Magnetic Enrichment Research Articles

DNA walker induced “signal off” electrochemical cytosensor strategy for ultrasensitive detection of tumor cells

The fragments of molecular motors can be transported and operated autonomously along a track. Most molecular walker systems are assembled from DNA building modules, exhibiting great potential applications in biocomputing, drug transport, sensing, etc. Hereby, we report a DNA walker driven by endonuclease Nt.BbvC I that can autonomously induce “signal off” sensing platform for highly sensitive detection of tumor cells. DNA density and trajectory conformation can affect DNA walker properties. Molybdenum selenide@gold nanoparticles (MoSe2@AuNPs) are modified on the electrode surface as a scaffold to accommodate more hairpin probe (H1) and provide a good interface for DNA walking. Magnetic beads are used to enrich target tumor cells, release single-stranded DNA (S1) and trigger DNA walker-based amplification strategy. Upon addition of tumor cells, more DNA-methylene blue (DNA-MB) is cracked from the electrode surface. Benefiting from efficient electrode modification, magnetic enrichment, DNA walker and enzyme-assisted amplification, an ultra-high sensitivity electrochemical cell analysis is realized, with a detection limit of 2 cells mL−1. More importantly, this DNA walker strategy is available for tumor cell analysis in clinical samples, suggesting the potential diagnostic applications.

Electrochemical detection of T4 polynucleotide kinase activity based on magnetic Fe3O4@TiO2 nanoparticles triggered by a rolling circle amplification strategy.

An ultrasensitive electrochemical detection of the activity and inhibition of T4 polynucleotide kinase (T4 PNK) was developed by using magnetic Fe3O4@TiO2 core-shell nanoparticles, which was triggered by a rolling circle amplification strategy (Fe3O4@TiO2-RCA). We used Fe3O4@TiO2 as a substrate to anchor a DNA primer. DNA S1 with 5'-OH termini was phosphorylated in the presence of T4 PNK and ATP, which was adsorbed on the surface of Fe3O4@TiO2 NPs and served as the primer for subsequent RCA reactions. After adding circular template DNA S2, RCA was initiated in the presence of phi29 DNA polymerase and dNTPs. Then, Fc-labeled DNA S3 (Fc-S3) was hybridized with RCA. The obtained Fe3O4@TiO2-RCA was adsorbed on the surface of a magnetic gold electrode (MGE) by magnetic enrichment, resulting in an enhanced electrochemical signal. The T4 PNK activity can be monitored by measuring the electrochemical signal generated. This electrochemical assay is sensitive to the activity of T4 PNK with a dynamic linear range of 0.00001-20 U/mL and a low detection limit of 3.0×10-6 U/mL. The proposed strategy can be used to screen the T4 PNK inhibitors, so it has great potential in the discovery of nucleotide kinase-target drug and early clinical diagnosis of cancer.

Microfluidic cell sorter sample preparation for genomic assays.

Single-cell RNA-Sequencing has led to many novel discoveries such as the detection of rare cell populations, microbial populations, and cancer mutations. The quality of single-cell transcriptomics relies heavily on sample preparation and cell sorting techniques that best preserve RNA quality while removing dead cells or debris prior to cDNA generation and library preparation. Magnetic bead cell enrichment is a simple process of cleaning up a sample but can only separate on a single-criterion. Droplet-based cell sorters, on the other hand, allows for higher purity of sorted cells gated on several fluorescent and scatter properties. The downside of traditional droplet-based sorters is their operational complexity, accessibility, and potential stress on cells due to their high-pressure pumps. The WOLF® Cell Sorter, and WOLF G2®, developed by NanoCellect Biomedical, are novel microfluidic-based cell sorters that use gentle sorting technology compatible with several RNA-sequencing platforms. The experiments highlighted here demonstrate how microfluidic sorting can be successfully used to remove debris and unwanted cells prior to genomic sample preparation resulting in more data per cell and improved library complexity.

