Biological Detection Methods Research Articles

Anti-inflammatory target prediction of Gentianae Radix et Rhizoma based on network pharmacology and construction of a bioassay method for its quality control

Based on the heat-clearing and detoxifying effects of Gentianae Radix et Rhizoma, the network pharmacology is mainly used to predict the potential targets of Gentianae Radix et Rhizoma for anti-inflammatory activity and to perform the experimental verification. A method for detecting the biological potency of Gentianae Radix et Rhizoma based on verifiable targets has been established to provide a reference for improving the quality evaluation and control standards of Gentianae Radix et Rhizoma. High performance liquid chromatography can be used to construct chemical fingerprints of different batches of Gentianae Radix et Rhizoma. Constructing a component-target-disease network of Gentianae Radix et Rhizoma for its anti-inflammatory activity was applied to screen potential anti-inflammatory components and related targets of Gentianae Radix et Rhizoma, and to verify the target of Gentianae Radix et Rhizoma by using biological evaluation methods. Detecting the biological potency of different batches of Gentianae Radix et Rhizoma extracts was used to inhibit COX-2 enzyme activity based the verifiable target cyclooxygenase-2(COX-2). The results showed that different batches of Gentianae Radix et Rhizoma accorded with the pharmacopoeia testing regulations, and the chemical fingerprints have a high similarity(similarity>0.93), suggesting that there is no significant difference in the characteristics of the chemical components. Based on network pharmacology predictions, 18 candidate targets were found to have potential direct interactions with the ingredients in Gentianae Radix et Rhizoma. Among them, the most important target is COX-2. Based on the experimental verification of recombinant human COX-2 protease activity inhibition, Gentianae Radix et Rhizoma can inhibit the COX-2 enzyme activity in a dose-dependent manner. It can function with a low concentration(0.75 mg·mL~(-1)), which preliminarily confirmed the accuracy of network pharmacology prediction. The biological potency detection method of Gentianae Radix et Rhizoma based on COX-2 inhibitory activity was optimized and established. The qualitative response parallel line method was used to calculate the biological potency of anti-inflammatory activity, which ranged from 23.04 to 46.60 U·mg~(-1). For network pharmacology prediction, it can screen and clarify the possible targets of traditional Chinese medicine rapidly, which can guide the establishment of a biological evaluation method for the quality of medicinal materials with related activities. Compared with chemical fingerprints, the biological potency testing can better detect quality fluctuations of traditional Chinese medicine.

Research on Biological Detection Based on Reflected Light Images of a Porous Silicon Bragg Mirror

In this paper, based on the gray value change of reflected light image of porous silicon (PSi) Bragg mirror, a fast and simple biological detection method is proposed. In this method, CdSe/ZnS quantum dots (QDs) are used as markers for refractive index amplification, and digital image method is used for detection. The detection light has the same wavelength as the lowest reflectivity of the edge of the Bragg mirror, and the reflected light radiated on the surface of the Bragg mirror is received by the detector. The theoretical simulation results show that the intensity of the reflected light increases with the increase of the refractive index caused by the biological reaction. According to the experimental results, the average gray value variation increases with the increase of the target DNA concentration and becomes a linear relationship in a certain range. Based on this method, the DNA detection limit is 20.74 pM. The method is low-cost, has a short detection time and can be used in the detection of biosensor microarray.

Review of NEDDylation inhibition activity detection methods

NEDDylation is a post-translational modification of a protein, which transfers Ubiquitin like protein NEDD8 (Neuronal Precursor Cell-expressed Developmentally Down-regulated Protein 8) to the lysine residue of the product through a three-stage enzymatic reaction, and widely regulates many biological processes, such as cell cycle signal transduction and immune recognition. In the past ten years, we have witnessed tremendous progress in the study of protein ubiquitination modification, from modification mechanisms to drug development. Which suggests that inhibition of NEDDylation is an effective way to inhibit tumor. A variety of biological detection methods have been developed during the development of the inhibitor. In this review, we briefly introduced the modification process and substrates of NEDDylation, and discussed detection methods of NEDDylation activity in detail. This review will provide an up-to-date and comprehensive review of the methods for detecting NEDDylation activity that will contribute to NEDDylation inhibitor development.

