SNP Array for Small-Shrimp (Genus Acetes) Origin Determination Using Machine Learning.

Accurate origin determination of seafood is crucial for consumer trust and safety. This study was performed to develop a machine learning-based single-nucleotide polymorphism (SNP) analysis technique to determine the origin of Acetes species in salted small-shrimp products. Mitochondrial DNA (COI and 16S rRNA) analysis revealed genetic variations among species and origins. Eight candidate SNPs were identified, six of which were developed into markers for genotyping analysis. Using the developed markers, an SNP array was created and SNP data from salted small-shrimp samples were obtained. Machine learning analysis using a supervised learning algorithm achieved 100% accuracy in classifying the origin of Acetes based on SNP data. This method offers a reliable method for regulatory bodies to combat food fraud and ensure product integrity. The approach can be further improved by expanding the data set to encompass a wider range of species and origins. This study highlights the potential of SNP analysis and machine learning for ensuring seafood authenticity and promoting sustainable practices.

In vivo evaluation and mechanism prediction of anti-diabetic foot ulcer based on component analysis of Ruyi Jinhuang powder.

BACKGROUND: Diabetes is a metabolic disease with a high complication rate. Diabetic foot ulcers (DFUs) seriously affect the quality of life of patients. A total of 15%-20% of diabetic patients develop DFUs, which heal with difficulty over a long time and can result in amputation and disability. Traditional Chinese medicine has a unique effect in the treatment of skin ulcerative diseases. Ruyi Jinhuang powder (RHP) is one of the classic prescriptions in traditional Chinese medicine and is widely used in clinical practice. AIM: To verify the ability of RHP to promote wound healing by electron microscopy analysis in animal models and hematoxylin-eosin (HE) staining. The effective components of RHP were extracted and identified by gas chromatography-mass spectrometry (GC-MS), and the obtained chemical components were analyzed by network pharmacology methods to predict its therapeutic mechanism. METHODS: Sprague Dawley rats were injected with streptozotocin to establish the DFU model. HE staining was used to observe the wound tissue under an electron microscope. The chemical constituents of RHP were extracted first by supercritical fluid extraction and alcohol extraction, and then, GC-MS and ultra-performance liquid chromatography-MS were used to separately identify the chemical constituents. In addition, the "herb-component-target" link was established through the Traditional Chinese Medicine Systems Pharmacology database to obtain the target information, and the molecular docking of important components and key targets was performed in Discovery Studio software. Cytoscape software was used to visualize and analyze the relationship between the chemical composition, targets and Traditional Chinese Medicine network. RESULTS: RHP promoted DFU healing in rats by affecting fibroblasts and nerve cells. A total of 89 chemical components were obtained by GC-MS. Network pharmacological analysis revealed that RHP was associated with 36 targets and 27 pathways in the treatment of DFU, of which the important components were luteolin, trans caryophyllene, ar-turmerone, palmitic acid, methyl palmitate, gallic acid, demethoxycurcumin, berberine, and rheic acid. The key targets were posttranscriptional silencing, topoisomerase II alpha, muscarinic acetylcholine receptor M2, interleukin 6, tumor necrosis factor and retinoic X receptor alpha, and the key pathways were the phosphoinositide 3-kinase-protein kinase B signaling pathway, neuroactive ligand-receptor interactions, and the forkhead box O signaling pathway. CONCLUSION: Our results indicated that RHP may play a role in the treatment of DFU through these target pathways by affecting insulin resistance, altering the nervous system and immune system, participating in inflammatory responses and regulating cell proliferation, differentiation and apoptosis through other specific mechanisms.

Heavy metal pollution characteristics and health risk assessment of dust fall related to industrial activities in desert steppes.

