Oral Administration of Antimicrobial Peptide NZ2114 Through the Microalgal Bait Tetraselmis subcordiformis (Wille) Butcher for Improving the Immunity and Gut Health in Turbot (Scophthalmus maximus L.).

Antibiotics are widely used in aquaculture to treat the bacterial diseases. However, the improper use of antibiotics could lead to environmental pollution and development of resistance. As a safe and eco-friendly alternative, antimicrobial peptides (AMPs) are commonly explored as therapeutic agents. In this study, a mutant strain of Tetraselmis subcordiformis containing AMP NZ2114 was developed and used as an oral drug delivery system to reduce the use of antibiotics in turbot (Scophthalmus maximus) aquaculture. The gut, kidney, and liver immune-related genes and their effects on gut digestion and bacterial communities in turbot fed with NZ2114 were evaluated in an 11-day feeding experiment. The results showed that compared with the group fed with wild-type T. subcordiformis, the group fed with T. subcordiformis transformants containing NZ2114 was revealed with decreased levels of both pro-inflammatory factors (TNF-α and IL-1ß), inhibitory effect on Staphylococcus aureus, Vibrio parahaemolyticus, and Vibrio splendidus demonstrated by the in vitro simulation experiments, and increased richness and diversity of the gut microbiota of turbot. In conclusion, our study provided a novel, beneficial, and low-cost method for controlling bacteria in turbot culture through the oral drug delivery systems.

Expression Patterns and Molecular Mechanisms Regulating Drought Tolerance of Soybean [Glycine max (L.) Merr.] Conferred by Transcription Factor Gene GmNAC19.

NAC transcription factors are commonly involved in the plant response to drought stress. A transcriptome analysis of root samples of the soybean variety 'Jiyu47' under drought stress revealed the evidently up-regulated expression of GmNAC19, consistent with the expression pattern revealed by quantitative real-time PCR analysis. The overexpression of GmNAC19 enhanced drought tolerance in Saccharomyces cerevisiae INVSc1. The seed germination percentage and root growth of transgenic Arabidopsis thaliana were improved in comparison with those of the wild type, while the transgenic soybean composite line showed improved chlorophyll content. The altered contents of physiological and biochemical indices (i.e., soluble protein, soluble sugar, proline, and malondialdehyde) related to drought stress and the activities of three antioxidant enzymes (i.e., superoxide dismutase, peroxidase, and catalase) revealed enhanced drought tolerance in both transgenic Arabidopsis and soybean. The expressions of three genes (i.e., P5CS, OAT, and P5CR) involved in proline synthesis were decreased in the transgenic soybean hairy roots, while the expression of ProDH involved in the breakdown of proline was increased. This study revealed the molecular mechanisms underlying drought tolerance enhanced by GmNAC19 via regulation of the contents of soluble protein and soluble sugar and the activities of antioxidant enzymes, providing a candidate gene for the molecular breeding of drought-tolerant crop plants.

Metabolic engineering and synthetic biology strategies for producing high-value natural pigments in Microalgae.

Microalgae are microorganisms capable of producing bioactive compounds using photosynthesis. Microalgae contain a variety of high value-added natural pigments such as carotenoids, phycobilins, and chlorophylls. These pigments play an important role in many areas such as food, pharmaceuticals, and cosmetics. Natural pigments have a health value that is unmatched by synthetic pigments. However, the current commercial production of natural pigments from microalgae is not able to meet the growing market demand. The use of metabolic engineering and synthetic biological strategies to improve the production performance of microalgal cell factories is essential to promote the large-scale production of high-value pigments from microalgae. This paper reviews the health and economic values, the applications, and the synthesis pathways of microalgal pigments. Overall, this review aims to highlight the latest research progress in metabolic engineering and synthetic biology in constructing engineered strains of microalgae with high-value pigments and the application of CRISPR technology and multi-omics in this context. Finally, we conclude with a discussion on the bottlenecks and challenges of microalgal pigment production and their future development prospects.

Promoting Heme and Phycocyanin Biosynthesis in Synechocystis sp. PCC 6803 by Overexpression of Porphyrin Pathway Genes with Genetic Engineering.

