantification of nucleic acids because the two-step assay performance may be compromised if the RPA and Cas13a reactions are simply unified in a single step. In this work, we first addressed the challenges associated with enzymatic incompatibility and the macromolecular crowding effect in the one-pot assay development, making the consolidated RPA-Cas13a assay a facile and robust diagnostic tool. Next, we found that the one-pot reaction cannot precisely quantify the targets at low concentrations. Thus, by leveraging droplet microfluidics, we converted the one-pot assay to a digital quantification format, termed Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA). Due to the droplet compartmentation, MEDICA greatly accelerates the reaction and enables relative detection in 10 min and the end-point quantification in 25 min. Moreover, MEDICA facilitates the droplet binarization for counting because of background-free signals generated by trans-cleavage reporting of Cas13a. Our clinical validation highlights that CRISPR-based isothermal assays are promising for the next generation of nucleic acid quantification methods.

Sequence-Specific Recognition of Double-Stranded DNA by Peptide Nucleic Acid Forming Double-Duplex Invasion Complex

Peptide nucleic acid (PNA) is an analog of natural nucleic acids, where the sugar-phosphate backbone of DNA is replaced by an electrostatically neutral N-(2-aminoethyl)glycine backbone. This unique peptide-based backbone enables PNAs to form a very stable duplex with the complementary nucleic acids via Watson–Crick base pairing since there is no electrostatic repulsion between PNA and DNA·RNA. With this high nucleic acid affinity, PNAs have been used in a wide range of fields, from biological applications such as gene targeting, to engineering applications such as probe and sensor developments. In addition to single-stranded DNA, PNA can also recognize double-stranded DNA (dsDNA) through the formation of a double-duplex invasion complex. This double-duplex invasion is hard to achieve with other artificial nucleic acids and is expected to be a promising method to recognize dsDNA in cellula or in vivo since the invasion does not require the prior denaturation of dsDNA. In this paper, we provide basic knowledge of PNA and mainly focus on the research of PNA invasion.

High-Frequency Variability of Bacterioplankton in Response to Environmental Drivers in Red Sea Coastal Waters.

Autotrophic and heterotrophic bacterioplankton are essential to the biogeochemistry of tropical ecosystems. However, the processes that govern their dynamics are not well known. We provide here a high-frequency assessment of bacterial community dynamics and concurrent environmental factors in Red Sea coastal waters. Weekly sampling of surface samples during a full annual cycle at an enclosed station revealed high variability in ecological conditions, which reflected in changes of major bacterioplankton communities. Temperature varied between 23 and 34°C during the sampling period. Autotrophic (Synechococcus, 1.7–16.2 × 104 cells mL−1) and heterotrophic bacteria (1.6–4.3 × 105 cells mL−1) showed two maxima in abundance in spring and summer, while minima were found in winter and autumn. Heterotrophic cells with high nucleic acid content (HNA) peaked in July, but their contribution to the total cell counts (35–60%) did not show a clear seasonal pattern. Actively respiring cells (CTC+) contributed between 4 and 51% of the total number of heterotrophic bacteria, while live cells (with intact membrane) consistently accounted for over 90%. Sequenced 16S rRNA amplicons revealed a predominance of Proteobacteria in summer and autumn (>40%) and a smaller contribution in winter (21–24%), with members of the Alphaproteobacteria class dominating throughout the year. The contribution of the Flavobacteriaceae family was highest in winter (21%), while the Rhodobacteraceae contribution was lowest (6%). Temperature, chlorophyll-a, and dissolved organic carbon concentration were the environmental variables with the greatest effects on bacterial abundance and diversity patterns.

Coating Silica Layer on Fe₃O₄ Magnetic Nanoparticles and Application in Extracting High Quality Nucleic Acids from Blood Sample.

