Increased serum extrachromosomal circular DNA SORBS1circle level is associated with insulin resistance in patients with newly diagnosed type 2 diabetes mellitus.

BACKGROUND: Extrachromosomal circular DNAs (eccDNAs) exist in human blood and somatic cells, and are essential for oncogene plasticity and drug resistance. However, the presence and impact of eccDNAs in type 2 diabetes mellitus (T2DM) remains inadequately understood. METHODS: We purified and sequenced the serum eccDNAs obtained from newly diagnosed T2DM patients and normal control (NC) subjects using Circle-sequencing. We validated the level of a novel circulating eccDNA named sorbin and SH3-domain- containing-1circle97206791-97208025 (SORBS1circle) in 106 newly diagnosed T2DM patients. The relationship between eccDNA SORBS1circle and clinical data was analyzed. Furthermore, we explored the source and expression level of eccDNA SORBS1circle in the high glucose and palmitate (HG/PA)-induced hepatocyte (HepG2 cell) insulin resistance model. RESULTS: A total of 22,543 and 19,195 eccDNAs were found in serum samples obtained from newly diagnosed T2DM patients and NC subjects, respectively. The T2DM patients had a greater distribution of eccDNA on chromosomes 1, 14, 16, 17, 18, 19, 20 and X. Additionally, 598 serum eccDNAs were found to be upregulated, while 856 eccDNAs were downregulated in T2DM patients compared with NC subjects. KEGG analysis demonstrated that the genes carried by eccDNAs were mainly associated with insulin resistance. Moreover, it was validated that the eccDNA SORBS1circle was significantly increased in serum of newly diagnosed T2DM patients (106 T2DM patients vs. 40 NC subjects). The serum eccDNA SORBS1circle content was positively correlated with the levels of glycosylated hemoglobin A1C (HbA1C) and homeostasis model assessment of insulin resistance (HOMA-IR) in T2DM patients. Intracellular eccDNA SORBS1circle expression was significantly enhanced in the high glucose and palmitate (HG/PA)-induced hepatocyte (HepG2 cell) insulin resistance model. Moreover, the upregulation of eccDNA SORBS1circle in the HG/PA-treated HepG2 cells was dependent on generation of apoptotic DNA fragmentation. CONCLUSIONS: These results provide a preliminary understanding of the circulating eccDNA patterns at the early stage of T2DM and suggest that eccDNA SORBS1circle may be involved in the development of insulin resistance.

Intrathecal Anesthesia Prevents Ventricular Arrhythmias in Rats with Myocardial Ischemia/Reperfusion.

INTRODUCTION: Ventricular arrhythmia is commonly provoked by acute cardiac ischemia through sympathetic exaggeration and is often resistant to anti-arrhythmic therapies. Thoracic epidural anesthesia has been reported to terminate fatal ventricular arrhythmia; however, its underlying mechanism is unknown. METHODS: Rats were randomly divided into four groups: sham, sham plus bupivacaine, ischemia/reperfusion (IR), and IR plus bupivacaine groups. Bupivacaine (1 mg/mL, 0.05 mL/100 g body weight) was injected intrathecally into the L5-L6 intervertebral space prior to establishing a myocardial IR rat model. Thereafter, cardiac arrhythmia, cardiac function, myocardial injury, and electrical activities of the heart and spinal cord were evaluated. RESULTS: Intrathecal bupivacaine inhibited spinal neural activity, improved heart rate variability, reduced ventricular arrhythmia score, and ameliorated cardiac dysfunction in IR rats. Furthermore, intrathecal bupivacaine attenuated cardiac injury and myocardial apoptosis and regulated cardiomyocyte autophagy and connexin-43 distribution during myocardial IR. CONCLUSION: Our results indicate that intrathecal bupivacaine blunts spinal neural activity to prevent cardiac arrhythmia and dysfunction induced by IR and that this anti-arrhythmic activity may be associated with regulation of autonomic balance, myocardial apoptosis and autophagy, and cardiac gap junction function.

