A microfluidic chip platform based on Pt nanozyme and magnetized phage composite probes for dual-mode detecting and imaging pathogenic bacteria viability.

The detection of pathogen viability is critically important to evaluate its infectivity. In the study, an integrated microfluidic chip based on dual-mode analytical strategy was developed to rapidly realize detection of bacteria activity (with Salmonella typhimurium, S.T, as a model analyte). Firstly, the composite probes, including deactivated phage modified magnetic beads and nano Pt-antimicrobial peptide (AMP) which can specifically recognize Gram-negative bacteria as nanozyme were prepared. When the composite probes are introduced into the chip together with target bacteria, after enrichment, oscillating and magnetic separation, they will conjugate with S.T and produce a magnetic sandwich complex. The complex can catalyze tetramethylbenzidine (TMB)-H2O2 to produce visible colorimetric signals which is correspondent to the total S.T content. Simultaneously, PtNPs in the complex can produce hydroxyl radical oxidation (∙OH) by decomposing H2O2. Under the synergistic action of ∙OH and AMP, the captured live S.T can be lysed to release ATP and emit bioluminescence signals which corresponds to the live S.T concentration. Therefore, the chip can simultaneously detect and image S.T at different viability in one test. The dual-mode assay demonstrated high sensitivity (≤33 CFU/mL), high specificity (identifying strain), signal amplification (5 folds) and short time (≤40min). The chip array can detect four samples in one test and exhibited advantages of high-integration, -sensitivity, -specificity and miniaturization, which are suitable to rapidly detect and image pathogen's viability in trace level. The replacement of phage probes can detect other bacteria. It has a wide prospect in pathogens screening.

Use of psychoactive substances by goods carriage drivers associated with Kerala, India.

OBJECTIVE: This study aimed to measure the prevalence of drugs of abuse (DOA) among the goods carriage drivers associated with the southern State of India, Kerala. METHODS: Point-of-collection testing (POCT) of oral fluid collected from the participants (n = 249) was done using the Evidence MultiSTAT DOA Oral Fluid II Assay kits and the Evidence MultiSTAT analyzer. RESULTS: Out of the total samples, 53 (21.29%) were positive for one or more DOA. A high prevalence of tetrahydrocannabinol (THC) (10.04%) and synthetic cannabinoids were detected in the samples. The use of ketamine, alpha-PVP, LSD, methamphetamine, opiate, 6-MAM, benzodiazepines I, methadone, PCP, tramadol, and amphetamine was also detected and their frequency of use ranged between 4.02 and 0.80%. An association between drug abuse and distance of travel was found in drivers in this study, χ2 (5, N = 249) = 123.5, p < 0.001. Confirmatory analysis using ultra-high performance liquid chromatography-tandem mass spectrometry showed excellent agreement with the results of the screening test. CONCLUSIONS: This was the first study conducted among drivers in India for the detection of DOA. Tetrahydrocannabinol (THC) was used more by the goods carriage drivers associated with Kerala State, India. The use of psychoactive substances significantly increased with the distance of travel. Point-of-collection testing (POCT) by the biochip array technology is an efficient method for the detection of these substances.

Evolution Toward Chip-Based Arrays in the Laboratory Diagnosis of Human Allergic Disease.

Multiplex-based specific IgE antibody assays have emerged into the clinical immunology laboratory through the combined use of pure, recombinant allergenic molecules and new methods to simultaneously and accurately analyze specific IgE antibodies to hundreds of allergens. This review traces the historical development and examines outstanding questions related to the strengths and limitations of these new molecular allergen multipex technologies for the assessment of human allergic sensitization. Multiplexed technologies are poised to provide the most cost-effective and comprehensive evaluation of patients with suspected allergy as compared with the commonly used singleplex autoanalyzers. How analytically sensitive and quantitative are the multiplex technologies, down to 0.1 kUA/L? Because each allergen is viewed as a unique assay, how will analytical and clinical performance be documented at the manufacturing and clinical laboratory levels to guarantee reproducibility and obtain government regulatory clearance? Will interference by naturally occurring allergen-specific IgG compromise analytical performance? Successful resolution of these and other questions covered in this review will position multiplex technologies to become the single most-effective means of screening patients for allergic sensitization, assessing IgE antibody cross-reactivity, and planning therapy directed at the patient with allergy.