Magnetic immunochromatographic assay with smartphone-based readout device for the on-site detection of zearalenone in cereals

In this study, a portable and quantitative immunochromatographic assay (ICA) was developed with a smartphone-based detection device as readout. The carboxyl group coated Fe 3 O 4 nanoparticles (MNPs) were synthesized by a one pot method and used as the labeled tracer. Initially, zearalenone monoclonal antibody (ZEN mAb) and MNPs conjugates were prepared and applied as the detection probe. MNPs probe produced brown band at the detection zone of the ICA. To increase the sensitivity, MNPs probe were used to enrich ZEN from sample solution, a portable detection device was designed and a user-friendly APP was self-written to quantify results. The cut-off value of the MNPs-ICA for ZEN in cereals was 2.5 μg/kg. The detection limit and quantification limit were 0.06 and 0.13 μg/kg, respectively. The recoveries ranged from 82.0 to 117.7%, with coefficient of variation from 1.3 to 10.6%. The parallel analysis results of 20 naturally contaminated cereal samples by LC-MS/MS showed that the proposed method has high accuracy. Moreover, the results of stability experiments revealed that the ICA products had a shelf life of up to 6 months. Although the feasibility is demonstrated using ZEN as a model analyte, this approach can be easily developed to be a universal analysis system and applied to detection of a wide variety of small molecule hazard factor. This study proposed a qualitative and quantitative analysis device for the food safety analysis. • MNPs was synthesized as the Ab-labeled tracer. • A portable, dual detection mode, multi-channel detection device was designed. • A user-friendly detection APP was self-programmed for quantitative analysis. • Magnetic enrichment was used to improve sensitivity and robustness of the method. • MNPs-ICA was developed to detect five zearalenone toxins in cereals.

Fc-MBL-modified Fe3O4 magnetic bead enrichment and fixation in Gram stain for rapid detection of low-concentration bacteria.

Functional bacterial enrichment magnetic beads (Fe3O4@SiO2@Fc-MBL) and Gram staining were combined for the fast diagnosis of infecting bacteria in meningitis. Fe3O4@SiO2@Fc-MBL has excellent microbial binding ability and can be used for bacterial enrichment from cerebrospinal fluid (CSF). The enriched bacteria are recognized by Gram stain at very low concentrations (10CFU·mL-1). The feasibility of this method was verified by five common bacteria in meningitis infection (Gram-positive: Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus capitis; Gram-negative: Klebsiella pneumoniae and Escherichia coli). The extraction efficiency of Fc-MBL-modified Fe3O4 magnetic beads was approximately 90% in artificialCSF for the selected bacteria, with the exception of E. coli (~ 60%). The bacteria were successfully recognized by Gram staining and microscopic observation. Fe3O4@SiO2@Fc-MBL acts by capturing and fixing the bacteria in a magnetic field throughout the experiment. Compared with traditional CSF Gram staining, this new method avoids interference by inflammatory cells and red blood cells during microscopic examination. Furthermore, the sensitivity of this method is much better than the centrifugation smear method. The whole process can be accomplished within 30min. This novel method may have potential as a clinical tool for analysis of bacteria in the CSF.

Detection of Directly Alloreactive Graft Antigen-Specific CD4+ T Cells in Peripheral Blood

<h3>Purpose</h3> Because the peptide antigens that activate directly alloreactive CD4+ T cells are not known, the phenotype and effects of graft antigen specific CD4+ T cells cannot be established, limiting the ability to accurately diagnose and treat allograft rejection. We hypothesize that a panel of human peptides presented by mismatched human leukocyte antigens (HLA) can be used to detect directly alloreactive CD4+ T cells in peripheral blood. <h3>Methods</h3> We developed a panel of 18 human peptides (18p) presented by HLA-DRB1*04(DR4) in order generate phycoerythrin(PE)- and allophycocyanin(APC)-labelled 18p:DR4 tetramers(18p:DR4^tet). After magnetic bead enrichment of 18p:DR4^tet+ cells, PBMC were stained for non-T lineage-, CD3+, CD8-, CD4+ T cells and quantified using flow cytometry. PBMC were also expanded <i>in vitro</i> for 7 days using 18p with K562 artificial antigen presenting cells (AAPC) with or without DR4 expression before 18p:DR4^tet enrichment and staining as above. <h3>Results</h3> 100-fold more 18p:DR4^tet+ CD4+ T cells were detected directly <i>ex vivo</i> in PBMC from DR4- individuals (Figure 1B, C) than in DR4+ individuals (Figure 1A, C), consistent with thymic deletion of 18p:DR4^tet+ CD4+ T cells in DR4+ individuals and existence of directly alloreactive 18p:DR4^tet+ CD4+ T cells in DR4- individuals. Ten-fold peptide-dependent expansion of 18p:DR4^tet+ CD4+ T cells was seen from PBMC from DR4- but not DR4+ individuals cultured with K562-DR4 expressing AAPC (but not with K562 lacking DR4)(Figure 1D) demonstrating peptide and alloHLA specificity of directly alloreactive CD4+ T cells. <h3>Conclusion</h3> Directly alloreactive CD4+ T cells specific for human peptides presented by alloHLA are detectable directly <i>ex vivo</i> in PBMC of HLA-mismatched individuals. This novel tool will be used to detect and characterize directly alloreactive CD4+ T cells in the PBMC, as well as bronchoalveolar lavage and pleural fluid, from lung transplant recipients in order to better diagnose allograft rejection.

