Optimization of the Preparation Process of Glucuronomannan Oligosaccharides and Their Effects on the Gut Microbiota in MPTP-Induced PD Model Mice.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, and accumulating evidence suggests a link between dysbiosis of the gut microbiota and the onset and progression of PD. In our previous investigations, we discovered that intraperitoneal administration of glucuronomannan oligosaccharides (GMn) derived from Saccharina japonica exhibited neuroprotective effects in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. However, the complicated preparation process, difficulties in isolation, and remarkably low yield have constrained further exploration of GMn. In this study, we optimized the degradation conditions in the preparation process of GMn through orthogonal experiments. Subsequently, an MPTP-induced PD model was established, followed by oral administration of GMn. Through a stepwise optimization, we successfully increased the yield of GMn, separated from crude fucoidan, from 1~2/10,000 to 4~8/1000 and indicated the effects on the amelioration of MPTP-induced motor deficits, preservation of dopamine neurons, and elevation in striatal neurotransmitter levels. Importantly, GMn mitigated gut microbiota dysbiosis induced by MPTP in mice. In particular, GM2 significantly reduced the levels of Akkermansia, Verrucomicrobiota, and Lactobacillus, while promoting the abundance of Roseburia and Prevotella compared to the model group. These findings suggest that GM2 can potentially suppress PD by modulating the gut microbiota, providing a foundation for the development of a novel and effective anti-PD marine drug.

Subcritical low temperature extraction of bioactive ingredients from foods and food by-products and its applications in the agro-food industry.

Bioactive ingredients are part of the food chain and are responsible for numerous health benefits. Subcritical low temperature extraction has been employed to acquire bioactive ingredients because of its excellent properties, such as energy conservation, low temperature, elimination of residual solvent, and high extraction yield and quality. This review aims to provide a clear picture of the basics of subcritical-temperature extraction, its bioactive ingredient extraction efficiency, and possible applications in the agro-food industry. This review suggested that the extraction temperature, time, co-solvents, solid-fluid ratio, and pressure impacted the extraction efficiency of bioactive ingredients from foods and food by-products. Subcritical solvents are appropriate for extracting low polar ingredients, while the inclusion of co-solvents could extract medium and high polar substances. Bioactive ingredients from foods and food by-products can be used as antioxidants, colorants, and nutritional supplements. Additionally, this technology could remove pesticide residues in tea, concentrate edible proteins, and reduce cigarette tar. A new trend toward using subcritical low temperature extraction in extracting bioactive ingredients will acquire momentum.

Ameliorative effect of mussel-derived ACE inhibitory peptides on spontaneous hypertension rats.

PURPOSE: The purpose of this study was to prepare the novel mussel-derived ACE inhibitory peptides (MEPs) by enzymatic hydrolysis of Mytilus edulis and investigate their antihypertensive effects in vivo. METHODS: After assessing the stability of MEPs in vitro, we investigated the effect of MEPs on hypertension using spontaneously hypertensive rats (SHRs). Subsequently, MEPs were purified and identified by ultrafiltration, gel filtration chromatography and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Our study demonstrated that MEPs could keep stable ACE inhibitory activity after treatment with heat, acid, alkali, metal ions and simulated gastrointestinal digestive fluid. Additionally, the animal experiments showed that both short-term and long-term treatment with MEPs resulted in a significant reduction in systolic and diastolic blood pressure in SHRs. Mechanistically, the results suggested that MEPs could reduce vascular remodeling, regulate renin-angiotensin system (RAS), and inhibit kidney and myocardial fibrosis. Finally, we isolated and identified five peptides from MEPs, with the peptide Ile-Leu-Thr-Glu-Arg showed the highest ACE inhibition rate. CONCLUSION: Our findings demonstrate the potential use of MEPs as active components in functional foods designed to lower blood pressure.

Immune Enhancement of Clam Peptides on Immunosuppressed Mice Induced by Hydrocortisone.