Identifying Parameters for Defining "Essentially Derived Varieties" of Maize Inbred Lines Using High-Throughput Genome-Wide SNP Markers.

Well-developed maize reference genomes and genotyping technology along with fast decreasing detection costs have enabled the chance of shifting essentially derived varieties (EDV) identification to high-throughput SNP genotyping technology. However, attempts of using high-throughput technologies such as SNP array on EDV identification and the essential baseline parameters such as genetic homozygosity and/or stability in EDV practices have not been characterized. Here, we selected 28 accessions of 21 classical maize inbreds, which definitely form a pedigree network from initial founders to derivatives that had made huge contribution to corn production, to demonstrate these fundamental analyses. Our data showed that average residual heterozygosity (RH) rate of these 28 accessions across genome was about 1.03%. However, the RH rate of some accessions was higher than 3%. In addition, some inbreds were found to have an overall RH rate lower than 2% but over 8% level at certain chromosomes. Genetic drift (GD) between two accessions from different years or breeding programs varied from 0.13% to 13.16%. Accessions with low GD level showed cluster distribution pattern and compared with RH distributions indicated that RH was not the only resource of GD. Both RH and GD data suggested that genetic purity analysis is an essential procedure before determining EDV. Eleven derivative lines were characterized with regard to their genome compositions and were inferred as their breeding histories. The backcross, bi-parental recycling, and mutation breeding records could be identified. The data provide insights of underlining fundamental parameters for defining EDV threshold and the results demonstrate the EDV identification process.

Human Papilloma Virus Frequency and Genotypes; Evaluation of the 4879 Screenings Made with Polymerase Chain Reaction and Chip Array Between 2001 and 2019 in Istanbul.

OBJECTIVES: The aim of this study is the documentation of human papilloma virus (HPV) frequency and types seen in the city of Istanbul, Turkey, as well as evaluation of the relationship between these subtypes and cytological and pathological diagnoses. METHODS: 4879 cases were studied in our molecular pathology department between 2001 and 2019 in Istanbul. Between 2001 and 2010, 1692 cases were screened for HPV 6, 11, 16, 18, 31, and 33 by conventional hybridization and polymerase chain reaction (PCR). Since 2011, up to 49 HPV typing has been performed for 3187 cases with chip array. The cases were referred to the pathology center and the hospital pathology department by clinicians for screening before HPV vaccination and on the observation of precancerous changes and koilocyts in cytological-histopathological evaluations. RESULTS: In this study, the frequency of HPV was found to be 10.8% (527 HPV-positive cases). Among these, 348 cases were high-risk groups, whether or not they were previously associated with a low-risk group. When we look at the distribution of the cases according to the high-risk HPV types, HPV 16 is the most common type. The frequencies of occurrence of other HPV types are as following: HPV-16: 41.7%, HPV-31: 11.7%, HPV-52: 7.9%, HPV-51: 7.1%, HPV-33: 6.9%, HPV-45: 6.5%, HPV-18: 6.3%, HPV-39: 6.1%, and HPV-58: 5.8%. It was further found that multiple infections were 28% of high grade squamous intraepithelial lesion cases. HPV frequency was 38% and 72%, respectively, in cases with cytologically or histopathological precancerous, low-grade squamous intraepithelial lesion, and HSIL changes. As a final note, HPV was detected in 9 of 10 cases with cervical cancer (90%). Only 1 adenocarcinoma case detected in the series was a double infection with HPV types 18 and 45. CONCLUSION: HPV 16 was the most common type found in this study. It is followed by types 31, 52, 51, 33, 45, 18, 39, and 58, respectively. The most common association observed in double infections was between HPV 16 and 58. It was also observed that the incidence of HPV in the city of Istanbul, Turkey, was similar to other developed countries. As a final note, in addition to screening tests, PCR and chip array studies should be conducted and the community should be informed about preventive medicine and the importance of condom use.