China's desert steppe is the transition zone between the grasslands in central China and the arid desert. Ecological security in this region has long been a subject of debate, both in the local and academic communities. Heavy metals and other pollutants are readily released during industrial production, combustion, and transportation, aggravating the vulnerability of the desert steppes. To understand the impact of industrial activiteis on the heavy metal content of dust fall in the desert steppe, a total of 37 dust fall samples were collected over 90 days. An inductively-coupled plasma mass spectrometer (NexION 350X) was used to measure the concentration of heavy metals Cu, Cd, Cr, Pb, Mn, Co, and Zn in the dust. Using comprehensive pollution index and multivariate statistical analysis methods, we explored the characteristics and sources of heavy metal pollution. We also quantitatively assessed the carcinogenic risks of heavy metals resulting from dust reduction with the help of health risk assessment models. The heavy metals' comprehensive pollution index values in the study area's dust fall were ranked as follows: Zn > Cd > Pb > Mn > Cu > Co > Cr. Among these, Zn, Cd, and Pb were significant pollution factors in the study area, and were affected by industrial production and transportation. The high pollution index was concentrated in the north of the research industrial park and on both sides of a highway. The seven heavy metals' total non-carcinogenic risk index (HI) values were ranked as follows: Mn > Co > Pb > Zn > Cr > Cu > Cd (only the HI of Mn was greater than one). Excluding Mn, the non-carcinogenic and carcinogenic risk index values of the other six heavy metals were within acceptable ranges. Previous studies have also shown that industrial transportation and production have had a significant impact on the heavy metal content of dust fall in the desert steppe.

Comprehensive evaluation of soil quality in a desert steppe influenced by industrial activities in northern China.

Desert steppe soil security issues have been the focus of attention. Therefore, to understand the impact of industrial activities on the soil quality of desert grasslands, this experiment investigated the Gaoshawo Industrial Concentration Zone in Yanchi County. Based on the distance and direction from the industrial park, sample plots were established at intervals of 1-2 km. A total of 82 surface soil samples (0-20 cm) representing different pollution sources were collected. The samples were analysed for pH, total nitrogen, total phosphorus, available phosphorus, available potassium, organic matter, copper (Cu), cadmium (Cd), chromium (Cr), lead (Pb), and zinc (Zn). The desert steppe soil quality was analysed based on the integrated fertility index (IFI) and the Nemerow pollution index (PN), followed by the calculation of the comprehensive soil quality index (SQI), which considers the most suitable soil quality indicators through a geostatistical model. The results showed that the IFI was 0.393, indicating that the soil fertility was relatively poor. Excluding the available potassium, the nugget coefficients of the fertility indicators were less than 25% and showed strong spatial autocorrelation. The average values of Cu, Cd, Cr, Pb and Zn were 21.64 ± 3.26, 0.18 ± 0.02, 44.99 ± 21.23, 87.18 ± 25.84, and 86.63 ± 24.98 mg·kg-1, respectively; the nugget coefficients of Cr, Pb and Zn were 30.79-47.35%. Pb was the main element causing heavy metal pollution in the study area. Higher PN values were concentrated north of the highway in the study area, resulting in lower soil quality in the northern region and a trend of decreasing soil quality from south to north. The results of this research showed that the average SQI was 0.351 and the soil quality was extremely low. Thus, industrial activities and transportation activities in the Gaoshawo Industrial Zone significantly impact the desert steppe soil quality index.

Sex Differences in Stroke Hospitalization Incidence, 30-Day Mortality, and Readmission in a Regional Medical Center in the Southwestern United States.

OBJECTIVES: This study explores sex differences in ischemic stroke hospitalization incidence, 30-day mortality, and 30-day readmission in a southwestern US medical center. METHODS: Ischemic stroke admissions in a regional medical center in the southwestern United States were obtained for a 6.5-year time frame (N = 1968). Logistic regression models examine the adjusted effects of sex on 30-day mortality and 30-day readmission outcomes among individuals hospitalized for ischemic stroke. RESULTS: Findings confirm that although women experience higher mortality than men (9.1% vs 6.7%), the sex disparity in mortality is explained by the age distribution of strokes. Women experience far more strokes and deaths because of stroke at older ages. No differences in principal procedure or 30-day readmission emerged. CONCLUSIONS: Men experienced higher stroke hospitalization incidence, although women exhibited higher 30-day mortality. Age composition explained sex differences in mortality, but higher male stroke hospitalization incidence represents a larger public health issue that suggests the need for behavioral change at the population level. No meaningful sex differences emerged in treatment, mortality, or readmission.

Population genetic structure of Semisulcospira gottschei: simultaneous examination of mtDNA and microsatellite markers.