Clam peptides, marine-derived biological peptides, have been broadly investigated and applied as health foods, among which immunomodulation is one of their biological activities that cannot be ignored in vivo. In this study, we concentrated on exploring the effects of Ruditapes philippinarum peptides (RPPs) on immunomodulation and the balance of intestinal microbiota in hydrocortisone (HC)-induced immunosuppressed mice. The results revealed that RPPs could increase the thymus and spleen indices and number of white blood cells, promote the secretion level of cytokines (IL-2, IL-6, TNF-α, and INF-γ), repair the morphology of the spleen and thymus, and enhance the proliferation of T-lymphocyte subsets in immunosuppressed mice. Moreover, RPPs improved the abundance of beneficial bacteria and preserved the ecological equilibrium of the gut microbiota. In conclusion, RPPs have significant immunomodulatory effects on immunosuppressed mice and may be developed as immunomodulators or immune adjuvants in functional foods and drugs; they are also beneficial to the utilization of the high value of marine shellfish.

Bioactive Peptides from Ruditapes philippinarum Attenuate Hypertension and Cardiorenal Damage in Deoxycorticosterone Acetate-Salt Hypertensive Rats.

Hypertension is a common disease that affects human health and can lead to damage to the heart, kidneys, and other important organs. In this study, we investigated the regulatory effects of bioactive peptides derived from Ruditapes philippinarum (RPP) on hypertension and organ protection in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We found that RPPs exhibited significant blood pressure-lowering properties. Furthermore, the results showed that RPPs positively influenced vascular remodeling and effectively maintained a balanced water-sodium equilibrium. Meanwhile, RPPs demonstrated anti-inflammatory potential by reducing the serum levels of inflammatory cytokines (TNF-α, IL-2, and IL-6). Moreover, we observed the strong antioxidant activity of RPPs, which played a critical role in reducing oxidative stress and alleviating hypertension-induced damage to the aorta, heart, and kidneys. Additionally, our study explored the regulatory effects of RPPs on the gut microbiota, suggesting a possible correlation between their antihypertensive effects and the modulation of gut microbiota. Our previous studies have demonstrated that RPPs can significantly reduce blood pressure in SHR rats. This suggests that RPPs can significantly improve both essential hypertension and DOAC-salt-induced secondary hypertension and can ameliorate cardiorenal damage caused by hypertension. These findings further support the possibility of RPPs as an active ingredient in functional anti-hypertensive foods.

Effects of total hip arthroplasty and hemiarthroplasty on hip function in patients with traumatic femoral neck fracture.

OBJECTIVE: Traumatic femoral neck fracture is a common disease that can be treated by hip arthroplasty, which is divided into hemiarthroplasty (HA) and total hip arthroplasty (THA). The difference between HA and THA are incompletely understood. The objective of this study was to investigate the effect of hip arthroplasty on hip function in patients with traumatic femoral neck fracture. METHODS: A total of 132 patients with traumatic femoral neck fracture admitted to our hospital from January 2019 to January 2021 were selected and divided into control group (HA group) and study group (THA group) with 66 cases in each group by random number table method. The duration of operation, intraoperative blood loss, postoperative drainage and length of hospital stay were compared between the two groups. The degree of pain before operation, 3 days after operation and 7 days after operation were observed, the hip joint function before operation, 6 months after operation and 12 months after operation was analyzed, and the occurrence of short-term and long-term complications was compared between the two groups. RESULT: Compared with the HA group, the operative time, intraoperative blood loss, postoperative drainage and hospital stay were higher in the THA group. The degree of pain in THA group was higher than that in HA group on 3 and 7 days after operation; At 6 and 12 months after surgery, the scores of pain, range of motion, joint function and deformity in the THA group were higher than those in the HA group with statistically significant. Compared with HA group, IGF-1 and Leptin in THA group were increased significantly, while inflammatory cytokines TNF-α was decreased in THA group. The total incidence of short-term and long-term complications was lower in THA group. CONCLUSION: Total hip arthroplasty can effectively restore hip joint function in patients with traumatic femoral neck fracture, with low incidence of short-term and long-term complications, high safety, and worthy of clinical application.

