Development of fluorescence/MR dual-modal manganese-nitrogen-doped carbon nanosheets as an efficient contrast agent for targeted ovarian carcinoma imaging.

BACKGROUND: Development of sensitive and specific imaging approaches for the detection of ovarian cancer holds great promise for improving the therapeutic efficacy and the lifespan of the patients. RESULTS: In this study, manganese-nitrogen doped carbon nanosheets (Mn-N-CNSs) coupled with Anti-HE4 monoclonal antibody (Mn-N-CNSs@Anti-HE4) were synthesized for the specific and targeted fluorescence/MR dual-modal imaging of ovarian carcinoma. The prepared Mn-N-CNSs revealed excellent aqueous dispersity, good colloidal stability, great optical properties and high longtudinal relaxivity rate (r1 = 10.30 mM-1 s-1). Encouraged by the tunable photoluminiscence of the nanoprobe and Anti-HE4 targeting ligand, the ovarian carcinoma cells were specifically labeled by the Mn-N-CNSs@Anti-HE4 nanoprobe with multi-color fluorescences. Benefiting from the high r1 relaxivity, the nanoprobe exhibited targeted and enhanced MR contrast effect in the ovarian carcinoma cells and tumor bearing mice model. Besides, the high biocompatibility and easy excretion from the body of the nanoprobe were further confirmed in vivo. CONCLUSION: The prepared Mn-N-CNSs@Anti-HE4 with excellent biocompatibility, high-performance and superior tumor-targeting ability provides a novel fluorescence/MR dual-modal nanoprobe for specific labeling and detection of ovarian carcinoma cells in vitro and in vivo.

NAD-Independent L-Lactate Dehydrogenase Required for L-Lactate Utilization in Pseudomonas stutzeri A1501.

UNLABELLED: NAD-independent L-lactate dehydrogenases (l-iLDHs) play important roles in L-lactate utilization of different organisms. All of the previously reported L-iLDHs were flavoproteins that catalyze the oxidation of L-lactate by the flavin mononucleotide (FMN)-dependent mechanism. Based on comparative genomic analysis, a gene cluster with three genes (lldA, lldB, and lldC) encoding a novel type of L-iLDH was identified in Pseudomonas stutzeri A1501. When the gene cluster was expressed in Escherichia coli, distinctive L-iLDH activity was detected. The expressed L-iLDH was purified by ammonium sulfate precipitation, ion-exchange chromatography, and affinity chromatography. SDS-PAGE and successive matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the purified L-iLDH indicated that it is a complex of LldA, LldB, and LldC (encoded by lldA, lldB, and lldC, respectively). Purified L-iLDH (LldABC) is a dimer of three subunits (LldA, LldB, and LldC), and the ratio between LldA, LldB, and LldC is 1:1:1. Different from the FMN-containing L-iLDH, absorption spectra and elemental analysis suggested that LldABC might use the iron-sulfur cluster for the L-lactate oxidation. LldABC has narrow substrate specificity, and only L-lactate and DL-2-hydrobutyrate were rapidly oxidized. Mg(2+) could activate L-iLDH activity effectively (6.6-fold). Steady-state kinetics indicated a ping-pong mechanism of LldABC for the L-lactate oxidation. Based on the gene knockout results, LldABC was confirmed to be required for the L-lactate metabolism of P. stutzeri A1501. LldABC is the first purified and characterized L-iLDH with different subunits that uses the iron-sulfur cluster as the cofactor. IMPORTANCE: Providing new insights into the diversity of microbial lactate utilization could assist in the production of valuable chemicals and understanding microbial pathogenesis. An NAD-independent L-lactate dehydrogenase (L-iLDH) encoded by the gene cluster lldABC is indispensable for the L-lactate metabolism in Pseudomonas stutzeri A1501. This novel type of enzyme was purified and characterized in this study. Different from the well-characterized FMN-containing L-iLDH in other microbes, LldABC in P. stutzeri A1501 is a dimer of three subunits (LldA, LldB, and LldC) and uses the iron-sulfur cluster as a cofactor.

Virtual monoenergetic imaging predicting Ki-67 expression in lung cancer.