Semisulcospira gottschei is an Asian endemic species inhabiting Korea and China. However, genetic structure analysis of the resource management of this species has not been performed. To investigate the genetic diversity among populations, microsatellites can be used to determine the geographic origins of marine and freshwater species. This study investigated the genetic structures of the Korean and Chinese populations of S. gottschei based on mitochondrial DNA (mtDNA) Cytochrome oxidase subunit I (COI) and polymorphic microsatellite loci developed from Semisulcospira coreana. Analysis of the mtDNA COI sequence revealed 43 haplotypes, which indicated no gene flow between the Korean and Chinese populations. To further elucidate the genetic structures of the Korean and Chinese populations, the population genetics of S. gottschei were analyzed using nine microsatellite markers. The genetic diversity analysis showed an average of 5.25 alleles per locus, with an average allelic richness of 4.02. Excessive homozygosity was found at all loci, which was expected to be due to the presence of null alleles at all loci. Populations of S. gottschei formed two separate clusters according to pairwise FST and AMOVA. Also, the UPGMA tree, PCA, STRUCTURE, and GeneClass indicated separation of the 11 populations into two clusters: Korea and China. These results have potential use in the management, restoration, and distinction of the origin country of populations.

Classification of Takifugu rubripes, T. chinensis and T. pseudommus by genotyping-by-sequencing.

Takifugu rubripes is more expensive than other species of the genus because of its high protein content and special flavor. However, it is easily confused with imported T. chinensis and T. pseudommus because they have similar morphological characteristics. We identified single nucleotide polymorphism (SNP) markers of T. rubripes by genotyping-by-sequencing (GBS) and evaluated their ability to distinguish among T. rubripes, T. chinensis, and T. pseudommus. In all, 18 polymorphic SNPs were subjected to phylogenetic analyses of the three Takifugu species. Additionally, we subjected a second set of samples to Sanger sequencing to verify that the polymorphic SNPs could be used to evaluate the genetic variation among the three Takifugu species. A phylogenetic tree that included the analyzed sequence of set A, which is referred to as the reference sequence, and a validation sequence of set B with 18 SNPs were produced. Based on this phylogenetic tree and STRUCTURE analyses, T. rubripes, T. chinensis and T. pseudommus have low genetic variation and should be considered the same gene pool. Our findings suggest that further studies are needed to estimate the genetic association of the three Takifugu species.

Characteristics and sources of heavy metal pollution in desert steppe soil related to transportation and industrial activities.

A study area was selected from the industrial region of Gaoshawo Town, Yanchi County, Ningxia, to explore the level of heavy metal pollution in desert grasslands due to industrial activities. A total of 82 surface soils were collected, and the concentration of heavy metals, namely, Cu, Cd, Cr, Pb, Zn, Mn, and Co, was determined by ICP-AES (atomic emission spectrometer) (HK-8100); the average values were 21.64 ± 3.26, 0.18 ± 0.02, 44.99 ± 21.23, 87.18 ± 25.84, 86.63 ± 24.98, 570.49 ± 171.57, and 17.96 ± 9.96 mg kg-1. The single-factor, Nemerow pollution, and potential ecological risk index methods were used to evaluate the status of soil heavy metal pollution and the contribution from the major sources identified by the receptor model. The results showed that 9.09% of the samples were slightly polluted, 32.47% of the samples were moderately polluted, and 58.41% of the samples were heavily polluted. The comprehensive potential ecological risk index indicated that 90.79% of the samples had moderate ecological risk. It was verified from the models and spatial distribution maps that Cr, Co, Zn, and Mn are mainly contributed by the industrial sources that account for 55.04%, 92.13%, 50.05%, and 48.77% of these heavy metals, respectively. The heavily contaminated areas are distributed around the industrial park. A total of 70.63% and 77.83% of Cu and Pb are contributed by transportation sources, respectively, with the concentrations decreasing from southwest to northeast. The contribution from agricultural activities to Cd is 77.02%, with concentrations largely distributed in the north of the highway. This study showed that the existence of the Gaoshawo Industrial Zone and the corresponding industrial and transportation activities have a significant impact on the grassland soil environment.

The N-terminus of MTRR plays a role in MTR reactivation cycle beyond electron transfer.

In eucaryotic cells, methionine synthase reductase (MSR/MTRR) is capable of dominating the folate-homocysteine metabolism as an irreplaceable partner in electron transfer for regeneration of methionine synthase. The N-terminus of MTRR containing a conserved domain of FMN_Red is closely concerned with the oxidation-reduction process. Maternal substitution of I22M in this domain can bring about pregnancy with high risk of spina bifida. A new variation of Arg2del was identified from a female conceiving a fetus with spina bifida cystica. Although the deletion is far from the N-terminal FMN_Red domain, the biochemical features of the variant had been seriously investigated. Curiously, the deletion of arginine(s) of MTRR could not affect the electron relay, if only the FMN_Red domain was intact, but by degrees reduced the ability to promote MTR catalysis in methionine formation. Confirmation of the interaction between the isolated MTRR N-terminal polypeptide and MTR suggested that the native MTRR N-terminus might play an extra role in MTR function. The tandem arginines at the end of MTRR N-terminus conferring high affinity to MTR were indispensable for stimulating methyltransferase activity perhaps via triggering allosteric effect that could be attenuated by removal of the arginine(s). It was concluded that MTRR could also propel MTR enzymatic reaction relying on the tandem arginines at N-terminus more than just only implicated in electron transfer in MTR reactivation cycle. Perturbance of the enzymatic cooperation due to the novel deletion could possibly invite spina bifida in clinics.