Highly Efficient Electrogenerated Chemiluminescence Quenching on Lipid-Coated Multifunctional Magnetic Nanoparticles for the Determination of Proteases.

This work reports a highly efficient electrogenerated chemiluminescence (ECL) quenching on lipid-coated multifunctional magnetic nanoparticles (MMNP) for the determination of proteases incorporating membrane-confined quenching with a specific cleavage reaction for the first time. A new ruthenium complex [Ru(bpy)2(ddcbpy)](PF6)2 (bpy = 2,2'-bipyridine, ddcbpy = 4,4'-didodecyl-carbonyl-2,2'-bipyridine with two hydrophobic long alkyl chains) was synthesized as a signal probe, while [cholesterol-(CH2)6-HSSKLQK(peptide)-ferrocene (quencher)] was designed as a specific peptide-quencher probe. The MMNP were prepared by inserting both the signal probe and the peptide-quencher probe into the cholesterol-phospholipid-coated Fe3O4 magnetic nanoparticles (Fe3O4 NP, ∼200 nm). When prostate specific antigen (PSA) taken as a model analyte was introduced into the suspension of MMNP, PSA cleaved the amide bond of SK in cholesterol-(CH2)6-HSSKLQK-Fc, and then the cleaved peptide-motif-Fc-quencher was deviated from the MMNP, resulting in the increase in the ECL intensity. It was found that the ECL quenching constant of [Ru(bpy)2(ddcbpy)]2+ on MMNP (KSV, NP/lipECL =2.68 × 107 M-1) is 137-folds higher than that on the lipid-coated electrode (KSV, lipECL=1.95 × 105 M-1) and 391-folds higher than that in the solution (KSV, aqECL =6.86 × 104 M-1). The ECL emission of Ru(bpy)32+ derivative-attached Fe3O4 NP was observed at ∼1.2 V, involving the tunnel-electron transfer pathway (TPA• + Ru(bpy)33+ = Ru(bpy)32+*). Based on the highly efficient ECL quenching of the ruthenium complex by ferrocene on the MMNP, a new ECL method was developed for PSA with a linear range from 0.01 to 1.0 ng/mL and a limit of detection of 3.0 pg/mL. This work demonstrates that the approach of ECL quenching by ferrocene on lipid-coated Fe3O4 NP is promising and could be easily extended to determine other proteases.

Combining single-cell RNA sequencing of peripheral blood mononuclear cells and exosomal transcriptome to reveal the cellular and genetic profiles in COPD.

BACKGROUND: It has been a long-held consensus that immune reactions primarily mediate the pathology of chronic obstructive pulmonary disease (COPD), and that exosomes may participate in immune regulation in COPD. However, the relationship between exosomes and peripheral immune status in patients with COPD remains unclear. METHODS: In this study, we sequenced plasma exosomes and performed single-cell RNA sequencing on peripheral blood mononuclear cells (PBMCs) from patients with COPD and healthy controls. Finally, we constructed competing endogenous RNA (ceRNA) and protein-protein interaction (PPI) networks to delineate the interactions between PBMCs and exosomes within COPD. RESULTS: We identified 135 mRNAs, 132 lncRNAs, and 359 circRNAs from exosomes that were differentially expressed in six patients with COPD compared with four healthy controls. Functional enrichment analyses revealed that many of these differentially expressed RNAs were involved in immune responses including defending viral infection and cytokine-cytokine receptor interaction. We also identified 18 distinct cell clusters of PBMCs in one patient and one control by using an unsupervised cluster analysis called uniform manifold approximation and projection (UMAP). According to resultant cell identification, it was likely that the proportions of monocytes, dendritic cells, and natural killer cells increased in the COPD patient we tested, meanwhile the proportions of B cells, CD4 + T cells, and naïve CD8 + T cells declined. Notably, CD8 + T effector memory CD45RA + (Temra) cell and CD8 + effector memory T (Tem) cell levels were elevated in patient with COPD, which were marked by their lower capacity to differentiate due to their terminal differentiation state and lower reactive capacity to viral pathogens. CONCLUSIONS: We generated exosomal RNA profiling and single-cell transcriptomic profiling of PBMCs in COPD, described possible connection between impaired immune function and COPD development, and finally determined the possible role of exosomes in mediating local and systemic immune reactions.