Detection of Listeria monocytogenes in Food Using the Proofman-LMTIA Assay.

Microbial factors, including bacteria, viruses, and other pathogens, are significant contributors to foodborne illnesses, posing serious food safety risks due to their potential for rapid growth and contamination. Listeria monocytogenes is one of the most common types of foodborne bacteria that can cause serious foodborne diseases or even fatalities. In this study, a novel nucleic acid amplification method called Proofman-LMTIA was employed to detect Listeria monocytogenes contamination in food. This method combines proofreading enzyme-mediated probe cleavage with ladder-shape melting temperature isothermal amplification. A positive recombinant plasmid was used as a control to ensure the accuracy of the detection results, and primers and Proofman probes were specifically designed for the LMTIA. Genomic DNA was extracted, the reaction temperature was optimized, and the primers' specificity was verified using foodborne pathogens like Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella. The sensitivity was assessed by testing serial dilutions of genomic DNA, and the method's applicability was confirmed by detecting artificially contaminated fresh pork. The established LMTIA method exhibited both high specificity and sensitivity. At the optimal reaction temperature of 63 °C, the primers specifically identified Listeria monocytogenes contamination in pork at a concentration of 8.0 ± 0.7 colony-forming units (CFUs) per 25 g. Furthermore, the Proofman-LMTIA method was applied to test Listeria monocytogenes DNA in 30 food samples purchased from a Chinese retail market, and reassuringly, all results indicated no contamination. Proofman-LMTIA can serve as a reliable and rapid method for detecting Listeria monocytogenes in food, contributing to public health by safeguarding consumers from foodborne illnesses, and strengthening food safety regulations.

Evaluation of Steaming and Drying of Black Sesame Seeds for Nine Cycles Using Grey-Correlation Analysis Based on Variation-Coefficient Weight.

This study aimed to improve the steaming process of black sesame seeds. A comprehensive evaluation was conducted using the grey-correlation method based on the variation-coefficient weight to observe the treatments of normal-pressure (NPS) and high-pressure (HPS) steaming (with/without soaking in water) for nine cycles. Their effects on the contents of water, protein, fat, ash, melanin, sesamin, and sesamolin of black sesame seeds, as well as the sensory score of the black sesame pill, were determined. We found that with varied steaming methods and increased steaming cycles, the contents of the nutritional and functional components of black sesame seeds and the sensory score of the black sesame pill differed. The results of the variation-coefficient method showed that water, protein, fat, ash, melanin, sesamin, sesamolin, and sensory score had different effects on the quality of black sesame seeds with weighting factors of 34.4%, 5.3%, 12.5%, 11.3%, 13.9%, 11.3%, 7.8%, and 3.5%, respectively. The results of two-factor analysis of variance without repeated observations indicated that the grey-correlation degree of HPS was the largest among the different steaming treatments, and the following sequence was HPS after soaking in water (SNPS), NPS, and SNPS. There was no significant difference between NPS and SNPS (p < 0.05). Moreover, with increased cycles, the value of the grey-correlation degree increased. The comprehensive score of the procedure repeated nine times was significantly higher than other cycles (p < 0.05). The results of the grey-correlation degree and grade analysis showed that the best steaming process of black sesame seeds was HPS for nine cycles, followed by HPS for eight cycles and NPS after soaking in water (SNPS) for nine cycles. These findings could provide a scientific basis for replacing SNPS with HPS to simplify steaming and realize the parametric steaming of black sesame seeds, and thus, ensure the quality of black-sesame products.

Micro-Executor of Natural Products in Metabolic Diseases.