Due to their unique biochemical and spectroscopic properties, both heme and phycocyanobilin are widely applied in the medical and food industries. Synechocystis sp. PCC 6803 contains both heme and phycocyanin, and is capable of synthesizing phycocyanin using heme as a precursor. The aim of this study was to uncover viable metabolic targets in the porphyrin pathway from Synechocystis sp. PCC 6803 to promote the accumulation of heme and phycocyanin in the recombinant strains of microalgae. A total of 10 genes related to heme synthesis pathway derived from Synechococcus elongatus PCC 7942 and 12 genes related to endogenous heme synthesis were individually overexpressed in strain PCC 6803. The growth rate and pigment content (heme, phycocyanin, chlorophyll a and carotenoids) of 22 recombinant algal strains were characterized. Quantitative real-time PCR technology was used to investigate the molecular mechanisms underlying the changes in physiological indicators in the recombinant algal strains. Among the 22 mutant strains, the mutant overexpressing the haemoglobin gene (glbN) of strain PCC 6803 had the highest heme content, which was 2.5 times higher than the wild type; the mutant overexpressing the gene of strain PCC 7942 (hemF) had the highest phycocyanin content, which was 4.57 times higher than the wild type. Overall, the results suggest that genes in the porphyrin pathway could significantly affect the heme and phycocyanin content in strain PCC 6803. Our study provides novel crucial targets for promoting the accumulation of heme and phycocyanin in cyanobacteria.

Characterizations of microRNAs involved in the molecular mechanisms underlying the therapeutic effects of noni (Morinda citrifolia L.) fruit juice on hyperuricemia in mice.

Background: Hyperuricemia is generally defined as the high level of serum uric acid and is well known as an important risk factor for the development of various medical disorders. However, the medicinal treatment of hyperuricemia is frequently associated with multiple side-effects. Methods: The therapeutic effect of noni (Morinda citrifolia L.) fruit juice on hyperuricemia and the underlying molecular mechanisms were investigated in mouse model of hyperuricemia induced by potassium oxonate using biochemical and high-throughput RNA sequencing analyses. Results: The levels of serum uric acid (UA) and xanthine oxidase (XOD) in mice treated with noni fruit juice were significantly decreased, suggesting that the noni fruit juice could alleviate hyperuricemia by inhibiting the XOD activity and reducing the level of serum UA. The contents of both serum creatinine and blood urine nitrogen of the noni fruit juice group were significantly lower than those of the model group, suggesting that noni fruit juice promoted the excretion of UA without causing deleterious effect on the renal functions in mice. The differentially expressed microRNAs involved in the pathogenesis of hyperuricemia in mice were identified by RNA sequencing with their target genes further annotated based on both Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases to explore the metabolic pathways and molecular mechanisms underlying the therapeutic effect on hyperuricemia by noni fruit juice. Conclusion: Our study provided strong experimental evidence to support the further investigations of the potential application of noni fruit juice in the treatment of hyperuricemia.

Molecular and biochemical investigations of the anti-fatigue effects of tea polyphenols and fruit extracts of Lycium ruthenicum Murr. on mice with exercise-induced fatigue.

Background: The molecular mechanisms regulating the therapeutic effects of plant-based ingredients on the exercise-induced fatigue (EIF) remain unclear. The therapeutic effects of both tea polyphenols (TP) and fruit extracts of Lycium ruthenicum (LR) on mouse model of EIF were investigated. Methods: The variations in the fatigue-related biochemical factors, i.e., lactate dehydrogenase (LDH), superoxide dismutase (SOD), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-2 (IL-2), and interleukin-6 (IL-6), in mouse models of EIF treated with TP and LR were determined. The microRNAs involved in the therapeutic effects of TP and LR on the treatment of mice with EIF were identified using the next-generation sequencing technology. Results: Our results revealed that both TP and LR showed evident anti-inflammatory effect and reduced oxidative stress. In comparison with the control groups, the contents of LDH, TNF-α, IL-6, IL-1ß, and IL-2 were significantly decreased and the contents of SOD were significantly increased in the experimental groups treated with either TP or LR. A total of 23 microRNAs (21 upregulated and 2 downregulated) identified for the first time by the high-throughput RNA sequencing were involved in the molecular response to EIF in mice treated with TP and LR. The regulatory functions of these microRNAs in the pathogenesis of EIF in mice were further explored based on Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses with a total of over 20,000-30,000 target genes annotated and 44 metabolic pathways enriched in the experimental groups based on GO and KEGG databases, respectively. Conclusion: Our study revealed the therapeutic effects of TP and LR and identified the microRNAs involved in the molecular mechanisms regulating the EIF in mice, providing strong experimental evidence to support further agricultural development of LR as well as the investigations and applications of TP and LR in the treatment of EIF in humans, including the professional athletes.