This study aimed to optimize slope and energy levels for evaluating Ki-67 expression in lung cancer using virtual monoenergetic imaging and compare the predictive efficiency of different energy spectrum slopes (λHU) for Ki-67. Forty-three patients with primary lung cancer confirmed via pathological examination were enrolled in this study. They underwent baseline arterial-phase (AP) and venous-phase (VP) energy spectrum computed tomography (CT) scanning before surgery. The CT values were 40-190 keV, with 40-140 keV indicating pulmonary lesions at AP and VP, and P < 0.05 indicating a statistically significant difference. An immunohistochemical examination was conducted, and receiver operating characteristic curves were used to analyze the prediction performance of λHU for Ki-67 expression. SPSS Statistics 22.0 (IBM Corp., NY, USA) was used for statistical analysis, and χ2, t, and Mann-Whitney U tests were used for quantitative and qualitative analyses of data. Significant differences were observed at the corresponding CT values of 40 keV (as 40-keV is considered the best for single-energy image for evaluating Ki-67 expression) and 50 keV in AP and at 40, 60, and 70 keV in VP between high- and low-Ki-67 expression groups (P < 0.05). In addition, the λHU values of three-segment energy spectrum curve in both AP and VP were quite different between two groups (P < 0.05). However, the VP data had greater predictive values for Ki-67. The areas under the curve were 0.859, 0.856, and 0.859, respectively. The 40-keV single-energy sequence was the best single-energy sequence to evaluate the expression of Ki-67 in lung cancer and to obtain λHU values using the energy spectrum curve in the VP. The CT values had better diagnostic efficiency.

Insight into the protein oxidation impact on the surface properties of myofibrillar proteins from bighead carp.

The objective of this study was to elucidate the influence of oxidative modifications of myofibrillar proteins (MPs) on their surface properties. Oxidative modifications (deamination, formation of disulfide bonds and Schiff bases), particle size, net surface charge, and binding ability of volatiles (2-enthylfuran, 1-octen-3-ol, hexanal, and octanal) of oxidized MPs was measured. Molecular docking of volatiles with actomyosin was performed using Qvina-W program and the specific oxidative modifications (monoxidation and deamination) of MPs were determined using LC-MS/MS. Results showed that oxidation of Cys (forming sulfinic, sulfonic, sulfenic acid, and disulfide bonds), monoxidation of Ala, Lys, Glu, and Asn, and deamination of Lys changed the surface properties of oxidized MPs including enhanced surface hydrophobicity and decreased affinity to volatile compounds and water. Overall, this study gives evidence of how protein oxidation affects the properties of MPs and therefore deteriorates fish meat quality.

Lactate utilization is regulated by the FadR-type regulator LldR in Pseudomonas aeruginosa.

NAD-independent L-lactate dehydrogenase (l-iLDH) and NAD-independent D-lactate dehydrogenase (D-iLDH) activities are induced coordinately by either enantiomer of lactate in Pseudomonas strains. Inspection of the genomic sequences of different Pseudomonas strains revealed that the lldPDE operon comprises 3 genes, lldP (encoding a lactate permease), lldD (encoding an L-iLDH), and lldE (encoding a D-iLDH). Cotranscription of lldP, lldD, and lldE in Pseudomonas aeruginosa strain XMG starts with the base, C, that is located 138 bp upstream of the lldP ATG start codon. The lldPDE operon is located adjacent to lldR (encoding an FadR-type regulator, LldR). The gel mobility shift assays revealed that the purified His-tagged LldR binds to the upstream region of lldP. An XMG mutant strain that constitutively expresses D-iLDH and L-iLDH was found to contain a mutation in lldR that leads to an Ile23-to-serine substitution in the LldR protein. The mutated protein, LldR(M), lost its DNA-binding activity. A motif with a hyphenated dyad symmetry (TGGTCTTACCA) was identified as essential for the binding of LldR to the upstream region of lldP by using site-directed mutagenesis. L-Lactate and D-lactate interfered with the DNA-binding activity of LldR. Thus, L-iLDH and D-iLDH were expressed when the operon was induced in the presence of L-lactate or D-lactate.

Genome sequence of Pseudomonas stutzeri SDM-LAC, a typical strain for studying the molecular mechanism of lactate utilization.