Obesity, diabetes, and cardiovascular diseases are the major chronic metabolic diseases that threaten human health. In order to combat these epidemics, there remains a desperate need for effective, safe, and easily available therapeutic strategies. Recently, the development of natural product research has provided new methods and options for these diseases. Numerous studies have demonstrated that microRNAs (miRNAs) are key regulators of metabolic diseases, and natural products can improve lipid and glucose metabolism disorders and cardiovascular diseases by regulating the expression of miRNAs. In this review, we present the recent advances involving the associations between miRNAs and natural products and the current evidence showing the positive effects of miRNAs for natural product treatment in metabolic diseases. We also encourage further research to address the relationship between miRNAs and natural products under physiological and pathological conditions, thus leading to stronger support for drug development from natural products in the future.

Rapid Discrimination of Panax quinquefolium and Panax ginseng Using the Proofman-Duplex-LMTIA Technique.

This study aims to establish a rapid identification method based on the Proofman-LMTIA technique for distinguishing between Panax quinquefolium and Panax ginseng. By targeting specific 18S rDNA sequences, suitable primers and Proofman probes labeled FAM or JOE were designed for LMTIA. Initially, single-species-primer Proofman-LMTIA assays were performed separately for each ginseng type to optimize reaction temperature, assess sensitivity and specificity, and determine the detection limit. Subsequently, both sets of primers and their corresponding probes were combined in the same reaction system to further optimize reaction conditions, evaluate sensitivity, and assess stability. Finally, the developed Proofman-duplex-LMTIA technique was employed to detect P. quinquefolium and P. ginseng slices available in the market. Single-plex Proofman-LMTIA assays revealed that the optimal reaction temperature for both P. quinquefolium and P. ginseng was 62 °C. The sensitivity was as low as 1 pg/µL, with a detection limit of 0.1%, and both showed excellent specificity. The optimal temperature for Proofman-duplex-LMTIA assays was 58 °C. This method could simultaneously identify P. quinquefolium and P. ginseng. Testing 6 samples of P. ginseng and 11 samples of P. quinquefolium from the market resulted in a 100% positive rate for all samples. This study successfully established a rapid, simple, sensitive, and specific Proofman-duplex-LMTIA identification method for P. quinquefolium and P. ginseng. It provides an effective means for quality control of P. quinquefolium, P. ginseng, and related products.

Detection of Soybean-Derived Components in Dairy Products Using Proofreading Enzyme-Mediated Probe Cleavage Coupled with Ladder-Shape Melting Temperature Isothermal Amplification (Proofman-LMTIA).

Food adulteration is a serious problem all over the world. Establishing an accurate, sensitive and fast detection method is an important part of identifying food adulteration. Herein, a sequence-specific ladder-shape melting temperature isothermal amplification (LMTIA) assay was reported to detect soybean-derived components using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. The results showed that, under the optimal temperature of 57 °C, the established Proofman-LMTIA method for the detection of soybean-derived components in dairy products was sensitive to 1 pg/µL, with strong specificity, and could distinguish soybean genes from those of beef, mutton, sunflower, corn, walnut, etc. The established Proofman-LMTIA detection method was applied to the detection of actual samples of cow milk and goat milk. The results showed that the method was accurate, stable and reliable, and the detection results were not affected by a complex matrix without false positives or false negatives. It was proved that the method could be used for the detection and identification of soybean-derived components in actual dairy products samples.

Impact of Ghrelin on Ventricular Arrhythmia and Related Mechanism After Myocardial Infarction.

INTRODUCTION: Ghrelin is an endogenous peptide with potential protective effects on ischemic heart. METHODS: Synthetic ghrelin was administered (100 µg·kg-1 subcutaneous injection, twice daily) for 4 weeks in a rat model of myocardial infarction (MI) with coronary artery occlusion. At the 5th week, electrocardiogram, monophasic action potentials and autonomic nerve function were evaluated. Cardiac tyrosine hydroxylase (TH) was determined by immunofluorescence staining. RESULTS: MI significantly increased sympathetic nerve activity (SNA) and ventricular arrhythmias, and prolonged APD dispersion and APD alternans (p < 0.01). Ghrelin treatment significantly increased ventricular fibrillation threshold (VFT), shortened APD dispersion and APD alternans, inhibited SNA and promoted vagus nerve activities (p < 0.01). Ghrelin also markedly reversed abnormal expression of TH in the peri-infarcted area of the heart (p < 0.01). DISCUSSION/CONCLUSION: Ghrelin provides a sustained electrophysiological protection by the increase of VFT and improvement of APD dispersion and APD alternans. The mechanism may be related to the regulation of autonomic nerve and sympathetic nerve remodeling. Thus, ghrelin represents a novel drug to prevent ventricular arrhythmia in ischemic heart disease.