First identification of Anaplasma phagocytophilum in both a biting tick Ixodes nipponensis and a patient in Korea: a case report

BackgroundHuman granulocytic anaplasmosis (HGA) is a tick-borne infectious disease caused by Anaplasma phagocytophilum. To date, there have been no reported cases of A. phagocytophilum infection found in both the biting tick and the patient following a tick bite.Case presentationAn 81-year-old woman presented with fever following a tick bite, with the tick still intact on her body. The patient was diagnosed with HGA. The tick was identified as Ixodes nipponensis by morphological and molecular biological detection methods targeting the 16S rRNA gene. The patient’s blood was cultured after inoculation into the human promyelocytic leukemia cell line HL-60. A. phagocytophilum growth was confirmed via culture and isolation. A. phagocytophilum was identified in both the tick and the patient’s blood by Anaplasma-specific groEL- and ankA-based nested polymerase chain reaction followed by sequencing. Moreover, a four-fold elevation in antibodies was observed in the patient’s blood.ConclusionWe report a case of a patient diagnosed with HGA following admission for fever due to a tick bite. A. phagocytophilum was identified in both the tick and the patient, and A. phagocytophilum was successfully cultured. The present study suggests the need to investigate the possible incrimination of I. nipponensis as a vector for HGA in Korea.

Biological functions of circRNAs and their progress in livestock and poultry.

CircRNAs are a new member of endogenous RNAs, which characterized by a closed-loop structure without terminated 5' caps and 3' tails via covalent bonding, which are evolutionarily conserved among different species and often exhibit tissue or developmental stage-specific expression. So far, thousands of circRNAs have been discovered in eukaryotic cells. CircRNAs are more stable due to its resistance to exonuclease implying important biological functions in all kingdoms of life. They could function as miRNA sponges, interfere with splicing and bind to protein to regulate the expression of host genes and so on. In addition, emerging evidence suggests that circRNAs are closely related to a series of physiological processes in livestock and poultry. In this review, we summarized the biogenesis mechanism, major biological function and detection methods and focused on research advance of circRNAs in livestock and poultry, aiming at providing certain reference value and novel techniques for the development of new molecular genetic markers and breeding of livestock and poultry. Meanwhile, we hope this review could show significant prospect for other researches.

Antarctic Adélie penguin feathers as bio-indicators of geographic and temporal variations in heavy metal concentrations in their habitats

Seabird feathers are effective indicators of heavy metal contamination. To propose a biological detection method suitable for the long-term monitoring of heavy metals in the Antarctic environment, eight heavy metals (As, Cd, Cr, Cu, Hg, Ni, Pb, and Zn) in Adélie penguin feathers from different body parts, years, and molting grounds in the Antarctic were investigated in this study. It is found that the concentrations of heavy metals in the feathers showed differences among the body part of origin. The levels of the eight elements in the abdomen and back feathers were approximately the same, but some elements of them were significantly higher than those in the tail feathers. The Hg and Cd concentrations in the abdomen and back feathers increased from root to tip, regardless of the sampling year or molting ground. Based on a structural analysis of the feathers, Hg and Cd were mainly distributed in the feather vane rather than the shaft. The methylmercury content accounted for 83%–97% of the total mercury content in all feather samples, regardless of location, year, or body part, thereby supporting the assumption that mercury entering the feather was predominantly methylated and indicating that a relatively toxic form of Hg is excreted through the feathers. The heavy metals in feathers from the same molting ground showed significant differences over the sampling years. The average concentrations of As, Cr, Cu, Ni, and Zn increased significantly from 2015 to 2019, whereas those of Cd, Hg, and Pb tended to decrease. The concentrations of the eight elements in Adélie feathers from three molting grounds varied greatly, possibly in relation to their living areas and dietary structures. Feathers from the Ross Sea site had the highest Cd, Cu, Hg, Ni, and Zn concentrations, the highest As and Cr concentrations were detected in feathers from the Zhongshan site, and feathers from the Great Wall site showed higher Pb concentrations than those from the other two sites. This study established a long-term, nondestructive, and efficient method for monitoring heavy metal contamination in the polar environment in the future.