IL-17 Triggers Invasive and Migratory Properties in Human MSCs, while IFNy Favors their Immunosuppressive Capabilities: Implications for the "Licensing" Process.

Mesenchymal stromal cells (MSCs) were first used as a source for cell therapy in 1995; however, despite their versatility and unambiguous demonstration of efficacy and safety in preclinical/phase I studies, the positive effect of MSCs in human phase III studies did not resemble the success obtained in mouse models of disease. This dissonance highlights the need to more thoroughly study the immunobiology of MSCs to make better use of these cells. Thus, we aimed to study the immunobiology of MSCs by using chip array analysis as a method for general screening to obtain a global picture in our model study and found IFNy and IL-17 signaling as the first two "top canonical pathways" involved in MSCs immunomodulation. The role of IFNy in triggering the immunosuppressive properties of MSCs is well recognized by many groups; however, the role of IL-17 in this process remains uncertain. Interestingly, in contrast to IFNy, which actively improved the MSCs-mediated immunosuppression, IL-17 did not improve directly the MSCs-mediated immunosuppression. Instead, IL-17 signaling induced the migration of MSCs and inflammatory cells, bringing these cell types together and increasing the likelihood of the lymphocytes sensing the immunosuppressive molecules produced by the MSCs. These effects also correlated with high levels of cytokine/chemokine production and metalloprotease activation by MSCs. Importantly, this treatment maintained the MSCs safety profile by not inducing the expression of molecules related to antigen presentation. In this way, our findings highlight the possibility of using IL-17, in combination with IFNy, to prime MSCs for cell therapy to improve their biological properties and thus their therapeutic efficacy. Finally, the use of preactivated MSCs may also minimize variations among MSCs to produce more uniform therapeutic products. In the not-so-distant future, we envisage a portfolio of MSCs activated by different cocktails specifically designed to target and treat specific diseases. Graphical abstract.

High density DNA methylation array is a reliable alternative for PCR-based analysis of the MGMT promoter methylation status in glioblastoma.

AIM: MGMT promoter methylation status is an important biomarker predicting survival and response to chemotherapy in patients suffering from glioblastoma. Since new diagnostic methods such as methylome-based classification of brain tumors are more and more frequently performed, we aimed at comparing the suitability of calculating the MGMT promoter methylation status in a quantitative manner from the methylome profiling as compared to the classic gold standard assessment by PCR. METHODS: Our cohort consisted of 39 cases diagnosed as "glioblastoma, IDH-wildtype" of which the MGMT promoter methylation status was analyzed with both methylation-specific PCR and high density DNA methylation array using the STP-27 algorithm. Contradictory results were validated by pyrosequencing. RESULTS: The inter-method reliability reached 77% (kappa-coefficient: 0.58) when also cases with an inconclusive result in one or the other method were taken into account. When only cases with conclusive results in both methods were considered, a very high inter-method reliability of 91% (kappa-coefficient: 0.86) could be achieved. For "methylated" cases, no contradictory results were obtained. For the remaining two cases with discrepant results subsequent pyrosequencing analyses spoke in favor of each previously applied method once. CONCLUSION: In addition to its benefits for molecular subgrouping and copy number analysis of brain tumors, DNA-methylation based classification is a highly reliable tool for the assessment of MGMT promoter methylation status in glioblastoma patients.

Altered Genome-Wide DNA Methylation in Peripheral Blood of South African Women with Gestational Diabetes Mellitus.

Increasing evidence implicate altered DNA methylation in the pathophysiology of gestational diabetes mellitus (GDM). This exploratory study probed the association between GDM and peripheral blood DNA methylation patterns in South African women. Genome-wide DNA methylation profiling was conducted in women with (n = 12) or without (n = 12) GDM using the Illumina Infinium HumanMethylationEPIC BeadChip array. Functional analysis of differentially methylated genes was conducted using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. A total of 1046 CpG sites (associated with 939 genes) were differentially methylated between GDM and non-GDM groups. Enriched pathways included GDM-related pathways such as insulin resistance, glucose metabolism and inflammation. DNA methylation of the top five CpG loci showed distinct methylation patterns in GDM and non-GDM groups and was correlated with glucose concentrations. Of these, one CpG site mapped to the calmodulin-binding transcription activator 1 (CAMTA1) gene, which have been shown to regulate insulin production and secretion and may offer potential as an epigenetic biomarker in our population. Further validation using pyrosequencing and conducting longitudinal studies in large sample sizes and in different populations are required to investigate their candidacy as biomarkers of GDM.