Разработка технологии комплексной переработки шлаков Златоустовского металлургического завода

Introduction. Currently, many metallurgical companies in Russia are experiencing difficulties with the supply of raw materials. One of the solutions to this problem is the involvement in the reuse of secondary resources that are formed in metallurgical processing. So there are more than 15 million tons of them on the territory of Zlatoust steel Mill. The development of metallurgical slag processing technology will partially solve the problem of the company’s own raw material base. Methodology. To develop an effective technology for complex processing of metallurgical slags of Zlatoust steel Mill four samples were taken from various sections of slag dumps. Research results and discussion. The work studied the chemical and mineralogical composition of samples, conducted studies on the enrichment of slags. To study the enrichment of metallurgical slags by the magnetic enrichment method, studies have been carried out, including separation of material with a size of -100 +40 mm on a drum separator and material with a size of -40+0 mm on a separator in a suspended state. The results show that products made of a material with a size of -100+40 mm meet the requirements for direct use in blast furnace production. and products obtained from a size class of -40+0 mm meet the requirements of agglomeration production. The chemical analysis of the obtained products after magnetic separation in a suspended state indicates significant nickel content in the magnetic separation tails of the sample with a size of -40+0 mm. The study of the microstructure of the sample showed that the size of nickel inclusions is on average 1 mm. In this work studies have been carried out on the grinding of tails to a size of less than 1 mm and enrichment on gravity apparatuses. Studies comparing the results of metallurgical slag enrichment on a concentration table and a centrifugal concentrator have shown significantly higher efficiency of slag separation on a centrifugal concentrator. Conclusions. The obtained results on the enrichment of metallurgical slags ofZlatoust steel Mill allows us to recommend a comprehensive processing technology. As a result of enrichment the following products are obtained: magnetic product 1 with a mass fraction of iron 88.4% when extracting 35.2%. magnetic product 2 with a mass fraction of iron 42.7% when extracting 12.4% and nickel-containing concentrate with a mass fraction of nickel 4.85% when extracting 67.9%.

Lipid Profiles of Human Serum Fractions Enhanced with CD9 Antibody-Immobilized Magnetic Beads.

Blood samples are minimally invasive and can be collected repeatedly, but they are far from the site of disease and the target molecules are diluted by the large amount of blood. Therefore, we performed lipidomics using immunoprecipitation as a method to enrich specific fractions of serum. In this study, a CD9 antibody was immobilized on magnetic beads to enrich CD9-containing components in the serum for lipidomics. The percentages of phospholipids recovered from serum by methanol and isopropanol extractions were not significantly different, but triglycerides were barely recovered from serum by methanol extraction, requiring the use of isopropanol. However, once the serum was enriched with CD9 magnetic beads, triglycerides, and phospholipids were recovered at similar levels in both methanol and isopropanol extractions. Therefore, it is possible that the triglyceride fraction of the whole serum and the triglyceride fraction were enriched in CD9 magnetic beads differ in localization and properties. In addition, the variation per disease was small in general serum lipidomics; however, the difference per disease appeared larger when CD9 magnetic bead enrichment was employed.