[Research advance in chemical compounds,pharmacological action and clinical application of Dengzhan Shengmai Capsules].

Dengzhan Shengmai Capsules( DZSMC),a well-known traditional Chinese medicine( TCM) formula,is comprised of the main drug of Erigeron breviscapus,and supplemented with Panax ginseng,Ophiopogon japonicus and Schisandra chinensis,with functions of supplementing Qi and nourishing Yin,promoting blood circulation and strengthening brain. DZSMC is the only Chinese patent drug with A-level evidence-based medicine in secondary prevention for stroke and ranks first among TCMs for neurological treatment. Modern studies indicate that the chemical constituents of DZSMC mainly include flavonoids,phenolic acids,lignans,saponins and so on. Pharmacological experimental studies have shown that DZSMC has such pharmacological effects as anti-oxidation,anti-inflammatory and anti-myocardial ischemia. DZSMC is mainly used in the convalescent care of ischemic cardiovascular and cerebrovascular diseases,and is often used in combination with various conventional therapeutic drugs to exert clinical efficacy through brain protection,neuroprotection,etc.,and improve clinical symptoms in patients. In this review,according to domestic and international related literature combined with research results obtained by our project,the research advances in the chemical constituents,pharmacological effects and clinical application of DZSMC have been systematically reviewed and summarized,providing reference and support for further study and secondary development of the formula.

Comprehensive characterization of multiple components and metabolites of Xiaojin Capsule based on ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Xiaojin Capsule, a classic traditional Chinese medicine formula, has been used to treat mammary cancer, thyroid nodules, and hyperplasia of the mammary glands. However, its systematic chemical information remained unclear, which hindered the interpretation of the pharmacology and the mechanism of action of this drug. In this research, an ultra high performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry method was developed to identify the complicated components and metabolites of Xiaojin Capsule. Two acquisition modes, including the MSEnergy mode and fast data directed acquisition mode, were utilized for chemical profiling. As a result, 156 compounds were unambiguously or tentatively identified by comparing their retention times and mass spectrometry data with those of reference standards or literature. After the oral administration of Xiaojin Capsule, 53 constituents, including 24 prototype compounds and 29 metabolites, were detected in rat plasma. The obtained results were beneficial for a better understanding of the therapeutic basis of Xiaojin Capsule. A high-resolution and efficient separation method was firstly established for systematically characterizing the compounds of Xiaojin Capsule and the associated metabolites in vivo, which could be helpful for quality control and pharmacokinetic studies of this medicine.

Identification and characterization of chemical constituents in Dengzhan Shengmai Capsule and their metabolites in rat plasma by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Dengzhan Shengmai Capsule (DZSMC) is a traditional Chinese medicine (TCM) formula with remarkable clinical effect in the treatment of stroke sequelae. Exploring the components of DZSMC and detecting the absorbed prototype constituents and metabolites in blood are of great significance to clarify the effective substances of this prescription. Here, a reliable method using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was established for the comprehensive analysis of chemical constituents of DZSMC and their metabolites in rat plasma after gastric perfusion. Two acquisition modes, including MSE mode and Fast DDA mode, were performed for acquiring more precursor ions and cleaner precursor-product ions background during the study of constituents of DZSMC. As a result, a total of 125 constituents were unambiguously characterized or tentatively identified. For the first time, a total of 92 components, including 44 prototype components and 48 metabolites were unambiguously or tentatively identified in rat plasma. The metabolic pathways included phase I reactions (hydration, hydrogenation, oxidation, demethylation and hydroxylation) and phase II reactions (conjugation with glucuronide, sulfate and methyl). Furthermore, the metabolites from caffeic acid and scutellarin were characterized and validated by phase II metabolic reactions in vitro, which could be established as a simulated in vivo environment of metabolites identification and verification of TCM formula. It is the first systematic study on metabolism of DZSMC in vivo and could also provide a valid analytical strategy for characterization of the chemical compounds and metabolites of TCM formula.