Sensitive and rapid determination of heat shock protein 70 using lateral flow immunostrips and upconversion nanoparticle fluorescence probes.

Heat shock protein 70 (Hsp70), belonging to the heat shock protein (HSP) family, is reported to be a potential diagnostic biomarker. In this work, a lateral flow immunostrip was fabricated for the sensitive and rapid determination of Hsp70 by the incorporation of fluorescence and upconversion nanoparticle probes. The upconversion nanoparticles (UCNPs, size ∼39 nm, λex = 980 nm; λem = 540 nm) consisting of a NaYF4:Yb/Er core and polyacrylic acid-modified shell were covalently coupled with Hsp70 antibodies to form the signal probe, which was characterized by dynamic light scattering and zeta potential analyses. The lateral flow assay (LFA) was constructed based on the sandwich-type immunoassay using a sample pad, a test pad, and an adsorption pad on a PVP backing. Hsp70 antibody, IgG antibody and the signal probe were separately dropped on the test zone, the control zone of the test pad, and the sample pad, respectively. In the sandwich LFA, since two antibodies bind to Hsp70 antigenic epitopes, i.e. specific binding, it provided superior specificity and high sensitivity, making it an ideal sensing platform for complex samples like serum Hsp70 samples. The important parameters for the preparation of the lateral flow immunostrips were optimized. Under the optimized conditions, Hsp70 can be detected using the increased fluorescence intensity of UCNPs with a wide linear range from 0.11 to 12 ng mL-1, low detection limit of 0.06 ng mL-1, small sample volume (120 µL), short assay time (15 min) and good reproducibility. The fluorescence method was successfully applied in the determination of Hsp70 in serum samples with good recovery. By combining the accessibility of the lateral flow immunostrips and upconversion nanoparticles, the fluorescence method can serve as a point-of-care testing method for protein assays with high sensitivity and fast detection.

Positive effect of compound amino acid chelated calcium from the shell and skirt of scallop in an ovariectomized rat model of postmenopausal osteoporosis.

BACKGROUND: Osteoporosis has become an important public health issue with the increase of aging population, and afflicts millions of people worldwide, particularly elderly or postmenopausal women. In the present study, we prepared compound amino acid chelated calcium (CAA-Ca) from processing by-products of Chlamys farreri, and evaluated its effect on postmenopausal osteoporosis with an ovariectomized (OVX) rat model. RESULTS: A 60-day treatment of OVX rats with CAA-Ca significantly enhanced the bone mineral density (BMD) and the bone calcium content. Meanwhile, some bone morphometric parameters, trabecular bone number (Tb.N), trabecular bone volume fraction (BV/TV), trabecular bone thickness (Tb.Th) and cortical bone wall thickness (Ct.Th), were also increased by 8.20%, 118.18%, 32.99% and 19.10%, respectively. In addition, the alkaline phosphatase (ALP) levels in serum were significantly reduced after CAA-Ca treatment, while the blood calcium levels were increased. Mechanistically, CAA-Ca down-regulated the levels of receptor activator of nuclear factor-κB (RANK) and receptor activator of nuclear factor-κB ligand (RANKL), and up-regulated osteoprotegerin (OPG) levels in osteoclasts, inhibiting bone resorption and bone loss. Meanwhile, CAA-Ca treatment raised ß-catenin levels and lowered Dickkopf1 (DKK1) levels in the Wnt signaling pathway of osteoblasts, which can promote calcium absorption and bone formation. CONCLUSION: The results suggested that CAA-Ca promoted bone formation, inhibited bone resorption and improved bone microstructure. Therefore, this study contributes to the potential application of CAA-Ca as a functional food resource in the treatment of postmenopausal osteoporosis. © 2021 Society of Chemical Industry.