One-pot synthesis of magnetic covalent organic frameworks for highly efficient enrichment of phthalate esters from fine particulate matter

Phthalate esters (PAEs) are a typical group of endocrine-disrupting chemicals (EDCs) and widely found in the environment, which have attracted great concern due to the potential risks to environment and human health. It is urgent to develop a high-efficiency and high-sensitivity analytical approach for enrichment and determination of ultra-trace PAEs. Herein, magnetic covalent organic frameworks (Fe3O4@TAPB-DVA) were fabricated via a one-pot approach at room temperature and explored as a magnetic enrichment probe with high surface area (275.50 m2/g), good crystallinity, regular pore size distribution (3.1 nm) and outstanding saturation magnetization (26.5 emu/g). Accordingly, a simple and sensitive method based on magnetic solid-phase extraction (MSPE) combined with HPLC-MS/MS was established to detect ultra-trace PAEs from fine particulate matter (PM2.5) samples. The proposed method showed wide linear ranges (1-400 ng/L), good linearity (r > 0.9978) and high enrichment factors (EFs, 6.99-95.01 folds) with low detection limits (LODs) ranged from 0.01 to 0.5 ng/L (S/N ≥ 3). On this basis, the proposed method was substantially employed for sensitive detection of low- abundance PAEs from PM2.5 samples, suggesting the promising application of the Fe3O4@TAPB-DVA in sample enrichment.

Magnetic Enrichment of SARS-CoV-2 Antigen-Binding B Cells for Analysis of Transcriptome and Antibody Repertoire

The ongoing COVID-19 pandemic has had devastating health impacts across the globe. The development of effective diagnostics and therapeutics will depend on the understanding of immune responses to natural infection and vaccination to the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While both B-cell immunity and T-cell immunity are generated in SARS-CoV-2-infected and vaccinated individuals, B-cell-secreted antibodies are known to neutralize SARS-CoV-2 virus and protect from the disease. Although interest in characterizing SARS-CoV-2-reactive B cells is great, the low frequency of antigen-binding B cells in human blood limits in-depth cellular profiling. To overcome this obstacle, we developed a magnetic bead-based approach to enrich SARS-CoV-2-reactive B cells prior to transcriptional and antibody repertoire analysis by single-cell RNA sequencing (scRNA-seq). Here, we describe isolation of SARS-CoV-2 antigen-binding B cells from two seropositive donors and comparison to nonspecific B cells from a seronegative donor. We demonstrate that SARS-CoV-2 antigen-binding B cells can be distinguished on the basis of transcriptional profile and antibody repertoire. Furthermore, SARS-CoV-2 antigen-binding B cells exhibit a gene expression pattern indicative of antigen experience and memory status. Combining scRNA-seq methods with magnetic enrichment enables the rapid characterization of SARS-CoV-2 antigen-binding B cells.

P052 Comparing T cell responses to food antigens in Inflammatory Bowel Disease patients and healthy donors

Abstract Background The role of nutrition in Inflammatory Bowel Disease (IBD) remains unclear. Studies about the effects of nutritional components on intestinal inflammation and the gut microbiome, as well as the concept of exclusive enteral nutrition in paediatric Crohn’s Disease (CD) are suggestive of an impact of nutrition on the pathophysiology. However, there is currently a lack of knowledge on how food components may affect IBD. Here, we investigated the T helper (Th) cell response against nutritional antigens in healthy controls and IBD patients. Methods We used a highly sensitive and specific method developed in our lab, the antigen reactive T cell enrichment (ARTE), to detect wheat and milk-reactive CD4+ T cells directly ex vivo in blood of healthy controls (n=18), CD (n=17) and Ulcerative Colitis (UC, n=15) patients. Peripheral blood mononuclear cells (PBMC) were incubated with whole antigen lysates and activated T helper cells were identified based on the up-regulation of the activation marker CD154. Magnetic enrichment of CD154 positive cells enabled to collect sufficient numbers of food-reactive T cells for their detailed characterization by multiparameter flow cytometry. Results We detected low frequencies of milk and wheat-reactive T cells in healthy individuals. Milk-reactive T cells were less frequent and expressed several markers of T cell exhaustion. In CD, but not UC patients, we identified higher frequencies of food-reactive T cells. These tended to be particularly increased in patients with disease manifestation in the upper gastrointestinal tract. In addition, we observed increased production of inflammatory cytokines of wheat-reactive T cells in CD, compared to healthy controls and UC patients. No ground-breaking differences were identified regarding the expression of exhaustion markers between IBD patients and controls. Conclusion Frequencies of food-reactive Th cells are increased in CD and show a proinflammatory cytokine pattern. The Th1-response of wheat-reactive T cells, characterized by high IFN-γ-secretion, might contribute to the perpetuation of chronic intestinal inflammation.

Rapid identification of bacterial mixtures in urine using MALDI-TOF MS-based algorithm profiling coupled with magnetic enrichment.