Pseudomonas stutzeri SDM-LAC is an efficient lactate utilizer with various applications in biocatalysis. Here we present a 4.2-Mb assembly of its genome. The annotated four adjacent genes form a lactate utilization operon, which could provide further insights into the molecular mechanism of lactate utilization.

Genome sequence of the lactate-utilizing Pseudomonas aeruginosa strain XMG.

Pseudomonas aeruginosa XMG, isolated from soil, utilizes lactate. Here we present a 6.45-Mb assembly of its genome sequence. Besides the lactate utilization mechanism of the strain, the genome sequence may also provide other useful information related to P. aeruginosa, such as identifying genes involved in virulence, drug resistance, and aromatic catabolism.

Rationally re-designed mutation of NAD-independent L-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli.

BACKGROUND: NAD-independent L-lactate dehydrogenase (L-iLDH) from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids. RESULTS: Val-108 of L-iLDH was changed to Ala by rationally site-directed mutagenesis. The L-iLDH mutant exhibited much higher activity than wide-type L-iLDH towards L-mandelate, an aromatic 2-hydroxycarboxylic acid. Using the engineered Escherichia coli expressing the mutant L-iLDH as a biocatalyst, 40 g·L(-1) of DL-mandelic acid was converted to 20.1 g·L(-1) of D-mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g·L(-1) of benzoylformic acid. CONCLUSIONS: A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally re-design mutagenesis. Two building block intermediates (optically pure D-mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system.

MicroRNA expression profile of cholesterol metabolism analysis mediated by Thelenota ananas desulfated holothurin A saponin in RAW264.7 macrophage-foam cells.

Saponins from the sea cucumber, Thelenota ananas, have been reported to modulate cholesterol metabolism and may be useful in treating atherosclerosis and related diseases, but the mechanism remains unclear. This study is designed to investigate the effect of the saponin desulfated holothurin A (desHA), prepared from Thelenota ananas, on cholesterol and lipid metabolism in oxidized low-density lipoprotein (oxLDL)-induced RAW264.7 macrophage-foam cells. The detection and prediction of miRNAs were conducted by high-throughput sequencing and associated bioinformatics analysis. We found that desHA could play an essential role in the species and expression of miRNAs. There were more abundant miRNAs in the desHA-treated groups. miR-365-2-5p, -125b-1-3p, -744-5p, -330-3p, -125a-3p and -3057-5p were extremely suppressed by desHA under the oxLDL treatment, while miR-212-3p and miR-132-3p were significantly upregulated by desHA. The clusters of orthologous groups (COG) analysis indicated that the detected miRNAs participated in lipid transport and metabolism (I) and secondary metabolite biosynthesis, transport, and catabolism (Q), which were closely linked with cholesterol metabolism. The network of the nine miRNAs with higher degree of differential expression (miR-125a-3p, -23b-5p, -3057-5p, -330-3p, -365-2-5p and -744-5p, -345-5p -212-3p and -132-3p) and their target genes further showed relationships with inflammatory response, cell proliferation and cholesterol metabolism. Due to miR-125a-3p and miR-365-2-5p being involved in the regulation of more genes, which are mostly related to the functions involved in cholesterol metabolism, they may be potential targets for desHA to prevent or treat atherosclerosis.

Serial CT changes in different components of lung cancer associated with cystic airspace in patients treated with neoadjuvant chemotherapy.

The aim of this study was to observe changes in different components (solid, cystic airspace, or entire tumor) in lung cancer associated with cystic airspace following treatment with neoadjuvant chemotherapy (NC), using computerized tomography (CT). We analyzed serial (baseline, first-time follow-up, and last-time follow-up) clinical data and CT imaging in six patients treated with NC. The diameters, areas, and volumes of different tumor components (solid, cystic airspace, and entire tumor) were measured. Delta (Δ) was used to represent changes in these parameters between two examinations: Δ1(%) represents the change from baseline to first follow-up after NC, and Δ2(%) represents the change from baseline to last follow-up after NC. We used the intra-group correlation coefficient (ICC) to test for consistency between parameters as measured by two radiologists. The diameter of solid components in all lesions showed a trend of continuous reduction compared with baseline (Δ1 ranged from - 8.3 to - 46.0%, Δ2 from - 30.8 to - 69.2%). For cystic airspace and entire tumors, different lesions showed different trends over the course of treatment. For diameter, area, and volume, Δ1 of changes in the solid component ranged from - 8.3 to - 46.9%, - 19.4 to - 70.8%, and - 19.1 to - 94.7%, respectively; Δ2 ranged from - 30.8 to - 69.2%, - 50.8 to - 92.1%, and - 32.7 to - 99.8% in diameter, area, and volume, respectively. Results were inconsistent between different components of lung cancer associated with cystic airspace that was treated with NC, but the diameter, area, and volume of solid components were continuously reduced during treatment. Furthermore, area and volume measurements showed more-significant variation than diameter measurements.