Electrogenerated Chemiluminescence Immunoassays on Nanoelectrode Ensembles Platform with Magnetic Microbeads for the Determination of Carbohydrate Antigen.

This work reports a gold nanoelectrode ensembles (Au-NEE) platform taken as a disposable electrogenerated chemiluminescence (ECL) platform with immunomagnetic microbeads for ECL immunoassays for the first time. The peak-shaped voltammograms were obtained at the Au-NEE, attributed to the total diffusional overlap. The ECL intensity at Au-NEE was 12.9 folds in the Ru(bpy)32+-tri-n-propylamine (TPA) ECL system and 19.6 folds in the luminol-H2O2 system, compared with that at the Au macroelectrode using the normalized active area of the electrodes, mainly attributed to the diffusion overlap of the Au-NEE and the edge effect of the individual gold nanodisks of the Au-NEE. The ECL immunoassay on the Au-NEE platform with magnetic microbeads for the determination of cancer biomarkers was developed. Carbohydrate antigen 19-9 (CA 19-9) was chosen as a model analyte while CA 19-9 antibody on the magnetic microbeads was taken as the capture probe, and ruthenium complex-labeled CA 19-9 antibody was used as the signal probe. A "sandwich" bioconjugates on the magnetic beads were transferred onto the ECL platform, and then the ECL measurements were performed in TPA solution. The developed method showed that the ECL peak intensity was directly in proportion to the concentration of CA 19-9 in the range from 0.5 to 20 U/mL with a limit of detection of 0.4 U/mL. This work demonstrates that the Au-NEE can be employed as a useful disposable ECL platform with the merits of cheapness, low nonspecific adsorption and practical application. The proposed approach will open a new avenue in the point-of-care test for the determination of protein biomarkers.

Effects of peanut meal extracts fermented by Bacillus natto on the growth performance, learning and memory skills and gut microbiota modulation in mice.

Recent studies have demonstrated that the nutritional properties of peanut meal (PM) can be improved after being fermented. The assessment of fermented PM has been reported to be limited to various physical and chemical evaluations in vitro. In the present study, PM was fermented by Bacillus natto to explore the effects of fermented PM extract (FE) on growth performance, learning and memory ability and intestinal microflora in mice. Ninety newly weaned male Kunming (KM) mice were randomly divided into seven groups: normal group (n 20), low-dose FE group (n 10), middle-dose FE group (MFE) (n 10), high-dose FE group (HFE) (n 20), unfermented extraction group (n 10), model group (10) and natural recovery group (10). Learning and memory skills were performed by the Morris water maze (MWM) test, and the variation in gut microbiota (GM) composition was assessed by 16S rDNA amplicon sequencing. The results show that HFE remarkably improved the growth performance in mice. In the MWM test, escape latency was shortened in both MFE and HFE groups, while the percentage of time, distance in target quadrant and the number crossing over the platform were significantly increased in the HFE group. Moreover, the FE played a preventive role in the dysbacteriosis of mice induced by antibiotic and increased the richness and species evenness of GM in mice.

Species-Specific Identification of Streptococcus based on DNA Marker in 16S-23S rDNA Internal Transcribed Spacer.

Streptococcus is closely correspondent to human. The accurate species-specific identification method of Streptococcus is important for the bacteria clinical diagnosis, molecular epidemiological analysis, and microecological study. In the last decades, DNA markers are widely utilized for identification of prokaryotic species. However, 16S rDNA, the most popular bacterial DNA marker, cannot properly distinguish closely related Streptococcus species. In present study, we employed 16S-23S rRNA gene internal transcribed spacer (ITS) sequence to explore the species-specific DNA marker. We predicted the secondary structure of Streptococcus ITS sequence transcribed products. Then we identified that the specific and consensus sequences in the primary structure can be found occupying an individual subunit in the secondary structure, which explained the foundation of the mosaic-like structure of ITS. We evaluated the specificity of ITS in Streptococcus, and found that the specificity can be detected by a further analysis of a BLAST result. Then, we developed an identification procedure based on the ITS sequence. We verified the procedure by 500 ITS sequence. The accuracy rate of this procedure was 100% for Streptococcus at genus level, and 99.3% at species level. It suggested that ITS can be utilized to accurately identify Streptococcus at the species level. This work suggests that further exploration of ITS could be applied in other bacterial genera for identification and classification, which may be a useful topic for future microbiology studies.

