Rapid Diagnosis by Microfluidic Techniques

Pathogenic bacteria in an aqueous or airborne environments usually cause infectious diseases in hospital or among the general public. One critical step in the successful treatment of the pathogen-caused infections is rapid diagnosis by identifying the causative microorganisms, which helps to provide early warning of the diseases. However, current standard identification based on cell culture and traditional molecular biotechniques often depends on costly or time-consuming detection methods and equipments, which are not suitable for point-of-care tests. Microfluidic-based technique has recently drawn lots of attention, due to the advantage that it has the potential of providing a faster, more sensitive, and higher-throughput identification of causative pathogens in an automatic manner by integrating micropumps and valves to control the liquid accurately inside the chips. In this chapter, microfluidic techniques for serodiagnosis of amebiasis, allergy, and rapid analysis of airborne bacteria are described. The microfluidic chips that integrate microcolumns, protein microarray, or a staggered herringbone mixer structure with sample to answer capability have been introduced and shown to be powerful in rapid diagnosis especially in medical fields

CRISPR-Cas- and Aptamer-based Systems for Diagnosing Pathogens: A Review

Pathogenic infections cause severe clinical illnesses in humans and animals. Increased encounters between humans and animals and constant environmental changes exacerbate the transmission of zoonotic infectious diseases. Recently, the World Health Organization has declared some zoonotic epidemics as public health emergencies of international concern. Hence, rapid and accurate detection of the causative pathogen is particularly essential in combating emerging and re-emerging infectious diseases. Traditional pathogen detection tools are time-consuming, costly, and require skilled personnel, which greatly hinder the development of rapid diagnostic tests, particularly in resource-constrained regions. Clustered regularly interspaced short palindromic repeats (CRISPR-)-Cas- and aptamer-based platforms have replaced traditional pathogen detection methods. Herein we review two novel next-generation core pathogen detection platforms that are utilized for clinical and foodborne pathogenic microorganisms: CRISPR-Cas-based systems, including dCas9, Cas12a/b, Cas13, and Cas14; and aptamer-based biosensor detection tools. We highlight CRISPR-Cas- and aptamer-based techniques and compare the strengths and weaknesses. CRISPR-Cas-based tools require cumbersome procedures, such as nucleic acid amplification and extraction, while aptamer-based tools require improved sensitivity. We review the combination of CRISPR-Cas- and aptamer-based techniques as a promising approach to overcome these deficiencies. Finally, we discuss Cas14-based tools as functionally stronger platforms for the detection of non-nucleic acid targets

Effects of an antibacterial membrane on osteoblast-like cells in vitro

Infection around membranes is often found in guided bone regeneration (GBR). The excellent antibacterial properties of Ag-nHA-nTiO2/polyamide-66 (PA66) nanocomposite membranes have been demonstrated previously. The aim of this study was to observe the microstructure of an Ag-nHA-nTiO2/PA66 membrane and its effects on osteoblast-like cells in vitro. An Ag-nHA-nTiO2/PA66 membrane was used in the experimental group, and both nHA/PA66 and expanded poly tetrafluroethylene (e-PTFE) membranes were set as control. MG63 osteoblast-like cells were cultured on the three kinds of membrane and tissue culture polystyrene (TCP). The microstructure of the above membranes and the cells adhered on them were detected by scanning electronic microscope (SEM). Cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell viability with a cell viability analyzer, and alkaline phosphatase (ALP) activity and Ca2+ concentration of osteoblast-like cell matrix by enzyme-linked immunosorbent assay. SEM showed that both Ag-nHA-nTiO2/PA66 membranes and nHA/PA66 membranes were composed of porous obverse face and smooth opposite face. The e-PTFE membranes showed elliptic surface structure with many tiny lined cracks. The MG63 cells adhered and proliferated well on all three kinds of membranes. Though cell viability on Ag-nHA-nTiO2/PA66 membranes was significantly lower than that of the control groups (P < 0.05), MTT values, ALP activity, and Ca2+ concentration did not differ significantly among the three kinds of membranes (P > 0.05). From these findings, it can be concluded that Ag-nHA-nTiO2/PA66 membranes are as biocompatible as nHA/ PA66 membranes and TCP, thus may be applied safely in GBR

Association of Base Excision Repair Gene Polymorphisms with ESRD Risk in a Chinese Population