Effect of MTGase on silver carp myofibrillar protein gelation behavior after peroxidation induced by peroxyl radicals.

The objective of this study was to explore the oxidative modification induced by AAPH (2,2'-azobis (2-amidinopropane) dihydrochloride) on the microbial transglutaminase (MTGase) cross-linking reaction and gelling properties of silver carp myofibril protein (SCMP). Compared to AAPH treatment, MTGase addition resulted further changes of protein properties as evidenced by the decreasing free amino and thiol group content, the decreased secondary and tertiary structure, and the increasing storage modulus (G') and gel strength (P < 0.05). The proper oxidation induced by AAPH (5 mM) promoted the glutamine-lysine and disulfide cross-linking due to MTGase (10 U/g). Therefore, the quality of the SCMP gel was improved with a good water-holding capacity (WHC), gel strength and G'. This results could lay a theoretical foundation for the development of silver carp surimi products with good quality. Chemical compounds: (2,2'-azobis(2-amidinopropane)dihydrochloride (PubChem CID:76344); O-Phthalaldehyde (PubChem CID:4807); 5,5'-Dithiobis(2-Nitrobenzoic Acid) (PubChem CID:6254); 8-Anilino-1-naphthalenesulfonic acid (PubChem CID:1369); Acrylamide (PubChem CID: 6579); ß-Mercaptoethanol (PubChem CID: 1567); Sodium dodecyl sulfate (PubChem CID:3423265).

Development of FL/MR dual-modal Au nanobipyramids for targeted cancer imaging and photothermal therapy.

Multifunctional nanodrugs have emerged as an effective platform to integrate multiple imaging and therapeutic functions for tremendous biomedical applications. However, the development of a simple potent theranostic nanoplatform is still an intractable challenge. Herein, a novel theranostic nanoplatform was developed by coupling prepared Au nanobipyramids with Gd2O3, Au nanoclusters and denatured bovine serum albumin (AuNBP-Gd2O3/Au-dBSA) for FL/MR dual-modal imaging guided photothermal therapy. AS1411 aptamers were conjugated to enhance its targetability towards breast cancer. The AS1411-AuNBP-Gd2O3/Au-dBSA suspension could be readily heated above 40 °C at a low concentration (2 mg/L) and NIR density (1 W/cm2). The AS1411-AuNBP-Gd2O3/Au-dBSA revealed a fluorescence quantum yield of 4.2% and higher longitudinal relaxivity rate of 6.75 mM-1 s-1 compared to Gd-DTPA of 4.45 mM-1 s-1. As a result, the AS1411-AuNBP-Gd2O3/Au-dBSA functions as a multimodal nanoprobe of photothermal, fluorescence and MR imaging for specific tumor diagnosis and guidance of therapy, which was validated via in vitro and in vivo tests. Moreover, AS1411-AuNBP-Gd2O3/Au-dBSA nanoparticles indicated excellent photothermal anticancer effect more than 95% in both in vitro and in vivo tests. Besides, the low toxicity of AS1411-AuNBP-Gd2O3/Au-dBSA nanocomposites was further confirmed in vitro and in vivo. Thus, these results demonstrated the AS1411-AuNBP-Gd2O3/Au-dBSA nanocomposites as a rational design of multifunctional nanoplatform to enable multimodal imaging guided photothermal therapy.

Tumor-targeted Gd-doped mesoporous Fe3O4 nanoparticles for T1/T2 MR imaging guided synergistic cancer therapy.