Urinary tract infections (UTIs) are a severe public health problem caused by mono- or poly-bacteria. Culture-based methods are routinely used for the diagnosis of UTIs in clinical practice, but those are time consuming. Rapid and unambiguous identification of each pathogen in UTIs can have a significant impact on timely diagnoses and precise treatment. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an alternative method for the identification of pathogens in clinical laboratories. However, a certain number of pure bacteria are required for MALDI-TOF MS analysis. Here, we explored a strategy combining magnetic enrichment and MALDI-TOF MS for the rapid identification of pathogenic bacterial mixtures in urine. Fragment crystallizable mannose-binding lectin-modified Fe3O4 (Fc-MBL@Fe3O4) was used for rapid enrichment and the individual-peak-based similarity model as the analytical tool. Within 30 min, a mixture of the four most prevalent UTI-causing bacteria, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa, was successfully identified using this method. This rapid MALDI-TOF MS-based strategy has potential applications in the clinical identification of UTI pathogens.

Affinity-Based Enrichment of Extracellular Vesicles with Lipid Nanoprobes.

Extracellular vesicles (EVs) are lipid-bilayer-enclosed vesicles with sub-micrometer size that are released by various cells. EVs contain a tissue-specific signature wherein a variety of proteins and nucleic acids are selectively packaged. Growing evidence has shown important biological roles and clinical relevance of EVs in diseases. For EV-related studies to thrive, rapid efficient isolation of pure EVs is a prerequisite. However, lengthy procedure, low yield, low throughput, and high contaminants stemmed from existing isolation approaches hamper both basic research and large-scale clinical implementation. We have shown that lipid nanoprobes (LNP) enable spontaneous labeling and rapid isolation of EVs by coupling with magnetic enrichment. Recently, we further developed a one-step EV isolation platform that utilizes EV size-matched silica nanostructures and surface-conjugated LNPs with an integrated microfluidic mixer. EVs, derived from up to 2-ml clinical plasma, can be processed with this point-of-care device using optimized flow rate. Subsequently, contents of isolated EVs can be extracted on-chip and eluted from the device for downstream molecular analyses. The LNP-functionalized microfluidic device combined with state-of-the-art analysis platforms could have great potential in promoting EV-centered research and clinical use in the future.

Fluorescence detection of the human angiotensinogen protein by the G-quadruplex aptamer.

Noncanonical G-quadruplex nucleic acid structures can be used as probes in biosensors for the detection of metal ions, proteins and nucleic acids. Angiotensinogen (AGT) is a glycosylated globulin found in serum, which can regulate blood pressure and body fluid homeostasis. AGT is an important part of the renin-angiotensin system (RAS) and can be potentially used as a biomarker of diseases with RAS alterations. G-quadruplex based biosensors can detect targets with high accuracy and speed and low cost. Employing the magnetic bead enrichment method we constructed a reliable and efficient fluorescent biosensor platform for G-quadruplex based detection of the human AGT protein. The primary antibody in our biosensor is recognized by fluorescently labeled secondary antibodies, which leads to the detection of AGT captured by the G-quadruplex aptamer coupled magnetic beads. This G-quadruplex based fluorescent biosensor designed with a detection limit of 5 × 10-5 mg mL-1 was used for the successful detection of AGT at the cellular level. Our G-quadruplex based fluorescent biosensor will contribute to the more reliable and efficient detection of AGT.

A Prospective Evaluation of Infant Cerebellar-Cerebral Functional Connectivity in Relation to Behavioral Development in Autism Spectrum Disorder