Comparison of protein-chip array analysis and traditional ELISAs for biomarker detection of diabetic limb arterial stenosis.

The aim of this study was to screen the biomarkers of diabetic limb arterial stenosis. Fasting blood samples of 40 patients with diabetic limb arterial stenosis (experimental group), 40 diabetes patients (diabetic control group), and 40 healthy individuals (healthy control group) were collected. Protein-chip assay analysis and ELISA were used to detect tumor necrosis factor α, interleukin-6, endothelin-1, calcitonin gene-related peptide and high-sensitivity C-reactive protein in the three groups. Protein-chip array analysis and ELISA found consistent results that endothelin-1, tumor necrosis factor α, interleukin-6 and high-sensitivity C-reactive protein in the experimental group were significantly up-regulated while the expression of calcitonin gene-related peptide was down-regulated compared with the healthy control group ( P < 0.01). When compared with the diabetic control group, only markedly increased calcitonin gene-related peptide and interleukin-6 were observed in the experimental group ( P < 0.01). The study suggests that high-throughput protein-chip may be a reliable method to screen biomarkers of diabetic limb arterial stenosis. Calcitonin gene-related peptide and interleukin-6 might be promising biomarkers for diabetic limb arterial stenosis.

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array.

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5'-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

Toll-like receptor 4 agonist, lipopolysaccharide, increases the expression levels of cytokines and chemokines in human peripheral blood mononuclear cells.

Toll-like receptors (TLRs) are members of the pattern recognition receptor family and are essential in the innate immune response. In total, 11 TLRs exist in humans, which are expressed in a variety of cells, including peripheral blood cells. TLR4 plays a significant role in the defense against gram-negative pathogens by recognizing the lipopolysaccharide (LPS) molecules in these bacteria. The aim of the present study was to detect the expression level variation of a number of major immune molecules in peripheral blood mononuclear cells (PBMCs) stimulated by LPS, in order to identify candidate genes involved in the biological functions mediated by TLR4. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis and an antibody chip were performed in the current study. The RT-qPCR results revealed a marked enhancement in the expression levels of various molecules, including cytokines, chemokines, growth factors and protein kinases. In addition, the antibody chip identified the increased secretion of crucial proinflammatory molecules in the supernatants collected from LPS-treated PBMCs. In conclusion, a large number of molecules were found to be involved in TLR4-mediated functions.

Chronic ethanol exposure alters the lung proteome and leads to mitochondrial dysfunction in alveolar type 2 cells.

The lungs can undergo irreversible damage from chronic alcohol consumption. Herein, we developed an animal model predisposed for edematous lung injury following chronic ingestion of alcohol to better understand the etiology of alcohol-related disorders. Using animal modeling, alongside high-throughput proteomic and microarray assays, we identified changes in lung protein and transcript in mice and rats, respectively, following chronic alcohol ingestion or a caloric control diet. Liquid chromatography-mass spectrometry identified several mitochondrial-related proteins in which the expression was upregulated following long-term alcohol ingestion in mice. Consistent with these observations, rat gene chip microarray analysis of alveolar cells obtained from animals maintained on a Lieber-DeCarli liquid alcohol diet confirmed significant changes in mitochondrial-related transcripts in the alcohol lung. Transmission electron microscopy revealed significant changes in the mitochondrial architecture in alcohol mice, particularly following lipopolysaccharide exposure. Chronic alcohol ingestion was also shown to worsen mitochondrial respiration, mitochondrial membrane polarization, and NAD(+)-to-NADH ratios in alveolar type 2 cells. In summary, our studies show causal connection between chronic alcohol ingestion and mitochondrial dysfunction, albeit the specific role of each of the mitochondrial-related proteins and transcripts identified in our study requires additional study.

Neurofilament light mutation causes hereditary motor and sensory neuropathy with pyramidal signs.