Lateral flow immunostrips for the sensitive and rapid determination of 8-hydroxy-2'-deoxyguanosine using upconversion nanoparticles.

Lateral flow immunostrips were newly designed and a sensitive and rapid fluorometric method for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a model target of small biomarker molecules was developed. The upconversion nanoparticles (UCNPs, NaYF4:Yb/Er core, and polyacrylic acid (PAA)-modified shell, size ~ 39 nm, excitation wavelength = 980 nm; emission wavelength = 540 nm) were employed as fluorescence signal material. The 8-OHdG antibody (Ab) was taken as the recognition probe while UCNP-labeled Ab was taken as the signal probe. Bovine serum albumin (BSA) was designed as carrier protein for 8-OHdG to form 8-OHdG-BSA conjugate as the capture probe. The lateral flow immunostrips were prepared by laminating a sample pad (glass fiber membrane), a test pad (nitrocellulose membrane), and adsorption pad (filter paper) on PVP backing. The capture probe was immobilized on the test zone while an IgG antibody taken as the control probe was immobilized on the control zone. When the signal probe and the sample were in sequence loaded on the sample pad, 8-OHdG analyte bound with the signal probe, and then the excess of the signal probe move along the strip and is collected by the capture probe on the test zone while the remnant signal probe is collected by the control probe on the control zone. The signal probe and capture probe were synthesized and characterized. The fluorescence intensity on the test zone was inversely proportional to the concentration of 8-OHdG for the quantitative determination while the fluorescence emission on the control zone was observed to validate the assay. The developed method showed a wide linear range from 0.10 to 10 nM, a quite low detection limit of 0.05 nM, small sample volume requirement (100 µL), short assay time (15 min), and good method reproducibility (RSD = 4.4%, nine immunostrips). Graphical abstract Schematic illustration of the configuration and measurement principle of lateral flow fluorescence immunostrip for 8-OHdG: (a) configuration; (b) preparation: load of capture probe (BSA-8-OHdG, 2 µL) on test zone; load of control probe (IgG Ab, 2 µL) on control zone; load of signal probe (UCNP-Ab, 16 µL) on sample pad; (c) measurement: load of sample (8-OHdG, 100 µL) on sample pad, collection, and measurement.

Homogeneous electrogenerated chemiluminescence immunoassay for the detection of biomarkers by magnetic preconcentration on a magnetic electrode.

A homogeneous electrogenerated chemiluminescence (ECL) immunoassay for highly sensitive quantification of specific biomarkers based on immunomagnetic beads and homogeneous detection on a magnetic electrode was developed, for the first time. The magnetic electrode is made of a glassy carbon electrode and a series of ring permanent magnets. D-Dimer antigen was taken as a model analyte while biotinylated D-dimer antibody bound on the streptavidin-coated magnetic beads was utilized as a magnetic capture probe and ruthenium complex-labeled D-dimer antibody was employed as an ECL probe. After a fixed amount of magnetic capture probe and the ECL probe was introduced into analyte D-dimer solution, the "sandwich" immunoconjugates on the magnetic beads were formed in tested solution and then magnetically concentrated on the surface of the magnetic electrode. The homogeneous ECL immunoassay for quantification of specific biomarker was directly carried out in the presence of co-reactant tripropylamine. The low detection limit of 1 ng/mL in magnetic enrich time of 2 min and the good magnetic regeneration for the detection of D-dimer were achieved. The magnetic bead shield ECL emission was extensively discussed. This work demonstrates that the homogeneous (separation-free) ECL immunoassay using magnetic beads and magnetic electrode is a promising approach to quantify the biomarkers with high sensitivity and selectivity and in a short time. This approach can be easily extended to ECL and electrochemical biosensing for other biomarkers. Graphical abstract.

