A Thermostable Cas12b-Powered Bioassay Coupled with Loop-Mediated Isothermal Amplification in a Customized "One-Pot" Vessel for Visual, Rapid, Sensitive, and On-Site Detection of Genetically Modified Crops.

Genetically modified crops (GMCs) have been discussed due to unknown safety, and thus, it is imperative to develop an effective detection technology. CRISPR/Cas is deemed a burgeoning technology for nucleic acid detection. Herein, we developed a novel detection method for the first time, which combined thermostable Cas12b with loop-mediated isothermal amplification (LAMP), to detect genetically modified (GM) soybeans in a customized one-pot vessel. In our method, LAMP-specific primers were used to amplify the cauliflower mosaic virus 35S promoter (CaMV35S) of the GM soybean samples. The corresponding amplicons activated the trans-cleavage activity of Cas12b, which resulted in the change of fluorescence intensity. The proposed bioassay was capable of detecting synthetic plasmid DNA samples down to 10 copies/µL, and as few as 0.05% transgenic contents could be detected in less than 40 min. This work presented an original detection method for GMCs, which performed rapid, on-site, and deployable detection.

Determination of Dencichine in Panax notoginseng in the Forest and Field Using High Performance Liquid Chromatography.

Dencichine is a nonprotein amino acid, an effective ingredient in Panax notoginseng with hemostatic and anti-inflammatory effects. There are few studies on the effects of regions and cultivation models on the accumulation of dencichine. In the current study, the content of dencichine in P. notoginseng collected from its global cultivation and trading center Yunnan, China, (>640 samples) was determined using an optimized high-performance liquid chromatography method coupled with a diode array detector but without derivatization. The recovery rate of this method was 80-110%, the relative standard deviation was <10%, and the limits of detection and quantification were 0.003% (w/w) and 0.01% (w/w), respectively. The content of dencichine in each part of P. notoginseng was as follows: rootlets (39.59%) > main roots (29.91%) > leaves (16.21%) > stems (14.29%). For leaves, P. notoginseng in the forest (5.52 ± 2.26 mg/g) was significantly higher than that in the field (3.93 ± 1.72 mg/g) but opposite for main roots. The origins and altitudes made different contributions to the accumulation of dencichine in P. notoginseng. This study provides an effective analytical method to determine dencichines in various parts of P. notoginseng from different origins and altitudes and supports quality control and product development of P. notoginseng.

Evaluation and verification of the characteristic peptides for detection of Staphylococcus aureus in food by targeted LC-MS/MS.

The traditional methods for detection of Staphylococcus aureus in food have some shortcomings, such as fussy operation and dependence on separation of pure colony. A novel strategy for detection of pathogenic bacteria bases on targeted analysis of the characteristic peptides by LC-MS/MS. The key to the success of this strategy is to identify a combination of reliable characteristic peptides that afford high specificity. In this study, the candidate characteristic peptides of S. aureus were evaluated bioformatically and then verified experimentally. UniProt protein database were used to perform BLAST analysis on the candidate characteristic peptides, and then their specificity were compared via targeted analysis in S. aureus and other Staphylococcus strains by LC-MS/MS. Finally, suitable S. aureus characteristic peptide was determined, by using which 100 cfu/mL S. aureus in milk samples were successfully detected, meeting the detection limit requirement in actual application. The results demonstrated the feasibility of the detection strategy of S. aureus based on characteristic peptides. The exploratory work is expected to promote the methodology research of food-borne pathogen inspection and further improve the inspection efficiency and quality.

Rapid Detection of SARS-CoV-2 Virus Using Dual Reverse Transcriptional Colorimetric Loop-Mediated Isothermal Amplification.

The outbreak and pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a public health emergency of international concern. The rapid and accurate detection of the virus is a critical means to prevent and control the disease. Herein, we provide a novel, rapid, and simple approach, named dual reverse transcriptional colorimetric loop-mediated isothermal amplification (dRT-cLAMP) assay, to accelerate the detection of the SARS-CoV-2 virus without using expensive equipment. The result of this assay is shown by color change and is easily detected by the naked eye. To improve the detection accuracy, we included two primer sets that specifically target the viral orf1ab and N genes in the same reaction mixture. Our assay can detect the synthesized SARS-CoV-2 N and orf1ab genes at a low level of 100 copies/µL. Sequence alignment analysis of the two synthesized genes and those of 9968 published SARS-CoV-2 genomes and 17 genomes of other pathogens from the same infection site or similar symptoms as COVID-19 revealed that the primers for the dRT-cLAMP assay are highly specific. Our assay of 27 clinical samples of SARS-CoV-2 virus and 27 standard-added environmental simulation samples demonstrated that compared to the commercial kits, the consistency of the positive, negative, and probable clinical samples was 100, 92.31, and 44.44%, respectively. Moreover, our results showed that the positive, but not negative, standard-added samples displayed a naked-eye-detectable color change. Together, our results demonstrate that the dRT-cLAMP assay is a feasible detection assay for SARS-CoV-2 virus and is of great significance since rapid onsite detection of the virus is urgently needed at the ports of entry, health care centers, and for internationally traded goods.