Development of a Ladder-Shape Melting Temperature Isothermal Amplification (LMTIA) Assay for the Identification of Cassava Component in Sweet Potato Starch Noodles.

Food authenticity has become increasingly important as a result of food adulteration. To identify the authenticity of sweet potato starch noodles, the ladder-shape melting temperature isothermal amplification (LMTIA) method of determining cassava (Manihot esculenta Crantz) DNA in sweet potato starch noodles was used. A set of primers targeted at the internal transcription spacer (ITS) of cassava was designed, genomic DNA was extracted, the LMTIA reaction temperature was optimized, and the specificity of the primer was verified with the genomic DNAs of cassava, sweet potato (Ipomoea batatas L.), Solanum tuberosum L., Zea mays L., Vigna radiate L., Triticum aestivum L., and Glycine max (L.) Merr. The sensitivity with the serially diluted genomic DNA of cassava and the suitability for the DNA extracted from sweet potato starch adulterated with cassava starch were tested. The LMTIA assay for identifying the cassava component in sweet potato starch noodles was established. At the optimal temperature of 52 °C, the primers could specifically distinguish a 0.01% (w/w) cassava component added to sweet potato starch. Additionally, the LMTIA method was applied to the cassava DNA detection of 31 sweet potato starch noodle samples purchased from retail markets in China. Of these, 14 samples were positive. The LMTIA assay could be a reliable method for the rapid detection of cassava components in sweet potato starch noodles, to protect the rights of consumers and to regulate the sale market order of starch noodles.

Cardiorenal Protective Effect of Costunolide against Doxorubicin-Induced Toxicity in Rats by Modulating Oxidative Stress, Inflammation and Apoptosis.

Doxorubicin (DXB) is one of the most commonly used anticancer agents for treating solid and hematological malignancies; however, DXB-induced cardiorenal toxicity presents a limiting factor to its clinical usefulness in cancer patients. Costunolide (COST) is a naturally occurring sesquiterpene lactone with excellent anti-inflammatory, antioxidant and antiapoptotic properties. This study evaluated the effect of COST on DXB-induced cardiorenal toxicity in rats. Rats were orally treated with COST for 4 weeks and received weekly 5 mg/kg doses of DXB for three weeks. Cardiorenal biochemical biomarkers, lipid profile, oxidative stress, inflammatory cytokines, histological and immunohistochemical analyses were evaluated. DXB-treated rats displayed significantly increased levels of lipid profiles, markers of cardiorenal dysfunction (aspartate aminotransferase, creatine kinase, lactate dehydrogenase, troponin T, blood urea nitrogen, uric acid and creatinine). In addition, DXB markedly upregulated cardiorenal malondialdehyde, tumor necrosis factor-α, interleukin-1ß, interleukin-6 levels and decreased glutathione, superoxide dismutase and catalase activities. COST treatment significantly attenuated the aforementioned alterations induced by DXB. Furthermore, histopathological and immunohistochemical analyses revealed that COST ameliorated the histopathological features and reduced p53 and myeloperoxidase expression in the treated rats. These results suggest that COST exhibits cardiorenal protective effects against DXB-induced injury presumably via suppression of oxidative stress, inflammation and apoptosis.

Small-Molecule Integrated Stress Response Inhibitor Reduces Susceptibility to Postinfarct Atrial Fibrillation in Rats via the Inhibition of Integrated Stress Responses.