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder diagnosed based on social impairment, restricted interests, and repetitive behaviors. Contemporary theories posit that cerebellar pathology contributes causally to ASD by disrupting error-based learning (EBL) during infancy. The present study represents the first test of this theory in a prospective infant sample, with potential implications for ASD detection. MethodsData from the Infant Brain Imaging Study (n= 94, 68 male) were used to examine 6-month cerebellar functional connectivity magnetic resonance imaging in relation to later (12/24-month) ASD-associated behaviors and outcomes. Hypothesis-driven univariate analyses and machine learning–based predictive tests examined cerebellar–frontoparietal network (FPN; subserves error signaling in support of EBL) and cerebellar–default mode network (DMN; broadly implicated in ASD) connections. Cerebellar-FPN functional connectivity was used as a proxy for EBL, and cerebellar-DMN functional connectivity provided a comparative foil. Data-driven functional connectivity magnetic resonance imaging enrichment examined brain-wide behavioral associations, with post hoc tests of cerebellar connections. ResultsCerebellar-FPN and cerebellar-DMN connections did not demonstrate associations with ASD. Functional connectivity magnetic resonance imaging enrichment identified 6-month correlates of later ASD-associated behaviors in networks of a priori interest (FPN, DMN), as well as in cingulo-opercular (also implicated in error signaling) and medial visual networks. Post hoc tests did not suggest a role for cerebellar connections. ConclusionsWe failed to identify cerebellar functional connectivity–based contributions to ASD. However, we observed prospective correlates of ASD-associated behaviors in networks that support EBL. Future studies may replicate and extend network-level positive results, and tests of the cerebellum may investigate brain-behavior associations at different developmental stages and/or using different neuroimaging modalities.

Deciphering the Plasmodium falciparum malaria-specific CD4+ T-cell response: ex vivo detection of high frequencies of PD-1+TIGIT+ EXP1-specific CD4+ T cells using a novel HLA-DR11-restricted MHC class II tetramer.

Relatively little is known about the ex vivo frequency and phenotype of the Plasmodium falciparum-specific CD4+ T-cell response in humans. The exported protein 1 (EXP1) is expressed by plasmodia at both, the liver stage and blood stage, of infection making it a potential target for CD4+ and CD8+ effector T cells. Here, a fluorochrome-labelled HLA-DRB1∗11:01-restriced MHC class II tetramer derived from the P. falciparum EXP1 (aa62-74) was established for ex vivo tetramer analysis and magnetic bead enrichment in 10 patients with acute malaria. EXP1-specific CD4+ T cells were detectable in 9 out of 10 (90%) malaria patients expressing the HLA-DRB1∗11 molecule with an average ex vivo frequency of 0.11% (0-0.22%) of total CD4+ T cells. The phenotype of EXP1-specific CD4+ T cells was further assessed using co-staining with activation (CD38, HLA-DR, CD26), differentiation (CD45RO, CCR7, KLRG1, CD127), senescence (CD57), and co-inhibitory (PD-1, TIGIT, LAG-3, TIM-3) markers as well as the ectonucleotidases CD39 and CD73. EXP1-specific tetramer+ CD4+ T cells had a distinct phenotype compared to bulk CD4+ T cells and displayed a highly activated effector memory phenotype with elevated levels of co-inhibitory receptors and activation markers: EXP1-specific CD4+ T cells universally expressed the co-inhibitory receptors PD-1 and TIGIT as well as the activation marker CD38 and showed elevated frequencies of CD39. These results demonstrate that MHC class II tetramer enrichment is a sensitive approach to investigate ex vivo antigen-specific CD4+ T cells in malaria patients that will aid further analysis of the role of CD4+ T cells during malaria.

Application of nanopore sequencing in diagnosis of secondary infections in patients with severe COVID-19.

To explore the application value of nanopore sequencing technique in the diagnosis and treatment of secondary infections in patients with severe coronavirus disease 2019 (COVID-19). A total of 77 clinical specimens from 3 patients with severe COVID-19 were collected. After heat inactivation, all samples were subjected to total nucleic acid extraction based on magnetic bead enrichment. The extracted DNA was used for DNA library construction, then nanopore real-time sequencing detection was performed. The sequencing data were subjected to Centrifuge software database species matching and R program differential analysis to obtain potential pathogen identification. Nanopore sequencing results were compared with respiratory pathogen qPCR panel screening and conventional microbiological testing results to verify the effectiveness of nanopore sequencing detection. Nanopore sequencing results showed that positive pathogen were obtained in 44 specimens (57.1%). The potential pathogens identified by nanopore sequencing included , , and , et al. , , were also detected in clinical microbiological culture-based detection; was detected in respiratory pathogen screening qPCR panel; was only detected by the nanopore sequencing technique. Comprehensive considerations with the clinical symptoms, the patient was treated with antibiotics against , and the infection was controlled. Nanopore sequencing may assist the diagnosis and treatment of severe COVID-19 patients through rapid identification of potential pathogens.