Improvement and evaluation of loop-mediated isothermal amplification combined with a chromatographic flow dipstick assay and utilization in detection of Vibrio cholerae.

Loop-mediated isothermal amplification (LAMP) is a specific, sensitive, and easy-to-perform nucleic acid analytical technique with wide application for diagnosis of disease. Recently, LAMP combined with use of a lateral chromatographic flow dipstick (LFD) has been widely used in nucleic acid detection. However, the LFD mechanism has not been systematically analyzed, and the optimal combination of labeled primers has not been adequately evaluated. We analyzed the LAMP mechanism and discovered that the labeled loop primers played a significant role in the LFD assay. To verify our hypothesis, we developed two LFD assays for Vibrio cholerae to detect the ctxA gene and the 16S-23S ribosomal DNA internal transcribed spacer (ITS). We labeled the inner primers [forward inner primer (FIP) and backward inner primer (BIP)] and loop primers [forward loop primer (LF) and backward loop primer (LB)]. Then the labeled and unlabeled primers were combined to form ten different primer sets. We assessed the specificity, sensitivity, and efficiency of LFD assays with use of different primer compositions. All triple-labeled primer sets resulted in false positive results in the LFD assay, as did the FIP and BIP double-labeled primer set. Other double-labeled-primer sets used in LFD assays showed higher sensitivity than the LAMP assays. Moreover, FIP and LF double-labeled and BIP and LB double-labeled sets had the highest sensitivity. In both cases, assays could be performed in 20 min. We also applied the ITS LFD assays in food samples. The enrichment broths of 112 oyster samples were tested, and the proportion that tested positive by the LFD assays was 6.25%, which was not lower than the rate for the conventional PCR method (5.36%). Graphical abstract ᅟ.

Data-Dependent Acquisition and Database-Driven Efficient Peak Annotation for the Comprehensive Profiling and Characterization of the Multicomponents from Compound Xueshuantong Capsule by UHPLC/IM-QTOF-MS.

The state of the art ion mobility quadrupole time of flight (IM-QTOF) mass spectrometer coupled with ultra-high performance liquid chromatography (UHPLC) can offer four-dimensional information supporting the comprehensive multicomponent characterization of traditional Chinese medicine (TCM). Compound Xueshuantong Capsule (CXC) is a four-component Chinese patent medicine prescribed to treat ophthalmic disease and angina. However, research systematically elucidating its chemical composition is not available. An approach was established by integrating reversed-phase UHPLC separation, IM-QTOF-MS operating in both the negative and positive electrospray ionization modes, and a "Component Knockout" strategy. An in-house ginsenoside library and the incorporated TCM library of UNIFITM drove automated peak annotation. With the aid of 85 reference compounds, we could separate and characterize 230 components from CXC, including 155 ginsenosides, six astragalosides, 16 phenolic acids, 16 tanshinones, 13 flavonoids, six iridoids, ten phenylpropanoid, and eight others. Major components of CXC were from the monarch drug, Notoginseng Radix et Rhizoma. This study first clarifies the chemical complexity of CXC and the results obtained can assist to unveil the bioactive components and improve its quality control.

Optimization and characterization of pullulan production by a newly isolated high-yielding strain Aureobasidium melanogenum.