Biological Toxicity Effects of Soil Pollution Caused by Galvanized Wastewater Based on Vibrio Qinghaiensis sp.-Q67.

Objective To establish a rapid diagnosis method for the biological toxicity of soil, accurately and rapidly evaluate the toxicity of contaminated sites and identify the dominant pollutants. Methods Take the soil pollution of a galvanized factory as an example, while the metal concentration level was analyzed and detected, a rapid biological toxicity detection method based on the acute toxicity test of luminescent bacteria （Vibrio qinghaiensis sp.-Q67） was established, and the dominant pollutants were identified by stepwise multiple regression. Results The pollutants came from wastewater and metal plating fragments directly discharged from the manufacturing line of the factory. The concentration of those pollutants was correlated with the acute toxicity of Vibrio qinghaiensis sp.-Q67. The dominant pollutants in the study were zinc （Zn）, aluminum （Al） and copper （Cu）. Conclusion The luminescent bacteria toxicity test method based on Vibrio qinghaiensis sp.-Q67 can conveniently and rapidly assess the degree of toxic damage of polluted soil and identify the dominant pollutants and can be applied to the acute toxicity evaluation of polluted soil.

Detection using a quantum dots/porous silicon optical biosensor based on digital fluorescence images

As a kind of nanostructured material, porous silicon (PSi) has many advantages, including a large specific surface area, easy surface modification, easy biological modification and so on, and it can be used to prepare biosensors with excellent performance. Based on the digital imaging of PSi surface fluorescence, a new low-cost biological detection method is proposed in this paper. Probe DNA that was coupled with semiconductor colloid CdSe/ZnS quantum dots (QDs) interacted with target DNA immobilized on the inner wall of modified PSi. The quantum dots in the reactants were excited in the PSi holes with a Bragg structure to produce fluorescence, which was enhanced by a Bragg mirror. The fluorescence image of the PSi surface was obtained by digital microscopy, and the target DNA was detected by calculating the average gray value of the image. The detection limit of this method for DNA detection is 88 pm, showing that the method can realize low-cost, fast and convenient biological detection.

Expression and antibody preparation of recombinant truncated glycoprotein of Guertu virus

Objective To express truncated glycoprotein (Gn, Gn1, Gn2, Gn3, Gc1 and Gc2) of Guertu virus (GTV) in Escherichia coli (E.coli) cells, and prepare polyclonal antibodies against recombinant proteins Gn-His, Gc1-His and Gc2-His after purification. Methods Gene fragments encoding Gn, Gn1, Gn2, Gn3, Gc1 and Gc2 of GTV DXM strain were amplified by RT-PCR, and cloned into the prokaryotic expression vector pET-32a (+ ) to construct recombinant expression plasmids. The transformed E. coli BL21(DE3) strains carrying expression plasmids were induced by IPTG to express target proteins, which were identified by SDS-PAGE. Recombinant proteins Gn-His, Gc1-His and Gc2-His were purified by nickel affinity chromatography and detected by Western blot using GTV-positive sheep serum for analysis of their antigenicity. New Zealand white rabbits were immunized with the purified recombinant proteins. The titers and specificity of serum antibodies were analyzed by ELISA. Meanwhile, eukaryotic expression vectors pcDNA3.1-Gn, pcDNA3.1-Gc1/Gc2 were constructed and transfected into mammalian Vero cells to evaluate the binding activity of rabbit polyclonal antibodies by indirect immunofluorescence method. The specific reactivity of serum antibodies to recombinant proteins was detected by Western blot. Results Restriction enzyme analysis and DNA sequencing confirmed that the recombinant expression vectors of pET-32a-Gn, pET-32a-Gn1/Gn2/Gn3, pET-32a-Gc1/Gc2, pcDNA3.1-Gn and pcDNA3.1-Gc1/Gc2 were constructed successfully. The relative molecular mass (Mr) of the expressed recombinant proteins Gn-His, Gn1/Gn2/Gn3-His, Gc1/Gc2-His were approximately 63.4×103, 37.1×103, 31.9×103, 30.8×103, 40×103 and 54.4×103, respectively. The recombinant proteins could be recognized by GTV-positive sheep serum. The titers of polyclonal antibodies against GTV Gn, Gc1 and Gc2 were 1∶409 600, 1∶204 800 and 1∶6 400, respectively. Indirect immunofluorescence assay and Western blot showed that the prepared rabbit polyclonal antibodies could specifically react with the proteins expressed in eukaryotic cells and the recombinant proteins. Conclusions The recombinant GTV glycoproteins Gn-His and Gc1/Gc2-His were efficiently expressed and purified and characterized with good immunity. The prepared polyclonal antibodies had high titers and good specificity. This study provided reference for further studying the biological function and detection methods of GTV glycoproteins and research on vaccines. Key words: Guertu Virus; Glycoprotein; Prokaryotic expression; Eukaryotic expression; Rabbit polyclonal antibody