Synergistic degradation of Azure B and sulfanilamide antibiotics by the white-rot fungus Trametes versicolor with an activated ligninolytic enzyme system.

The treatment of complex polluted wastewater has become an increasingly critical concern for the various types of hazardous organic compounds, including synthetic dyes and pharmaceuticals. Due to their efficient and eco-friendly advantages, the white-rot fungi (WRF) have been applied to degrade environmental pollutants. This study aimed to investigate the removal ability of WRF (i.e., Trametes versicolor WH21) in the co-contamination system composed of Azure B dye and sulfacetamide (SCT). Our study discovered that the decolorization of Azure B (300 mg/L) by strain WH21 was significantly improved (from 30.5% to 86.5%) by the addition of SCT (30 mg/L), while the degradation of SCT was also increased from 76.4% to 96.2% in the co-contamination system. Transcriptomic and biochemical analyses indicated that the ligninolytic enzyme system was activated by the enhanced enzymatic activities of MnPs and laccases, generating higher concentration of extracellular H2O2 and organic acids in strain WH21 in response to SCT stress. Purified MnP and laccase of strain WH21 were revealed with remarkable degradation effect on both Azure B and SCT. These findings significantly expanded the existing knowledge on the biological treatment of organic pollutants, indicating the strong promise of WRF in the treatment of complex polluted wastewater.

Saline-Alkali Soil Property Improved by the Synergistic Effects of Priestia aryabhattai JL-5, Staphylococcus pseudoxylosus XW-4, Leymus chinensis and Soil Microbiota.

Two saline-alkali-tolerant bacterial strains, Priestia aryabhattai JL-5 and Staphylococcus pseudoxylosus XW-4, were isolated, with high capabilities of hydrolyzing phosphate and producing cellulase, respectively. The molecular mechanisms regulating the saline-alkali tolerance in the strain JL-5 were further investigated using transcriptome analysis. The contents of lactic acid and proline and the enzymatic activity of glutamine synthetase in the strain JL-5 were significantly increased. The properties of saline-alkali soils were significantly improved by the enhanced growth of the indicator plant Leymus chinensis under the combined applications of the strains JL-5 and XW-4 mixed with corn straw. The contents of catalase, peroxidase, superoxide dismutase and proline of L. chinensis were significantly increased, and the content of malondialdehyde was significantly decreased in the combined treatment of both bacterial strains. The contents of available nitrogen, phosphorus and potassium and organic matters in the soil treated with both strains were significantly increased, as well as the diversity and abundance of the soil microbiota. Our study evidently demonstrated the synergistic effects of the strains JL-5 and XW-4, indicator plants and the local microbiota in terms of improving the saline-alkali soil properties, providing strong experimental evidence to support the commercial development of the combined application of both strains to improve the properties of saline-alkali soils.

Improved biosynthesis of heme in Bacillus subtilis through metabolic engineering assisted fed-batch fermentation.

BACKGROUND: Heme is an iron/porphyrin complex compound, widely used in the health care, food, and pharmaceutical industries. It is more advantageous and attractive to develop microbial cell factories to produce heme by fermentation, with lower production costs and environmentally more friendly procedures than those of the traditional extraction based on animal blood. In this study, Bacillus subtilis, a typical industrial model microorganism of food safety grade, was used for the first time as the host to synthesize heme. RESULTS: The heme biosynthetic pathway was engineered as four modules, the endogenous C5 pathway, the heterologous C4 pathway, the uroporphyrinogen (urogen) III synthesis pathway, and the downstream synthesis pathway. Knockout of hemX encoding the negative effector of the concentration of HemA, overexpression of hemA encoding glutamyl-tRNA reductase, and knockout of rocG encoding the major glutamate dehydrogenase in the C5 pathway, resulted in an increase of 427% in heme production. Introduction of the heterologous C4 pathway showed a negligible effect on heme biosynthesis. Overexpression of hemCDB, which encoded hydroxymethylbilane synthase, urogen III synthase, and porphobilinogen synthase participating in the urogen III synthesis pathway, increased heme production by 39%. Knockouts of uroporphyrinogen methyltransferase gene nasF and both heme monooxygenase genes hmoA and hmoB in the downstream synthesis pathway increased heme production by 52%. The engineered B. subtilis produced 248.26 ± 6.97 mg/L of total heme with 221.83 ± 4.71 mg/L of extracellular heme during the fed-batch fermentation in 10 L fermenter. CONCLUSIONS: Strengthening endogenous C5 pathway, urogen III synthesis pathway and downstream synthesis pathway promoted the biosynthesis of heme in B. subtilis. The engineered B. subtilis strain has great potential as a microbial cell factory for efficient industrial heme production.