Hybrid multidimensional data acquisition and data processing strategy for comprehensive characterization of known, unknown and isomeric compounds from the compound Dan Zhi Tablet by UPLC-TWIMS-QTOFMS.

The compound Dan Zhi Tablet (DZT), a reputable traditional Chinese medicine prescription, is widely used for the treatment of ischemic stroke in clinic. However, its systematic chemical constituents have rarely been elucidated, which hampers its quality evaluation, the study of bioactive constituents and the mechanism of action interpretation. In this study, we developed a combination of multidimensional data acquisition and data processing strategy with the aim to globally and comprehensively identify the chemical constituents in DZT based on UPLC-TWIMS-QTOFMS. First, multidimensional acquisition modes (MSE, Fast DDA and HDMSE) were performed on UPLC-TWIMS-QTOFMS. Second, targeted characterizations of the known compounds and their analogues present in DZT were carried out on the basis of the corresponding commercial standards or Mass2Motifs. Third, untargeted identification of unknown compounds in DZT was performed by extracting shared Mass2Motifs from the raw fragmentation spectra. Finally, the coeluting isomers were characterized using a precursor and/or product ion mobility. Consequently, 202 compounds were detected from DZT: 29 of them were unambiguously identified by comparison with reference compounds, 29 unknown compounds were discovered in specific medicinal materials, and ten pairs of coeluting isomers, which could not be distinguished using conventional MSE or Fast-DDA, were resolved using HDMSE only. This strategy was successfully used for the rapid and global identification of complex compounds including known, unknown and coeluting isomeric compounds in DZT and provided helpful chemical information for further quality control, pharmacology and active mechanism research on DZT.

Metabolite profiling of traditional Chinese medicine formula Dan Zhi Tablet: An integrated strategy based on UPLC-QTOF/MS combined with multivariate statistical analysis.

Metabolites derived from traditional Chinese medicine (TCM) are becoming active substances of pharmacologically as well as promising sources for discovering new drugs. However, detection and identification of constituents in vivo remains a challenge for TCM, due to massive endogenous interference and low abundance of metabolites in biological matrix. Traditional Chinese medicine formula Dan Zhi Tablet (DZT), a well-established TCM formula developed based on years of clinical experiences, was widely used to treat cerebral infraction disease. In this study, an integrated strategy based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was adopted to comprehensively identify the prototype and metabolite constituents of DZT. The potential constituents were screened by cross orthogonal partial least-squares discriminant analysis (OPLS-DA). Automatic matching analysis was performed on UNIFI platform based on the function of predicting metabolites. Using this strategy, a total of 170 compounds, including 51 prototype constituents and 119 metabolites were unambiguously or tentatively identified in rat plasma. Furthermore, 31 compounds have also been detected in rat cerebrospinal fluid. The metabolism reactions included phase I reactions (hydroxylation, hydrolysis, deglycosylation, hydrogenation, demethylation and dehydroxylation) and phase II reactions (conjugation with glutatione, cysteine, acetylcysteine, glucuronide, sulfate). It is the first systematic metabolic study of DZT in vivo and some metabolites were also reported for the first time, which could provide a scientific basis for explaining the multiple functions of DZT. More importantly, the integrated strategy also shows promising perspectives in the identification of the metabolites in TCM from a complicated biological matrix.

Rapid characterization and identification of the chemical constituents and rat metabolites of Deng-Zhan-Xi-Xin injection using ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Deng-Zhan-Xi-Xin injection is a well-known traditional Chinese medicine prescription for the treatment of cardiovascular and cerebral vessel diseases. However, there have been few reports on its chemical constituents and metabolic pathway, which has blocked its further quality control and studies on its pharmacology and mechanism of action. In this study, an integrative method was established to rapidly explore the chemical constituents and metabolites of Deng-Zhan-Xi-Xin injection using ultra high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry and the UNIFI™ software combined with multiple data processing approaches. As a result, a total of 40 compounds, including 9 flavonoids and 31 phenolic acids were identified or tentatively characterized, and five compounds were first reported in Deng-Zhan-Xi-Xin injection. Under the same analysis conditions, 70 compounds have been detected in rats, including 25 prototypes and 45 metabolites. This was the first systematic research study on the metabolic profiling of Deng-Zhan-Xi-Xin injection. This study provides valuable chemical information for the quality control and research on pharmacology and mechanism of action of Deng-Zhan-Xi-Xin injection. Moreover, it provides a valuable strategy for analyzing the chemical components and metabolites of other traditional Chinese medicine prescriptions.