Research advances in animal models of cow′s milk protein allergy

Cow′s milk protein allergy(CMPA)is the most common food allergy among infants and young children, which has an increasing incidence in recent years.Performing suitable animal experiments is a key to study CMPA, but currently there aren′t proper models consistent with human physiological sensitization process.Ideal animal models can serve as critical tools to reveal the mechanisms of CMPA and develop preventive and therapeutic approaches.Animal species and strains, different types of the models, biological detection methods and evaluation are reviewed in this paper.It also summarizes characteristics of animal models, which will provide a reference for basic and applied researches of CMPA. Key words: Cow′s milk protein allergy; Animal models

2D Graphical Representation of DNA Sequences Based on Variant Map

Mining and analyzing the information and structure of DNA sequences is an important method of exploring and explaining the mysteries of biology. In addition to traditional biological detection methods for DNA sequences, technological advances have allowed researchers to utilize computers to analyze DNA sequences, such as in DNA visualization. However, DNA visualization techniques have disadvantages, such as difficult operation and complex calculation. Therefore, in this paper, we propose an algorithm called VARCH based on a variant map that is a part of variant logic construction. VARCH is a high-efficiency and concise visualization algorithm for DNA sequences. The principle of this approach is that the number of base combinations in each DNA subsequence is counted, and a matrix is constructed based on the full arrangement of different base combinations. Finally, the matrix is mapped to the 2D coordinate plane, obtaining the image of DNA sequences. The experimental results demonstrate that VARCH can effectively display features of DNA sequences.

Effect of Moxibustion on Intestinal Microbiome in Acute Gastric Ulcer Rats.

In Traditional Chinese Medicine (TCM), moxibustion had been used for thousands of years. Many clinical case reports and scientific studies had proved that moxibustion had a good effect in treating acute gastric ulcer (AGU). Some studies had shown that the relative content and species of bacteria in the intestinal would be changed when gastric mucosal injury happened. However, there was little research on the effect of intestinal microbiome with AGU rats that were treating by moxibustion. This study is aimed at analyzing the effect of fecal microbiome in rats with AGU by the 16S rDNA sequencing technology. Male SD rats were established by orally feeding once with 70% ethanol at 4 ml/kg except the control group, then treated by moxibustion in the stomach meridian group (“Liangmen,” “Zusanli”) and the gallbladder meridian group (“Riyue,” “Yanglingquan”) for 5 days. The 16S rDNA sequencing technology analysis of feces combined with histopathological methods and molecular biological detection methods was used to evaluate the therapeutic mechanism of moxibustion on AGU. AGU brought cause changes in the number and species of intestinal bacteria. Moxibustion on stomach meridian group could reduce the area of gastric mucosal injury and regulate the relative content of GAS and EGF. Moreover, moxibustion on the stomach meridian group could increase the relative content and species of beneficial bacteria in the intestine of rats with AGU. The relative abundance of intestinal probiotics was significantly upregulated in Alphaproteobacteria, Actinomycetales, and Bacillales. In addition, moxibustion might promote the repair of gastric mucosal injury by increasing the number and species of beneficial bacteria in the intestine.