The given research revealed that the size of Fe₃O₄ magnetic nanoparticles (MNPs) could be controlled by varying the pre-mixing conditions in the solvothermal method. Scanning electron microscopy (SEM) showed that the size of the MNPs gradually increased with increasing the initial temperature at which reaction components were mixed while the reaction component's mixing time was kept constant. The smallest sized MNPs were achieved among the five treatments (25, 50, 75, 100, and 125 °C) when reaction components were mixed at 25 °C, while the larger sized MNPs were synthesized among the five treatments when reaction components were mixed at 125 °C. Then, Stöber method was followed for coating silica layer onto the MNPs. However, ammonium hydroxide was replaced with potassium hydroxide as a catalyst, which significantly increased the speed of silica coating onto MNPs. The Fourier transform infrared (FTIR) spectrometer revealed that the MNPs were successfully covered with silica in five minutes. FTIR spectra exhibited a peak about 1088.8 cm-1, which belonged to the asymmetry stretching vibration of Si-O-Si. Transmission electronic microscopy (TEM) analysis was conducted to confirm the presence of silica layer onto MNPs. Thus, potassium hydroxide was successfully employed as a catalyst for quick silica layer coating onto MNPs. Furthermore, these silica coated MNPs were used to extract high quality nucleic acids from blood sample.

Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection.

The COVID-19 pandemic has brought great challenges to traditional nucleic acid detection technology. Thus, it is urgent to develop a more simple and efficient nucleic acid detection technology. CRISPR-Cas12 has signal amplification ability, high sensitivity and high nucleic acid recognition specificity, so it is considered as a nucleic acid detection tool with broad development prospects and high application value. This review paper discusses recent advances in CRISPR-Cas12-based nucleic acid detection, with an emphasis on the new research methods and means to improve the nucleic acid detection capability of CRISPR-Cas12. Strategies for improving sensitivity, optimization of integrated detection, development of simplified detection mode and improvement of quantitative detection capabilities are included. Finally, the future development of CRISPR-Cas12-based nucleic acids detection is prospected.

Distribution of human papillomavirus genotypes in suspected women cytological specimens from Tehran, Iran.

Background and Objectives:The human papillomavirus (HPV) is associated with more than 70% of the cervical neoplasm. The current study aims to evaluate the distribution of HPV genotypes in suspected women cytological specimens from Tehran, Iran.Materials and Methods:In the current cross-sectional study, HPV genotype prevalence was investigated in 433 subject women. DNA extraction was performed by High Pure Viral Nucleic Acid kit. A semi-automatically hybriSpot 24™ (HS24) setting was used for HPV typing and data interpreted by hybriSoft™ software according to instructions.Results:Pathologic data showed 181 (41.8%) had non-malignant lesions, 212 (49%) had inflammation and 40 (9.2%) reported LSIL in primary Pap-smear result. HPV was found in 143 (33%) specimens and the most comment high-risk and low-risk HPV types were HPV-16 and −6, respectively. Also, 62 (43%) were co-infected with multiple genotypes includes, 34 (24%) cases had co-infection with two HPV types, 17 (12%) cases had co-infection with three HPV types, 6 (4%) cases had co-infection with four HPV types and 5 (3%) cases had co-infection with five HPV types. There was statistically different domination on high-risk genotype in most of the co-infected samples (p<0.01).Conclusion:Current study indicates that the lesion pathology assessment was significantly associated with the HPV infection (p<0.01). Furthermore, the age group assessment shows that most of the HPV positive cases were 21 to 40 (p<0.01). The HPV infection prevalence in the current study was 33% and the most frequently reported high-risk and low-risk HPV types were 16 and 6, respectively.

Elemental and macromolecular composition of the marine Chloropicophyceae, a major group of oceanic photosynthetic picoeukaryotes

AbstractChloropicophyceae (Prasinophyte Clade VII) are small nonmotile coccoid cells with cell diameters ranging from 1 to 3 μm. Molecular surveys indicate they are relatively high in abundance in moderately oligotrophic oceanic waters and may substantively contribute to biogeochemical cycling in the sea. Here, we quantify the elemental and macromolecular composition of three subtropical Chloropicophyceae strains: Chloropicon mariensis, Chloropicon maureeniae, and Chloropicon roscoffensis under nutrient‐sufficient exponential growth and nitrate starvation. Under nutrient‐sufficient conditions the Chloropicophyceae are high in C : N and quite low in C : P and N : P relative to the canonical Redfield ratio, reflecting their relatively high nucleic acid composition compared to many other phytoplankton taxa. Nitrate starvation causes increases in C : N and C : P and decreases in N : P, primarily due to increases in carbohydrate and lipid and decreases in protein and RNA. There is genetic evidence that unlike most other green algae, Chloropicophyceae are diploid. The high nucleic acid content in the Chloropicon is consistent with the hypothesis that the nucleus, as a nonscalable component, takes up a larger and substantial proportion of cell mass in diploid picoeukaryotes. The elemental and macromolecular composition of these Chloropicophyceae, and relatively homeostatic response to N‐starvation compared to diatoms, provides some insight into their success in the moderately oligotrophic ocean.