Phosphorylation of the eukaryotic translation initiation factor 2 α-subunit, which subsequently upregulates activating transcription factor 4 (ATF4), is the core event in the integrated stress response (ISR) pathway. Previous studies indicate phosphorylation of eukaryotic translation initiation factor 2 ɑ-subunit in atrial tissue in response to atrial fibrillation (AF). This study investigated the role of ISR pathway in experimental AF by using a small-molecule ISR inhibitor (ISRIB). Accordingly, rats were subjected to coronary artery occlusion to induce myocardial infarction (MI), or sham operation, and received either trans-ISRIB (2 mg/kg/d, i.p.) or vehicle for seven days. Thereafter, animals were subjected to the AF inducibility test by transesophageal rapid burst pacing followed by procurement of left atrium (LA) for assessment of atrial fibrosis, inflammatory indices, autophagy-related proteins, ISR activation, ion channel, and connexin 43 expression. Results showed a significant increase in the AF vulnerability and the activation of ISR in LA as evidenced by enhanced eukaryotic translation initiation factor 2 ɑ-subunit phosphorylation. ISRIB treatment suppressed upregulation of ATF4, fibrosis as indexed by determination of α-smooth muscle actin and collagen levels, inflammatory macrophage infiltration (i.e., CD68 and inducible nitric oxide synthase/CD68-positive macrophage), and autophagy as determined by expression of light chain 3. Further, ISRIB treatment reversed the expression of relevant ion channel (i.e., the voltage-gated sodium channel 1.5 , L-type voltage-dependent calcium channel 1.2, and voltage-activated A-type potassium ion channel 4.3) and connexin 43 remodeling. Collectively, the results suggest that the ISR is a key pathway in pathogenesis of AF, post-MI, and represents a novel target for treatment of AF. SIGNIFICANCE STATEMENT: The activation of integrated stress response (ISR) pathway as evidenced by enhanced eukaryotic translation initiation factor 2 ɑ-subunit phosphorylation in left atrium plays a key role in atrial fibrillation (AF). ISR inhibitor (ISRIB) reduces AF occurrence and atrial proarrhythmogenic substrate. The beneficial action of ISRIB may be mediated by suppressing ISR pathway-related cardiac fibrosis, inflammatory macrophage infiltration, autophagy, and restoring the expression of ion channel and connexin 43. This study suggests a key dysfunctional role for ISR in pathogenesis of AF with implications for novel treatment.

Recent development in friction of 2D materials: from mechanisms to applications.

Two-dimensional (2D) materials with a layered structure are excellent candidates in the field of lubrication due to their unique physical and chemical properties, including weak interlayer interaction and large specific surface area. For the last few decades, graphene has received lots of attention due to its excellent properties. Besides graphene, various new 2D materials (including MoS2, WS2, WSe2, NbSe2, NbTe2, ReS2, TaS2and h-BN etc.) are found to exhibit a low coefficient of friction at the macro- and even micro-scales, which may lead to widespread application in the field of lubrication and anti-wear. This article focuses on the latest development trend in 2D materials in the field of tribology. The review begins with a summary of widely accepted nano-scale friction mechanisms contain surface friction mechanism and interlayer friction mechanism. The following sections report the applications of 2D materials in lubrication and anti-wear as lubricant additives, solid lubricants, and composite lubricating materials. Finally, the research prospects of 2D materials in tribology are presented.

Long non-coding RNA AK085865 ablation confers susceptibility to viral myocarditis by regulating macrophage polarization.