An Inframe Trinucleotide Deletion in MTRR Exon 1 is Associated with the Risk of Spina Bifida.

Maternal genetic variants of enzymes in folate-homocysteine metabolic network are significantly correlative with the risk of spina bifida. To survey the genetic causality, the genotypes of three women having spina bifida fetuses from two unrelated Chinese families were screened in candidate alleles. Polymerase chain reaction, capillary electrophoresis and Sanger sequencing were employed to recognize the allelic variation. A trinucleotide deletion (c.4_6delAGG) was identified in the first exon of MTRR. All the three women showed the novel clinical variation including one heterozygous and two homozygous. The siblings who had healthy babies from the same families did not harbor the variation. In the unaffected control individuals, the variant was also not observed. Eukaryotic expression and bioinformatics techniques were utilized to explore the molecular pathogenesis of the potential genetic risk of developing spina bifida. Exceptionally, the functional examination revealed that the Arg2del variant kept subcellular localization unaltered with catalytic activity intact, but failed to efficiently activate MTR compared with the wild type. Genetic disorder of folate and homocysteine metabolism during pregnancy is believed to be associated with folate-sensitive neural tube defects. The report highlights that the inframe deletion in MTRR exon 1 could be a high risk factor susceptibility to spina bifida.

Intracellular DNA sensing pathway of cGAS-cGAMP is decreased in human newborns and young children.

Newborns are highly susceptible to DNA virus infections, which may result from the characteristics of neonatal innate immune systems. Here we analyzed for the first time the development of innate immune sensing and signaling of intracellular DNA virus infection in human newborns and young children. Both mRNA and protein expression of cGAS, an intracellular DNA sensor, were shown to be significantly reduced in neonatal peripheral blood mononuclear cells (PBMCs). In addition, cGAS expression in neonatal PBMCs could be induced upon herpes simplex virus type 1 (HSV-1) or interferon-α (IFNα) stimulation. Furthermore, production of the second messenger cGAMP and activation of the transcriptional factor IRF3 was severely decreased in neonatal cord blood mononuclear cells (CBMCs) or PBMCs compared with adults. In contrast, the downstream signaling STING-TBK1-IRF3 appeared to be functional in neonatal PBMCs, as demonstrated by the fact that IRF3 phosphorylation and IFNß production in these cells could be activated by cGAMP. Intriguingly, decreased expression of cGAS in neonatal cells can be rescued by DNA demethylation, with concomitant enhancement in IFNß induction by HSV-1. Thus, cGAS restoration or STING stimulation by small molecules during infancy might improve the age-dependent susceptibility to DNA virus infection.

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

Bacterial virulence analysis using brine shrimp as an infection model in relation to the importance of quorum sensing and proteases.

Brine shrimp are aquatic crustaceans belonging to a genus of Artemia. This organism is widely used for testing the toxicity of chemicals. In this study, brine shrimp were evaluated as an infection model organism to study bacterial virulence. Artemia nauplii were infected with various pathogenic bacteria, such as Vibrio vulnificus, Pseudomonas aeruginosa, Burkholderia vietnamiensis, Staphylococcus aureus, and Escherichia coli, and the susceptibility to these bacteria was investigated by counting the survival of the infected nauplii. While all of the tested bacteria have significant virulence to brine shrimp, killing the nauplii in a few days, V. vulnificus showed the strongest virulence. P. aeruginosa also showed a dose-dependent virulence to brine shrimp, but the virulence was weaker than that of V. vulnificus. The virulence tests using the virulence-attenuated mutants of V. vulnificus and P. aeruginosa, such as quorum sensing (QS) mutants or protease-deficient mutants showed a significant attenuation of virulence, demonstrating that the QS mechanism is important in the virulence of these bacteria to brine shrimp. B. vietnamiensis, S. aureus, and E. coli were also virulent to brine shrimp and the virulence was correlated with dosage within 24 h under our conditions. Salmonella enterica Typhimurium and Bacillus subtilis were also virulent to brine shrimp, but the virulence was weak and slowly exerted compared with that of other bacteria. Taken together, we suggest that brine shrimp are a good infection model to assay bacterial virulence, especially for V. vulnificus and P. aeruginosa, and QS is important in the bacterial virulence to brine shrimp.