Pullulan is an extracellular water-soluble polysaccharide with wide applications. In this study, we screened strains that could selectively produce high molecular weight pullulan for application in industrial pullulan production. A new fungus strain A4 was isolated from soil and identified as Aureobasidium melanogenum based on colony characteristics, morphology, and internally transcribed spacer analysis. Thin-layer chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance analysis suggested that the dominant exopolysaccharide produced by this strain, which presented a molecular weight of 1.384 × 106 Dalton in in-gel permeation chromatography, was pullulan. The culture conditions for A. melanogenum A4 were optimized at 30 °C and 180 rpm: carbon source, 50 g/L maltose; initial pH 7; and 8 g/L Tween 80. Subsequently, batch fermentation was performed under the optimized conditions in a 5-L stirred-tank fermentor with a working volume of 3 L. The fermentation broth contained 303 g/L maltose, which produced 122.34 g/L pullulan with an average productivity of 1.0195 g/L/h and 82.32 g/L dry biomass within 120 h. The conversion efficiency of maltose to pullulan (Y%) and specific production rate (g/h/g dry cells) (Qs) reached 40.3% and 0.0251 g/L/g dry cells, respectively. The results showed strain A4 could be a good candidate for industrial production.

Marinobacter bohaiensis sp. nov., a moderate halophile isolated from benthic sediment of the Bohai Sea.

A Gram-stain-negative, motile, aerobic and rod-shaped bacterial strain, designated T17T, was isolated from benthic sediment sampled at Jiaozhou Bay, Bohai Sea, China, and its taxonomic position was investigated. The 16S rRNA gene sequence of strain T17T exhibited the highest similarity values to those of the type strain Marinobacter lacisalsi FP2.5 (96.2 %) and Marinobacter koreensis DD-M3T (96.2 %). Strain T17T grew optimally at 35 °C, pH 7.0-8.0 and in the presence of 6.0-10.0 % (w/v) NaCl. The predominant ubiquinone in strain T17T was identified as Q-9. The major fatty acids of strain T17T were C12 : 0, C16 : 0 and C16 : 0 10-CH3. The major polar lipids of strain T17T were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidglycerol, an unidentified aminolipid and an unidentified phospholipid. The DNA G+C content of strain T17T was 63.0 mol%. The draft genome sequence of strain T17T includes 4 755 891 bp in total (N50=2 856 325 bp) with a medium read coverage of 100.0x and 11 scaffolds. In silico DNA-DNA hybridization with the three type strains showed 20.3, 19.7 and 19.9 % relatedness to Marinobacter santoriniensis NKSG1T, Marinobacter segnicrescens SS11B1-4T and Marinobacter daqiaonensis CGMCC 1.9167T, respectively. On the basis of the phenotypic, phylogenetic, genomic and chemotaxonomic properties, strain T17T is considered to represent a novel species within the genus Marinobacter, for which the name Marinobacterbohaiensis sp. nov. is proposed. The type strain is T17T (=KCTC 52710T=MCCC 1K03282T).

Thoracic Inlet Parameters for Degenerative Cervical Spondylolisthesis Imaging Measurement.

BACKGROUND The aim of this study was to explore the diagnostic value of sagittal measurement of thoracic inlet parameters for degenerative cervical spondylolisthesis (DCS). MATERIAL AND METHODS We initially included 65 patients with DCS and the same number of health people as the control group by using cervical radiograph evaluations. We analyzed the x-ray and computer tomographic (CT) data in prone and standing position at the same time. Measurement of cervical sagittal parameters was carried out in a standardized supine position. Multivariate logistic regression analysis was performed to evaluate these parameters as a diagnostic index for DCS. RESULTS There were 60 cases enrolled in the DCS group, and 62 cases included in the control group. The T1 slope and thoracic inlet angle (TIA) were significantly greater for the DCS group compared to the control group (24.33±2.85º versus 19.59±2.04º, p=0.00; 76.11±9.82º versus 72.86±7.31º, p=0.03, respectively). We observed no significant difference for the results of the neck tilt (NT), C2-C7 angle in the control and the DSC group (p>0.05). Logistic regression analysis and receiver operating characteristic (ROC) curve revealed that preoperative T1 slope of more than 22.0º showed significantly diagnostic value for the DCS group (p<0.05). CONCLUSIONS Patients with preoperative sagittal imbalance of thoracic inlet have a statistically significant increased risk of DCS. T1 slope of more than 22.0º showed significantly diagnostic value for the incidence of DCS.