Chemical composition and anti-inflammatory activities of Castanopsis honey.

Castanopsis is diffusely spread in tropical and subtropical regions and is an important nectar source plant in China. The Castanopsis honey (CH) is characterized by its bitter taste. However, its composition and functions remain unclear. In this study, the physicochemical parameters, chemical composition, and antioxidant capacity of CH were comprehensively investigated, with the anti-inflammatory effects of the Castanopsis honey extract (CHE) evaluated based on the RAW 264.7 cell inflammatory model. The results revealed a high level of quality in CH based on the quality standards. Among a total of 84 compounds identified in CH, 5 high response compounds and 29 phenols were further quantified by UPLC-Q/TOF-MS. The high content of phenylethylamine (117.58 ± 64.81 mg kg-1) was identified as a potential marker of CH. Furthermore, the CH showed evident antioxidant activities, and the anti-inflammatory activities of CHE were observed to inhibit the release of nitric oxide (NO) and reduce the content of tumor necrosis factor alpha (TNF-α) and improve the content of interleukin-10 (IL-10) by regulating the NF-κB pathway. Our study indicates that CH has sound physicochemical properties and biological activities with a high level of quality, providing strong experimental evidence to support the further economic and agricultural development and application of CH.

Sustainable production of astaxanthin in microorganisms: the past, present, and future.

Astaxanthin (3,3'-dihydroxy-4,4'-diketo-ß-carotene) is a type of C40 carotenoid with remarkable antioxidant characteristics, showing significant application prospects in many fields. Traditionally, the astaxanthin is mainly obtained from chemical synthesis and natural acquisition, with both approaches having many limitations and not capable of meeting the growing market demand. In order to cope with these challenges, novel techniques, e.g., the innovative cell engineering strategies, have been developed to increase the astaxanthin production. In this review, we first elaborated the biosynthetic pathway of astaxanthin, with the key enzymes and their functions discussed in the metabolic process. Then, we summarized the conventional, non-genetic strategies to promote the production of astaxanthin, including the methods of exogenous additives, mutagenesis, and adaptive evolution. Lastly, we reviewed comprehensively the latest studies on the synthesis of astaxanthin in various recombinant microorganisms based on the concept of microbial cell factory. Furthermore, we have proposed several novel technologies for improving the astaxanthin accumulation in several model species of microorganisms.

Molecular Characterization and Drought Resistance of GmNAC3 Transcription Factor in Glycine max (L.) Merr.

Soybean transcription factor GmNAC plays important roles in plant resistance to environmental stresses. In this study, GmNAC3 was cloned in the drought tolerant soybean variety "Jiyu47", with the molecular properties of GmNAC3 characterized to establish its candidacy as a NAC transcription factor. The yeast self-activation experiments revealed the transcriptional activation activity of GmNAC3, which was localized in the nucleus by the subcellular localization analysis. The highest expression of GmNAC3 was detected in roots in the podding stage of soybean, and in roots of soybean seedlings treated with 20% PEG6000 for 12 h, which was 16 times higher compared with the control. In the transgenic soybean hairy roots obtained by the Agrobacterium-mediated method treated with 20% PEG6000 for 12 h, the activities of superoxide dismutase, peroxidase, and catalase and the content of proline were increased, the malondialdehyde content was decreased, and the expressions of stress resistance-related genes (i.e., APX2, LEA14, 6PGDH, and P5CS) were up-regulated. These expression patterns were confirmed by transgenic Arabidopsis thaliana with the overexpression of GmNAC3. This study provided strong scientific evidence to support further investigation of the regulatory function of GmNAC3 in plant drought resistance and the molecular mechanisms regulating the plant response to environmental stresses.

Disulfiram enhances chemotherapeutic effects of doxorubicin liposomes against human hepatocellular carcinoma via activating ROS-induced cell stress response pathways.