The base excision repair (BER) pathway, containing OGG1, MTH1 and MUTYH, is a major protector from oxidative DNA damage in humans, while 8-oxoguanine (8-OHdG), an index of DNA oxidation, is increased in maintenance hemodialysis (HD) patients. Four polymorphisms of BER genes, OGG1 c.977C > G (rs1052133), MTH1 c.247G > A (rs4866), MUTYH c.972G > C (rs3219489), and AluYb8MUTYH (rs10527342), were examined in 337 HD patients and 404 healthy controls. And the 8-OHdG levels in leukocyte DNA were examined in 116 HD patients. The distribution of MUTYH c.972 GG or AluYb8MUTYH differed between the two groups and was associated with a moderately increased risk for end-stage renal disease (ESRD) (P = 0.013 and 0.034, resp.). The average 8-OHdG/106 dG value was significantly higher in patients with the OGG1 c.977G, MUTYH c.972G or AluYb8MUTYH alleles (P < 0.001 via ANOVA). Further analysis showed that combination of MUTYH c.972GG with OGG1 c.977GG or AluYb8MUTYH increased both the risk for ESRD and leukocyte DNA 8-OHdG levels in HD patients. Our study showed that MUTYH c.972GG, AluYb8MUTYH, and combination of OGG1 c.977GG increased the risk for ESRD development in China and suggested that DNA oxidative damage might be involved in such process

IgG Fc-binding motif-conjugated HIV-1 fusion inhibitor exhibits improved potency and in vivo half-life: Potential application in combination with broad neutralizing antibodies

The clinical application of conventional peptide drugs, such as the HIV-1 fusion inhibitor enfuvirtide, is limited by their short half-life in vivo. To overcome this limitation, we developed a new strategy to extend the in vivo half-life of a short HIV-1 fusion inhibitory peptide, CP24, by fusing it with the human IgG Fc-binding peptide (IBP). The newly engineered peptide IBPCP24 exhibited potent and broad anti-HIV-1 activity with IC50 values ranging from 0.2 to 173.7 nM for inhibiting a broad spectrum of HIV-1 strains with different subtypes and tropisms, including those resistant to enfuvirtide. Most importantly, its half-life in the plasma of rhesus monkeys was 46.1 h, about 26- and 14-fold longer than that of CP24 (t1/2 = 1.7 h) and enfuvirtide (t1/2 = 3 h), respectively. IBP-CP24 intravenously administered in rhesus monkeys could not induce significant IBP-CP24-specific antibody response and it showed no obvious in vitro or in vivo toxicity. In the prophylactic study, humanized mice pretreated with IBPCP24 were protected from HIV-1 infection. As a therapeutic treatment, coadministration of IBP-CP24 and normal human IgG to humanized mice with chronic HIV-1 infection resulted in a significant decrease of plasma viremia. Combining IBP-CP24 with a broad neutralizing antibody (bNAb) targeting CD4-binding site (CD4bs) in gp120 or a membrane proximal external region (MPER) in gp41 exhibited synergistic effect, resulting in significant dose-reduction of the bNAb and IBP-CP24. These results suggest that IBP-CP24 has the potential to be further developed as a new HIV-1 fusion inhibitor-based, long-acting anti-HIV drug that can be used alone or in combination with a bNAb for treatment and prevention of HIV-1 infection

Normative Analysis of Individual Brain Differences Based on a Population MRI-Based Atlas of Cynomolgus Macaques

The developmental trajectory of the primate brain varies substantially with aging across subjects. However, this ubiquitous variability between individuals in brain structure is difficult to quantify and has thus essentially been ignored. Based on a large-scale structural magnetic resonance imaging dataset acquired from 162 cynomolgus macaques, we create a species-specific 3D template atlas of the macaque brain, and deploy normative modeling to characterize individual variations of cortical thickness (CT) and regional gray matter volume (GMV). We observed an overall decrease in total GMV and mean CT, and an increase in white matter volume from juvenile to early adult. Specifically, CT and regional GMV were greater in prefrontal and temporal cortices relative to early unimodal areas. Age-dependent trajectories of thickness and volume for each cortical region revealed an increase in the medial temporal lobe, and decreases in all other regions. A low percentage of highly individualized deviations of CT and GMV were identified (0.0021%, 0.0043%, respectively, P \u3c 0.05, false discovery rate [FDR]-corrected). Our approach provides a natural framework to parse individual neuroanatomical differences for use as a reference standard in macaque brain research, potentially enabling inferences regarding the degree to which behavioral or symptomatic variables map onto brain structure in future disease studies

Metabolomics-Based Identification of Bleached Shellac in Liquid Fruit Wax

The purpose of this study was to identify whether liquid fruit waxes could be added with bleached shellac. The compounds of bleached shellac from different sources were analyzed by targeted metabolomics based on liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) combined with liquid chromatography with evaporative light scattering detection. Twelve target compounds were selected by multivariate statistical analysis including unsupervised principal component analysis (PCA) and supervised partial least squares discriminant analysis (PLS-DA). According to variable importance in the projection (VIP) value, five differential compounds and seven shared compounds were identified. Finally, the viability of this method in the detection of commercial liquid fruit wax products was verified using the shared compounds as biomarkers. These results showed that chromatography combined with mass spectrometry can identify whether bleached shellac could be added in liquid fruit wax