Cross Species use of Human Microarray Genotyping Technology for Bornean Orangutan (Pongo pygmaeus) SNP Discovery

Previous genetic studies of orangutans (Pongo spp.) have relied mainly upon mitochondrial DNA or microsatellite short tandem repeats (STR) for genomic genotyping analysis. Scientists have yet to take advantage of the genetic closeness of the great apes to humans for genomic analysis by using advanced techniques available for human genotyping. To genotype orangutans at Tanjung Puting National Park, we developed a novel combination of a methyl-based magnetic enrichment capture of genomic fecal DNA with genotyping on a human targeted single nucleotide polymorphism (SNP) microarray, and compared this to additional microsatellite (STR) micro-capillary genotyping. We successfully isolated 125 known human genomic SNP loci (0.08% of those targeted) which hybridized orangutan DNA on the human targeted Illumina Infinium QC array. We estimated genetic diversity and relatedness (r) using three estimators for a total of 32 (21 female and 9 male) wild orangutans at the Camp Leakey study site. Average TrioML relatedness within the sample, estimated from our combo SNP/STR dataset, was at a range consistent with half and first cousins (r = .082). All sampled males and females had relatives within the study site indicating we have verified a local, closely related community of wild orangutans at Camp Leakey.

Controlling Droplet Marangoni Flows to Improve Microscopy-Based TB Diagnosis.

In developing countries, the most common diagnostic method for tuberculosis (TB) is microscopic examination sputum smears. Current assessment requires time-intensive inspection across the microscope slide area, and this contributes to its poor diagnostic sensitivity of ≈50%. Spatially concentrating TB bacteria in a smaller area is one potential approach to improve visual detection and potentially increase sensitivity. We hypothesized that a combination of magnetic concentration and induced droplet Marangoni flow would spatially concentrate Mycobacterium tuberculosis on the slide surface by preferential deposition of beads and TB–bead complexes in the center of an evaporating droplet. To this end, slide substrate and droplet solvent thermal conductivities and solvent surface tension, variables known to impact microfluidic flow patterns in evaporating droplets, were varied to select the most appropriate slide surface coating. Optimization in a model system used goniometry, optical coherence tomography, and microscope images of the final deposition pattern to observe the droplet flows and maximize central deposition of 1 μm fluorescent polystyrene particles and 200 nm nanoparticles (NPs) in 2 μL droplets. Rain-X® polysiloxane glass coating was identified as the best substrate material, with a PBS-Tween droplet solvent. The use of smaller, 200 nm magnetic NPs instead of larger 1 μm beads allowed for bright field imaging of bacteria. Using these optimized components, we compared standard smear methods to the Marangoni-based spatial concentration system, which was paired with magnetic enrichment using iron oxide NPs, isolating M. bovis BCG (BCG) from samples containing 0 and 103 to 106 bacilli/mL. Compared to standard smear preparation, paired analysis demonstrated a combined volumetric and spatial sample enrichment of 100-fold. With further refinement, this magnetic/Marangoni flow concentration approach is expected to improve whole-pathogen microscopy-based diagnosis of TB and other infectious diseases.

A Sediment Magnetic Record in the North Pacific Across the Mid-Pleistocene Transition and its Implication on Asian Dust Evolution

Eolian dust deposited in the North Pacific is an important archive of the evolutionary history of Asian interior source regions and climate system. Here, we present a ∼1 Myr sediment magnetic record from the central North Pacific to characterize eolian dust properties since the middle Pleistocene. For the studied sediments, magnetic components are mainly identified as biogenic magnetite and detrital magnetic minerals (dust and volcanic origins) based on coercivity analysis, microscopic observations, and sedimentological information. The detrital magnetic component is characterized by high coercivity (&amp;gt;100 mT) and shows a long-term increase in concentration since ∼1 Ma. In particular, the concentration shows a considerable increase at ∼0.8–0.7 Ma compared to the inorganic silicate fraction, indicative of magnetic mineral enrichment in detrital sediment fraction. At the same time, the coercivity distribution of the detrital component also decreases, which can be attributed to an increase in the ferrimagnetic mineral contribution. As the detrital sediments are primarily wind-blown particles, such ferrimagnetic enrichment implies a change in dust source materials after ∼0.8 Ma, which could be explained by the reorganization of atmospheric circulation and/or regional aridification in source regions across the mid-Pleistocene transition. The dust property change in source areas is likely to be synchronized across the North Pacific based on the similarity of the long-term trend of magnetic signals.