Accumulating evidence indicates that regulators of macrophage polarization may exert pivotal functions in the development of coxsackievirus B3 (CVB3)-induced viral myocarditis (VM). However, the mechanisms underlying macrophage polarization remain to be explored. Here, we sought to identify novel and functionally important long non-coding RNAs (lncRNAs) during macrophage polarization and to investigate their function and contribution to VM. In this study, we identified the lncRNA AK085865 as an important regulator of macrophage polarization. Knock-down of AK085865 diminished phenotypical expression of M2 macrophages while promoting polarization to the M1 phenotype. Moreover, AK085865-/- mice had increased susceptibility to CVB3-induced VM. We observed striking bias towards M1 macrophages, whereas the M2 population was decreased in AK085865-/- VM mice. Collectively, our findings uncover a critical role of AK085865 in the regulation of macrophage polarization in vitro and in vivo, identifying a new player in the development of VM and providing a potential clinically significant therapeutic target.

Inspection of disbonds in titanium alloy honeycomb sandwich structures with different skin thicknesses using linear frequency-modulated thermography.

This paper reports the linear frequency-modulated thermography inspection of disbonds in titanium alloy honeycomb sandwich structures with different skin thicknesses. A three-dimensional finite element model of a titanium alloy honeycomb sandwich structure is built. The maximum value of the phase difference between the disbond defect region and the nondefective region is used to optimize the heating duration and frequency bandwidth. Three titanium alloy honeycomb sandwich structure specimens, with a skin thickness of 0.6 mm, 0.85 mm, and 1.2 mm, respectively, are manufactured, in which skin-to-core disbond defects are prepared. The linear frequency-modulated thermography experiments are carried out on the specimens. The correlation algorithm is used to process the infrared image sequences. The experimental results show that linear frequency-modulated thermography can realize the fast and efficient inspection of the disbonds in titanium alloy honeycomb sandwich structures with different skin thicknesses. For skin thickness ranges from 0.6 mm to 1.2 mm, a heating duration of 22 s and a frequency range of 0.01 Hz-0.21 Hz are recommended.

Motor unit number index detects the effectiveness of surgical treatment in improving distal motor neuron loss in patients with incomplete cervical spinal cord injury.

BACKGROUND: Recovery of motor dysfunction is important for patients with incomplete cervical spinal cord injury (SCI). To enhance the recovery of muscle strength, both research and treatments mainly focus on injury of upper motor neurons at the direct injury site. However, accumulating evidences have suggested that SCI has a downstream effect on the peripheral nervous system, which may contribute to the poor improvement of the muscle strength after operation. The aim of this study is to investigate the impact of early vs. delayed surgical intervention on the lower motor neurons (LMNs) distal to the injury site in patients with incomplete cervical SCI. METHODS: Motor unit number index (MUNIX) was performed on the tibialis anterior (TA), extensor digitorum brevis (EDB) and abductor hallucis (AH) in 47 patients with incomplete cervical SCI (early vs. delayed surgical-treatment: 17 vs. 30) and 34 healthy subjects approximately 12 months after operation. All patients were further assessed by American spinal injury association (ASIA) motor scales and Medical Research Council (MRC) scales. RESULTS: There are no difference of both ASIA motor scores and MRC scales between the patients who accepted early and delayed surgical treatment (P > 0.05). In contrast, the patients undergoing early surgical treatment showed lower MUSIX values in both bilateral EDB and bilateral TA, along with greater MUNIX values in both right-side EDB and right-side TA, compared to the patients who accepted delayed surgical treatment (P < 0.05). CONCLUSIONS: Cervical SCI has a negative effect on the LMNs distal to the injury site. Early surgical intervention in Cervical SCI patients may improve the dysfunction of LMNs distal to the injury site, reducing secondary motor neuron loss, and eventually improving clinical prognosis.

Nanomechanical Properties of Ti3C2 Mxene.