Nuclear magnetic resonance biosensor based on streptavidin–biotin system and poly-l-lysine macromolecular targeted gadolinium probe for rapid detection of Salmonella in milk

An accurate biosensor for rapid detection of Salmonella via low-field nuclear magnetic resonance (NMR) technology was developed. First, poly-l-lysine (PLL) containing several amino groups was modified with diethylene triamine pentacetate acid (DTPA) and gadolinium (Gd). The antibody was bound to Gd–DTPA–PLL through streptavidin and biotin interaction to obtain the probes. After the capture of Salmonella using the probes, the mixture was filtered to separate the excess probes. Finally, the filtrate was collected and measured by NMR. This method was successfully used in the simultaneous detection of Salmonella in pure culture and milk. The detection sensitivity was 2.4 × 104 cfu mL−1, and the overall detection process could be finished in 2 h. Because of the remarkable specificity of the probe and strong anti-interference, it was suitable for the direct detection of the complex matrix. The NMR biosensor has great potential to become a very promising biological molecular detection method.

Cyclospora cayetanensis infection in humans: biological characteristics, clinical features, epidemiology, detection method and treatment.

Cyclospora cayetanensis, a coccidian parasite that causes protracted and relapsing gastroenteritis, has a short recorded history. At least 54 countries have documented C. cayetanensis infections and 13 of them have recorded cyclosporiasis outbreaks. Cyclospora cayetanensis infections are commonly reported in developing countries with low-socioeconomic levels or in endemic areas, although large outbreaks have also been documented in developed countries. The overall C. cayetanensis prevalence in humans worldwide is 3.55%. Among susceptible populations, the highest prevalence has been documented in immunocompetent individuals with diarrhea. Infections are markedly seasonal, occurring in the rainy season or summer. Cyclospora cayetanensis or Cyclospora-like organisms have also been detected in food, water, soil and some other animals. Detection methods based on oocyst morphology, staining and molecular testing have been developed. Treatment with trimethoprim-sulfamethoxazole (TMP-SMX) effectively cures C. cayetanensis infection, whereas ciprofloxacin is less effective than TMP-SMX, but is suitable for patients who cannot tolerate co-trimoxazole. Here, we review the biological characteristics, clinical features, epidemiology, detection methods and treatment of C. cayetanensis in humans, and assess some risk factors for infection with this pathogen.

Graft virus infection following corneal transplantation

Graft rejection and endothelial cell loss were believed to be the main reasons for graft edema following keratoplasty. With the application of various molecular biological detection methods in ophthalmology, virus infection has emerged and become an important contributing factor for graft failure. This review focused on the etiology, clinical manifestation and diagnosis methods for virus infection following keratoplasty, and discussed possible prevention and treatment.(Chin J Ophthalmol, 2019, 55: 713-716).

Analysis and inspection techniques for mouse liver injury based on terahertz spectroscopy.

At present, researchers are exploring biological tissue detection method using terahertz techniques. In this paper, techniques to inspect mouse liver injury by using terahertz spectroscopy were studied. The boxplots were applied to remove abnormal data, and the maximal information coefficient was employed to select crucial features from the absorption coefficient and refractive index spectra. Random Forests and AdaBoost were applied to recognize different levels of liver injury. We found that AdaBoost had better performance on low-level injury classification. This work suggests that terahertz techniques have the potential to detect liver injury at an early stage and evaluate liver treatment strategies.

Ultrasensitive aptamer fluorometric detection of IFN-γ by dual atom transfer radical polymerization amplification

Exploiting biological detection methods with high selectivity and sensitivity has been an urgent demand for low abundance biomolecules. Herein, an ultrasensitive aptamer fluorometric method of protein detection was fabricated via a sequential dual atom transfer radical polymerization (ATRP). For the first time, surface-initiated dual ATRP was utilized to make the fluorometric signal amplification for gamma-interferon (IFN-γ) protein assay. Two aptamers are used as protein probes, which can form a high stable and selective “aptamer/ protein/aptamer” sandwich structure. One aptamer probe is attached on superparamagnetic nanoparticle via surface self-assembled, the other one is linked with initiators of dual ATRP amplification by click chemistry formed triazole group. With large numbers of fluorescein-o-acrylate grown directly on a nanoparticle surface, a convenient “aptamer/protein/aptamer-polymer” supersandwich sensor was fabricated. Under optimal conditions, IFN-γ can be assayed with detection limit down to 0.178 fM in PBS buffer (equals to 1.072 ˣ103 IFN-γ molecules). Even in complex human serum, IFN-γ can also be detected with the limit of 1.54 fM, equals to 9.275 ˣ103 IFN-γ molecules. Results show a great potential in real ultrasensitive protein detection for its high selectivity, efficiency and stability. Besides, this proposed sensing method, which combines the advantages of superparamagnetic nanomaterial and chemically catalyzed surface-initiated dual ATRP polymerization, compared to the traditional signal amplify methods, is novel, more convenient, simpler and enzyme-free cascade amplification.