To identify novel mutations causing hereditary motor and sensory neuropathy (HMSN) with pyramidal signs, a variant of Charcot-Marie-Tooth disease (CMT), we screened 28 CMT and related genes in four members of an affected Japanese family. Clinical features included weakness of distal lower limb muscles, foot deformity, and mild sensory loss, then late onset of progressive spasticity. Electrophysiological studies revealed widespread neuropathy. Electron microscopic analysis showed abnormal mitochondria and mitochondrial accumulation in the neurons and Schwann cells. Brain magnetic resonance imaging (MRI) revealed an abnormally thin corpus callosum. In all four, microarrays detected a novel heterozygous missense mutation c.1166A>G (p.Y389C) in the gene encoding the light-chain neurofilament protein (NEFL), indicating that NEFL mutations can result in a HMSN with pyramidal signs phenotype.

Gene Expression Profiles of Peripheral Blood Mononuclear Cells Reveal Transcriptional Signatures as Novel Biomarkers for Cardiac Remodeling in Rats with Aldosteronism and Hypertensive Heart Disease.

OBJECTIVES: In searching for a noninvasive surrogate tissue having mimicry with the prooxidant/-proinflammatory hypertensive heart disease (HHD) phenotype, we turned to peripheral blood mononuclear cells (PBMC). We tested whether iterations in [Ca2+]i, [Zn2+]i and oxidative stress in cardiomyocytes and PBMC would complement each other eliciting similar shifts in gene expression profiles in these tissues demonstrable during preclinical (wk 1) and pathologic (wk 4) stages of aldosterone/salt treatment (ALDOST). BACKGROUND: Inappropriate neurohormonal activation contributes to pathologic remodeling of myocardium in HHD associated with aldosteronism. In rats receiving chronic ALDOST, evidence of reparative fibrosis replacing necrotic cardiomyocytes and coronary vasculopathy appears at wk 4 associated with the induction of oxidative stress by mitochondria that overwhelms endogenous, largely Zn2+-based, antioxidant defenses. Biomarker-guided prediction of risk prior to the appearance of cardiac pathology would prove invaluable. METHODS: In PBMC and cardiomyocytes, quantitation of cytoplasmic free Ca2+ and Zn2+, H2O2 and 8-iosprostane levels, as well as isolation of RNA and gene expression, together with statistical and clustering analyses, and confirmation of genes by in situ hybridization and RT-PCR, were performed. RESULTS: Compared to controls, at wk 1 and 4 ALDOST, we found comparable: increments in [Ca2+]i, [Zn2+]i and 8-isoprotane coupled to increased H2O2 production in cardiac mitochondria and PBMC, together with the common networks of expression profiles dominated by genes involved in oxidative stress, inflammation and repair. These included three central Ingenuity pathway-linked genes: p38MAPK, a stress-responsive protein; NFκB, a redox-sensitive transcription factor and a proinflammatory cascade it regulates; and TGF-ß1, a fibrogenic cytokine involved in tissue repair. CONCLUSIONS: Significant overlapping demonstrated in the molecular mimicry of PBMC and cardiomyocytes during preclinical and pathologic stages of ALDOST implicates that transcriptional signatures of PBMC may serve as early noninvasive and novel sentinels predictive of impending pathologic remodeling in HHD.

The COE Program at University of Toyama / 日本東洋医学雑誌

The outline of COE program at University of Toyama entitled “Advanced Approach to Personalized Medicine Based on Oriental Philosophy” was presented. This program consists of two parts, i.e., basic research group and clinical research group. On the basic researches, we performed field work for natural resources of medicinal plants both in Mongolia and China. And we revealed the differences of the chemical ingredients in rhubarb family. Furthermore, we developped a new DNA microarray to detect the panax ginseng family. In this paper, we also reported that the chemical ingredients of medicinal herbs convert new substances by the action of intestinal bacterias. In the clinical research group, we revealed that the characteristic protein pattern of “blood stagnant (Oketsu) syndrome” in Kampo medicine by means of protein chip array system. We showed the result of the midway evaluation by Japan Society for the Promotion of Science.