PURPOSE: Increasing evidences have revealed the anti-cancer effect of disulfiram. Current disulfiram-based cancer therapies still have limitations, such as poor tumor-targeting ability and insufficient studies on anti-tumor mechanisms. METHODS: In the present study, tumor-targeting liposomes were prepared as drug carriers to increase retention of disulfiram in tumor cells. Then, anti-tumor efficacy of liposomes and the underlying mechanisms were investigated in in vitro, in vivo, and transcriptomic level. RESULTS: The results showed that disulfiram enhanced sensitivity of human hepatocellular carcinoma cells to doxorubicin by 15-27-fold, and increased reactive oxygen species (ROS) production as well as caspase-dependent apoptosis. Inhibition of tumor migration and invasion by doxorubicin were further enhanced by disulfiram. In vivo study showed that disulfiram additive doxorubicin liposomes had better performance in suppressing tumor growth than single doxorubicin liposomes. Gene expression profiling found that cellular components destruction, cell stress, check point regulation, and immunoregulation were the main anti-tumor mechanisms of disulfiram. More importantly, disulfiram possessed a great potential to be a protein ubiquitination and murine double minute 4 (MDM4) targeting compound. CONCLUSIONS: Due to its low price and good safety, it is worth to repurposing disulfiram as a chemotherapeutic drug. Furthermore, MDM4 may act as a biomarker for observation the clinical effect of disulfiram-based treatment.

Transcriptome Analysis of the Accumulation of Astaxanthin in Haematococcus pluvialis Treated with White and Blue Lights as well as Salicylic Acid.

Haematococcus pluvialis is the most commercially valuable microalga for the production of natural astaxanthin, showing enhanced production of astaxanthin with the treatments of high-intensity light and hormones. The molecular mechanisms regulating the biosynthesis of astaxanthin in H. pluvialis treated with white light, blue light, and blue light with salicylic acid (SA) were investigated based on the transcriptome analysis. Results showed that the combined treatment with both blue light and SA generated the highest production of astaxanthin. A total of 109,443 unigenes were identified to show that the genes involved in the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway (PPP), and the astaxanthin biosynthesis were significantly upregulated to increase the production of the substrates for the synthesis of astaxanthin, i.e., pyruvate and glyceraldehyde-3-phosphate generated in the TCA cycle and PPP, respectively. Results of transcriptome analysis were further verified by the quantitative real-time PCR (qRT-PCR) analysis, showing that the highest content of astaxanthin was obtained with the expression of the bkt gene significantly increased. Our study provided the novel insights into the molecular mechanisms regulating the synthesis of astaxanthin and an innovative strategy combining the exogenous hormone and physical stress to increase the commercial production of astaxanthin by H. pluvialis.

MicroRNAs Involved in the Therapeutic Functions of Noni (Morinda citrifolia L.) Fruit Juice in the Treatment of Acute Gouty Arthritis in Mice Induced with Monosodium Urate.

We investigated the functions of microRNAs in the therapeutic effects of noni (Morinda citrifolia L.) fruit juice on mouse models of acute gouty arthritis induced with monosodium urate (MSU). Compared with the model group (treated with MSU), mice in both the positive control group (treated with both MSU and colchicine) and noni fruit juice group (treated with MSU and noni fruit juice) showed a significantly decreased degree of paw swelling in 5 days, as well as the contents of two types of proinflammatory cytokines (i.e., NALP3 and TNF-α). Based on the next-generation sequencing technology, a total of 3896 microRNAs (234 known and 3662 novel) were identified in mice treated with noni fruit juice. A large amount of differentially expressed miRNAs were identified in the noni fruit juice group, suggesting the significant effects of noni fruit juice on the mice with acute gouty arthritis, while the different patterns of change in the numbers of both upregulated and downregulated miRNAs in both noni fruit juice and positive control groups indicated that the mice of acute gouty arthritis may be regulated by differential mechanisms between the treatments of noni fruit juice and colchicine. The target genes of microRNAs involved in the pathogenesis and pathology of acute gouty arthritis in mice were identified and further annotated by both Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Our results revealed the therapeutic effects of noni fruit juice on acute gouty arthritis in mice with a group of microRNAs involved in the pharmacological mechanisms of noni fruit juice, providing scientific evidence to support both the agricultural cultivation and pharmacological significance of noni plants.

Improved Q-Learning Algorithm Based on Approximate State Matching in Agricultural Plant Protection Environment.