Use of biological detection methods to assess dioxin-like compounds in sediments of Bohai Bay, China

Bohai Bay, in the western region of northeastern China's Bohai Sea, receives water from large rivers containing various pollutants including dioxin-like compounds (DLCs). This study used the established zebrafish (Danio rerio) model, its known developmental toxicity endpoints and sensitive molecular analyses to evaluate sediments near and around an industrial effluent site in Bohai Bay. The primary objective was to assess the efficacy of rapid biological detection methods as an addition to chemical analyses. Embryos were exposed to various concentrations of sediment extracts as well as a 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) positive control. Exposure to sediment extract nearest the discharge site (P1) resulted in the most severe- and highest rates of change in embryos and larvae, suggesting that DLC contaminated sediment probably did not occur much beyond it. P1 extract resulted in concentration dependent increases in mortality and pericardial edema. Its highest concentration caused up-regulation of P-450 (CYP)-1A1(CYP1A) mRNA expression at 72 h post fertilization (hpf), an increase in its expression in gill arches as observed by whole mount in situ hybridization, and an increased signal in the Tg(cyp1a: mCherry) transgenic line. The pattern and magnitude of response was very similar to that of TCDD and supported the presence of DLCs in these sediment samples. Follow-up chemical analysis confirmed this presence and identified H7CDF, O8CDF and O8CDD as the main components in P1 extract. This study validates the use of biological assays as a rapid, sensitive, and cost-effective method to evaluate DLCs and their effects in sediment samples. Additionally, it provides support for the conclusion that DLCs have limited remobilization capacity in marine sediments.

Integration of a Gold-Specific Whole E. coli Cell Sensing and Adsorption Based on BioBrick.

Detection and recovery of heavy metals from environmental sources is a major task in environmental protection and governance. Based on previous research into cell-based visual detection and biological adsorption, we have developed a novel system combining these two functions by the BioBrick technique. The gold-specific sensory gol regulon was assembled on the gold-chaperone GolB (Gold-specific binding protein), which is responsible for selectively absorbing gold ions, and this led to an integration system with increased probe tolerance for gold. After being incorporated into E. coli, this system featured high-selective detection and recycling of gold ions among multi-metal ions from the environment. It serves as an efficient method for biological detection and recovery of various heavy metals. We have developed modular methods for cell-based detection and adsorption of heavy metals, and these offer a quick and convenient tool for development in this area.

Detecting reactive oxygen species in biological fluids by platinum nanoelectrode applying amperometric method

Reactive oxygen species (ROS) are vital metabolites in numerous biological functions. Disorders of cellular mechanisms can cause overproduction of ROS and, subsequently, oxidative damage to DNA, proteins, cells and tissues, which is associated with the pathogenesis of a number of neurodegenerative and inflammatory diseases. Development of highly sensitive, relatively simple and fast-to-implement innovative methods to detect oxidative stress requires understanding of how such disorders relate to the level of ROS. This research aimed to apply the biological fluids' ROS detection method we have developed (using the stable platinum nanoelectrode that allows assessing the level of hydrogen peroxide (H2O2) down to 1 μM) and determine the level of H2O2 in lacrimal and intraocular fluids of rabbits, as well as to investigate how the level of H2O2 changes under the influence of antioxidant therapy. The effect superoxide dismutase (SOD) nanoparticles produce on biological fluids' ROS level was shown. The level of H2O2 in lacrimal fluid increased 10 and 30 min after instillation of SOD nanoparticles. As for the intraocular fluid, H2O2 concentration starts to grow only 30 min after instillation of SOD nanoparticles, which suggests that the they penetrate the internal structures of the eye gradually. The method seems to be of value in the context of eye diseases diagnosing and treatment.