MXene is a new type of two-dimensional (2D) material that finds wide applications; however, its adhesion and tribological properties have not yet been fully revealed. Here, the chemical structure of MXene was revealed by X-ray diffraction, and the adhesion and friction behaviors of MXene were studied with atomic force microscopy (AFM) Nanoman technology. It was found in AFM tests that MXene exhibited enhanced adhesion and friction at higher pressure but exhibited reduced adhesion and friction at elevated temperature, while the sliding speed and delay time of the AFM probe have negligible effects on the friction. X-ray photoelectron spectroscopy was used to reveal the change in the chemical structure of MXene at different temperatures. We found increased oxidation, and replacement of C by O atoms occurred on MXene at higher temperatures, which has a profound impact on the adhesion and friction between MXene and the probe tip. This discovery lays the foundation for understanding the adhesion mechanism and the dynamic adhesion behavior in friction of MXene, rendering MXene a much broader application prospect.

Detection of Cassava Component in Sweet Potato Noodles by Real-Time Loop-mediated Isothermal Amplification (Real-time LAMP) Method.

Sweet potato (Ipomoea batatas) noodles are a traditional Chinese food with a high nutritional value; however, starch adulteration is a big concern. The objective of this study was to develop a reliable method for the rapid detection of cassava (Manihot esculenta) components in sweet potato noodles to protect consumers from commercial adulteration. Five specific Loop-mediated Isothermal Amplification (LAMP) primers targeting the internal transcribed spacer (ITS) of cassava were designed, genomic DNA was extracted, the LAMP reaction system was optimized, and the specificity of the primers was verified with genomic DNA of cassava, Ipomoea batatas, Zea mays, and Solanum tuberosum; the detection limit was determined with a serial dilution of adulterated sweet potato starch with cassava starch, and the real-time LAMP method for the detection of the cassava-derived ingredient in sweet potato noodles was established. The results showed that the real-time LAMP method can accurately and specifically detect the cassava component in sweet potato noodles with a detection limit of 1%. Furthermore, the LAMP assay was validated using commercial sweet potato noodle samples, and results showed that 57.7% of sweet potato noodle products (30/52) from retail markets were adulterated with cassava starch in China. This study provides a promising solution for facilitating the surveillance of the commercial adulteration of sweet potato noodles from retail markets.

Analysis of pesticide residues in tobacco with online size exclusion chromatography with gas chromatography and tandem mass spectrometry.

An ultrasensitive method for the simultaneous analysis of pesticides residues in tobacco was developed with online size exclusion chromatography with gas chromatography and tandem mass spectrometry. Tobacco samples were extracted with the solvent mixture of cyclohexane and acetone (7:3, v/v) and centrifuged. Then, the supernatant liquors were injected directly into the online size exclusion chromatography with gas chromatography and tandem mass spectrometry without any other purification procedures after being filtered with a 0.22 µm organic phase filter. The matrix interferences were effectively removed and recoveries of most pesticides were in the range of 72-121%. Especially, for chlorothalonil, the analysis efficiency of this method was much more favorable than that of the general method, in which dispersive solid-phase extraction was used as an additional purified procedure. In addition, the limits of quantitation of this method were from 1 to 50 µg/kg. Therefore, a rapid, cost-effective, labor-saving method was proposed in the present work, which was suitable for the analysis of 41 pesticide residues in tobacco.

Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification.

The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assay.

A Comparison of In-House Real-Time LAMP Assays with a Commercial Assay for the Detection of Pathogenic Bacteria.

Molecular detection of bacterial pathogens based on LAMP methods is a faster and simpler approach than conventional culture methods. Although different LAMP-based methods for pathogenic bacterial detection are available, a systematic comparison of these different LAMP assays has not been performed. In this paper, we compared 12 in-house real-time LAMP assays with a commercialized kit (Isothermal Master Mix) for the detection of Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121, E. coli O145 and Streptococcus agalactiae. False-positive results were observed in all 12 in-house real-time LAMP assays, while all the negative controls of Isothermal Master Mix remained negative after amplification. The detection limit of Isothermal Master Mix for Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121 and Streptococcus agalactiae was 1 pg, whereas the sensitivity of the commercialized kit for E. coli O145 was 100 pg. In conclusion, the 12 in-house real-time LAMP assays were impractical to use, while the commercialized kit Isothermal Master Mix was useful for the detection of most bacterial pathogens.