An Unmanned Aerial Vehicle (UAV) can greatly reduce manpower in the agricultural plant protection such as watering, sowing, and pesticide spraying. It is essential to develop a Decision-making Support System (DSS) for UAVs to help them choose the correct action in states according to the policy. In an unknown environment, the method of formulating rules for UAVs to help them choose actions is not applicable, and it is a feasible solution to obtain the optimal policy through reinforcement learning. However, experiments show that the existing reinforcement learning algorithms cannot get the optimal policy for a UAV in the agricultural plant protection environment. In this work we propose an improved Q-learning algorithm based on similar state matching, and we prove theoretically that there has a greater probability for UAV choosing the optimal action according to the policy learned by the algorithm we proposed than the classic Q-learning algorithm in the agricultural plant protection environment. This proposed algorithm is implemented and tested on datasets that are evenly distributed based on real UAV parameters and real farm information. The performance evaluation of the algorithm is discussed in detail. Experimental results show that the algorithm we proposed can efficiently learn the optimal policy for UAVs in the agricultural plant protection environment.

Genetic Divergence and Population Structure in Weedy and Cultivated Broomcorn Millets (Panicum miliaceum L.) Revealed by Specific-Locus Amplified Fragment Sequencing (SLAF-Seq).

Broomcorn millet (Panicum miliaceum L.) is one of the earliest domesticated crops in the world. Weedy broomcorn millet [Panicum ruderale (Kitag.) Chang or Panicum miliaceum subsp. ruderale (Kitag.) Tzvel] is thought to be the descendant of the wild ancestor or the feral type of this cereal. The genealogical relationships and genetic divergence among these taxa have not been clarified. In this study, the genetic diversity and population structure of weedy and cultivated broomcorn millets were investigated by using the high-throughput sequencing technology, i.e., the specific-locus amplified fragment sequencing (SLAF-seq). Our analyses consistently revealed both the wild and the feral genotypes in the weedy broomcorn millets. The single nucleotide polymorphisms (SNPs) at the genomic level provided useful evidence to distinguish the wild and the endoferal/exoferal types of weedy broomcorn millets. The genetic divergence revealed between the cultivated broomcorn millet from eastern Eurasia and those from central-western Eurasia was probably derived from either the genetic introgression from weedy broomcorn millets along the spread routes or the founder effect, while the limited gene flow of broomcorn millets from eastern and central-western Eurasia was probably due to the different uses of broomcorn millets and eating habits of the local people.

Menzerath-Altmann's Law of Syntax in RNA Accretion History.

RNA evolves by adding substructural parts to growing molecules. Molecular accretion history can be dissected with phylogenetic methods that exploit structural and functional evidence. Here, we explore the statistical behaviors of lengths of double-stranded and single-stranded segments of growing tRNA, 5S rRNA, RNase P RNA, and rRNA molecules. The reconstruction of character state changes along branches of phylogenetic trees of molecules and trees of substructures revealed strong pushes towards an economy of scale. In addition, statistically significant negative correlations and strong associations between the average lengths of helical double-stranded stems and their time of origin (age) were identified with the Pearson's correlation and Spearman's rho methods. The ages of substructures were derived directly from published rooted trees of substructures. A similar negative correlation was detected in unpaired segments of rRNA but not for the other molecules studied. These results suggest a principle of diminishing returns in RNA accretion history. We show this principle follows a tendency of substructural parts to decrease their size when molecular systems enlarge that follows the Menzerath-Altmann's law of language in full generality and without interference from the details of molecular growth.

Rapid Detection of Clostridium botulinum in Food Using Loop-Mediated Isothermal Amplification (LAMP).

Botulinum neurotoxins are considered as one of the most potent toxins and are produced by Clostridium botulinum. It is crucial to have a rapid and sensitive method to detect the bacterium Clostridium botulinum in food. In this study, a rapid detection assay of C. botulinum in food using loop-mediated isothermal amplification (LAMP) technology was developed. The optimal primers were identified among three sets of primers designed specifically based on the partial ntnh gene encoding nontoxic-nonhaemagglutinin (NTNH) for rapid detection of the target DNA in plasmids. The optimal temperature and reaction time of the LAMP assay were determined to be 64 °C and 60 min, respectively. The chemical kit could be assembled based on these optimized reaction conditions for quick, initial high-throughput screening of C. botulinum in food samples. The established LAMP assay showed high specificity and sensitivity in detecting the target DNA with a limit of 0.0001 pg/ul (i.e., ten times more sensitive than that of the PCR method) and an accuracy rate of 100%. This study demonstrated a potentially rapid, cost-effective, and easy-operating method to detect C. botulinum in food and clinical samples based on LAMP technology.