Modeling polar marine ecosystem functions guided by bacterial physiological and taxonomic traits

Abstract. Heterotrophic marine bacteria utilize organic carbon for growth and biomass synthesis. Thus, their physiological variability is key to the balance between the production and consumption of organic matter and ultimately particle export in the ocean. Here we investigate a potential link between bacterial traits and ecosystem functions in the rapidly warming West Antarctic Peninsula (WAP) region based on a bacteria-oriented ecosystem model. Using a data assimilation scheme, we utilize the observations of bacterial groups with different physiological traits to constrain the group-specific bacterial ecosystem functions in the model. We then examine the association of the modeled bacterial and other key ecosystem functions with eight recurrent modes representative of different bacterial taxonomic traits. Both taxonomic and physiological traits reflect the variability in bacterial carbon demand, net primary production, and particle sinking flux. Numerical experiments under perturbed climate conditions demonstrate a potential shift from low nucleic acid bacteria to high nucleic acid bacteria-dominated communities in the coastal WAP. Our study suggests that bacterial diversity via different taxonomic and physiological traits can guide the modeling of the polar marine ecosystem functions under climate change.

Hydrogel-compartmentalized heterogeneous amplification for viral digital genotyping

Accurate viral genotyping is significant for exploring clinical manifestations and treatment options. The classical nucleic acid amplification within homogeneous system merely reflects the average characteristics of all molecules in samples, which prevents the simultaneous analysis for sample with viruses of different genotypes. Herein, we propose hydrogel-compartmentalized nucleic acid amplification and introduce restriction enzyme to establish a competitive amplification reaction. This system localizes high molecular-weight viral nucleic acids and allows single-molecule heterogeneous amplification in response to various endonuclease digestion. With this strategy, we could provide accurate information for digital genotyping of Hepatitis B virus. Considering its well-performance and easy-to-use, the hydrogel-compartmentalized heterogeneous amplification system should be a promising tool for digital genotyping, and will be of great help to other genetic analysis.

Effect of the array of amines on the transfection efficiency of cationic peptidomimetic lipid molecules into neural cells.

The amino groups in the head group of a cationic lipid play a determinative role regarding the nucleic acid delivery efficiency of the LNP formulated from lipids. Herein, we designed four types of lipid bearing different amine-containing branched head groups to investigate the influence of type and number of amines on the neural cell targeted nuclei acid delivery. Conjugation of an ethylamino group at selected positions of a lysine-based cationic lipid resulted in 4 distinct lipids with 3 (denoted N3 lipid), 4 (denoted N4 lipid), 5 (denoted N5 lipid) and 6 (denoted N6 lipid) amino groups, respectively. Comparative analysis by flow cytometry revealed that the N3 lipid had the highest nucleic acid (plasmid and siRNA) transfection efficiency to neural cell lines (BV2 cells and N2a cells). Furthermore, the N3 lipid mediated delivery of siRNA against Toll Like Receptor 4 (TLR4) into oxygen glucose deprivation (OGD)-treated BV2 cells resulted in remarkable silencing of TLR4, inducing alternative polarization (M2) of the cells. Collectively, our data suggest that the N3 lipid is a promising siRNA delivery agent in neural cells.

Quantitative Real-Time RT-PCR Systems to Detect SARS-CoV-2.

Since the outbreak of coronavirus disease 2019 (COVID-19) on the Diamond Princess cruise ship docked at Yokohama Port on February 3, 2020, real-time reverse transcription-polymerase chain reaction (RT-PCR) testing using nasopharyngeal swab samples from symptomatic and asymptomatic COVID-19 individuals has been the main way to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in almost all clinical laboratories in Japan. With the diffusion of sets containing the primers and probe, the gold standard real-time RT-PCR test has permeated throughout the country. To prevent the spread of infection, real-time RT-PCR testing is important to confirm whether people are positive, asymptomatic, or negative for COVID-19. Now, in addition to pharyngeal swab, saliva and blood samples can be used to detect SARS-CoV-2 RNA. Here, we introduce a clinical laboratory test performed using the High Pure viral nucleic acid kit and subsequent real-time RT-PCR system to detect SARS-CoV-2 RNA in serum, plasma, or whole blood in a hospital in Yokohama, Japan.