[Progress in functions and mechanisms of immunosuppressive neutrophil subsets: An update].

Neutrophils are the most abundant circulating leukocytes in healthy adults, serving as the first-line defense against infections. In the last few years, scientists have reported neutrophils of immune suppressive properties, such as tumor-associated neutrophils (TAN) and low-density neutrophils (LDN). These neutrophils are found to be involved in many physiological and pathological conditions owing to their ability of regulating T cell functions and promoting angiogenesis and tumor growth through a number of mechanisms, including PD-1/PD-L1, antigen presentation, reactive oxygen species (ROS), arginase-1 (Arg1), inducible nitric oxide synthase (iNOS), etc. This review summarizes the recent advances in investigating the function and mechanism of immune suppressive neutrophils.

Recent advances in unraveling the molecular mechanisms and functions of HOXA11­AS in human cancers and other diseases (Review).

A large number of previously published research articles have demonstrated that the expression levels of long noncoding RNAs (lncRNAs) are generally dysregulated, either through overexpression or underexpression, in cancer and other types of disease. As a recently discovered lncRNA, HOXA11 antisense RNA (HOXA11­AS) is able to serve as an oncogenic or tumor­suppressor gene and serves a vital role in the processes of proliferation, invasion, and migration of cancer cells. HOXA11­AS appears to be a major factor contributing to epigenetic modification, and exerts transcriptional, post­transcriptional, translational and post­translational regulatory effects on genes through a variety of mechanisms; for example, by competing endogenous RNA (ceRNA) and a molecular scaffold mechanism. A number of reports have demonstrated that HOXA11­AS functions as a protein scaffold for polycomb repressive complex 2 (PRC2), lysine­specific histone demethylase 1 (LSD1) and DNA methyltransferase 1 (DNMT1) to perform epigenetic modifications on chromosomes in the nucleus. Furthermore, HOXA11­AS is also located in the cytoplasm and can act as a ceRNA, which sponges miRNAs. In addition, HOXA11­AS may be useful as a biomarker for the diagnosis and prognosis of cancer. In the present review article, the clinical value, phenotype and mechanism of HOXA11­AS in a variety of tumors types are briefly summarized, as well as its clinical value in certain additional diseases. The perspective of the authors is that HOXA11­AS may represent an effective tumor marker and therapeutic target for cancer diagnosis and therapy.

Identification of camel species in food products by a polymerase chain reaction-lateral flow immunoassay.

With an increased demand for camel meat, camel meat-related food products are susceptible to food fraud. To effectively authenticate camel-containing foods, a novel analytical technique based on polymerase chain reaction (PCR)-lateral flow immunoassay (LFI) was developed. The camel-specific PCR primers were designed to target at the mitochondrial COI gene. Both of the in silico and in vitro tests confirmed that the PCR-LFI was specific. A limit of detection of 0.1% w/w of camel meat in beef was achieved for both the raw and cooked (i.e. boiling and deep frying) meat samples. This novel method was used to authenticate 20 processed camel-meat products purchased from local grocery stores in China and online. Two products purchased online were identified as containing no camel meat. Overall, this novel PCR-LFI method is ideal for governmental laboratories to rapidly authenticate camel-meat containing food products.

Identification of donkey meat in foods using species-specific PCR combined with lateral flow immunoassay.

Food authenticity is a global issue and has raised increasing concerns in the past decades. DNA-based methods are more favourable than the conventional protein-based techniques and have been applied to species identification and meat fraud detection. To effectively identify donkey meat for meat product authentication, a highly specific and robust method that coupled polymerase chain reaction (PCR) with lateral flow immunoassay (LFI) was developed. Donkey-specific PCR primers were designed by targeting at the mitochondrial D-loop gene and the specificity was verified in silico and in vitro against 22 species involved in meat authentication. A limit of detection of 0.0013 ng µL-1 DNA extract was achieved and as low as 0.001% w/w (raw) and 0.01% w/w (cooked) donkey meat in beef were successfully detected using the developed PCR-LFI. LFI strip-based visualization of PCR products allowed for a 10-fold higher sensitivity than conventional gel electrophoresis and significantly reduced the analysis time for the post-PCR analysis. This PCR-LFI is highly suitable for rapid identification of donkey or incorporating into multiplex screening protocol for other meat authentication in the laboratories of both regulatory agencies and commercial services.

[Separation of proteins by gradient pressurized capillary electrochromatography].

The separation of four proteins including RNase A, cytochrome C, lysozyme and myoglobin was investigated by reversed-phase gradient pressurized capillary electrochromatography (p-CEC) with 1.5 microm non-porous silica C18 stationary phase. This mode was compared with micro-high performance liquid chromatography (mu-HPLC) and the effects of applied voltage, stationary phase and concentration of ion-pairing agent (trifluoroacetic acid, TFA) on the gradient p-CEC were also studied. This separation was performed rapidly on a new CEC instrument Trisep 2010 GV. The results showed that the retention mechanism of proteins in p-CEC mode is based on both chromatographic partitioning and electrophoretic migration. The results also demonstrated that p-CEC may have great potential for fast and efficient separation of proteins.