In this study, a novel intelligent nanoplatform to integrate multiple imaging and therapeutic functions for targeted cancer theranostics. The nanoplatform, DOX@Gd-MFe3O4 NPs, was constructed Gd-doped mesoporous Fe3O4 nanoparticles following with the doxorubicin (DOX) loading in the mesopores of the NPs. The DOX@Gd-MFe3O4 NPs exhibited good properties in colloidal dispersity, photothermal conversion, NIR triggered drug release, and high T1/T2 relaxicity rate (r1=9.64 mM-1s-1, r2= 177.71 mM-1s-1). Benefiting from the high MR contrast, DOX@Gd-MFe3O4 NPs enabled simultaneous T1/T2 dual-modal MR imagining on 4T1 bearing mice in vivo and the MR contrast effect was further strengthened by external magnetic field. In addition, the DOX@Gd-MFe3O4 NPs revealed the strongest inhibition to the growth of 4T1 in vitro and in vivo under NIR irradiation and guidance of external magnetic field. Moreover, biosafety was also validated by in vitro and in vivo tests. Thus, the prepared DOX@Gd-MFe3O4 NPs would provide a promising intelligent nanoplatform for dual-modal MR imagining guided synergistic therapy in cancer theranostics.

Cloning, expression, purification, and activity assay of proteins related to D-lactic acid formation in Lactobacillus rhamnosus.

Two proteins that might be responsible for D-lactic acid (D-LA) formation were screened from the genome database of Lactobacillus rhamnosus GG. The coding genes of the two proteins in L. rhamnosus CASL, ldhD1 and ldhD2, were cloned and expressed in Escherichia coli Rosetta with an inducible expression vector pETDuet-1 (Novagen, Darmstadt, Germany), respectively. The two purified proteins, LdhD-1 and LdhD-2, migrated as a single protein band separately, both corresponding to an apparent molecular mass between 35 kDa and 45 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activities of LdhD-1 and LdhD-2 catalyzing pyruvate to LA were 0.02 U/mg and 0.21 U/mg, respectively. The configuration of LA converted from pyruvate was determined using high-performance liquid chromatography equipped with a chiral column. Only D-LA was detected when LdhD-1 and LdhD-2 were tested. In summary, the two proteins cloned and expressed in this study were most probably responsible for D-LA formation during fermentation of L. rhamnosus CASL.

Tumor Targeted Multifunctional Magnetic Nanobubbles for MR/US Dual Imaging and Focused Ultrasound Triggered Drug Delivery.

The development of multifunctional nanoplatforms that are safe and have multiple therapeutic functions integrated with dual- or multi-imaging modality is one of the most urgent medical requirements for active cancer therapy. In our study, we prepared multifunctional magnetic nanobubbles (MF-MNBs) by co-encapsulating superparamagnetic iron oxide nanoparticles (SPIONs) and doxorubicin into polylactideco-glycolide-polyethylene glycol-folate (PLGA-PEG-FA) polymer-based nanobubbles for tumor-targeted ultrasound (US)/magnetic resonance (MR) imaging and focused ultrasound (FUS)-triggered drug delivery. Hydrophobic SPIONs were successfully embedded into MF-MNBs by a typical double emulsion process. The MF-MNBs were highly dispersed with well-defined spherical morphology and an average diameter of 208.4 ± 12.58 nm. The potential of MF-MNB as a dual-modal contrast agent for US and MR imaging was investigated via in vitro study, and the MF-MNB exhibits promising US/MR contrast ability. Moreover, tumor targeting ability was further enhanced by folate conjugation and assessed through in vitro cell test. Furthermore, FUS, as a non-invasive and remote-control technique, was adopted to trigger the release of doxorubicin from MF-MNB and generate the sonoporation effect to enhance drug release and cellular uptake of MF-MNBs. The 4T1 cell viability was significantly decreased by FA ligand-receptor-mediated targeting and FUS sonication. In addition, the developed MF-MNB also exhibits enhanced accumulation in tumor site by FA ligand-receptor-mediated tumor targeting, in which the accumulation of MF-MNB was further enhanced by FUS sonication. Hence, we believe that the MF-MNB could be a promising drug nanocarrier for US/MR-guided anticancer drug delivery to improve cancer treatment efficacy.

The predictive value of energy spectral CT parameters for assessing Ki-67 expression of lung cancer.

BACKGROUND: To investigate the predictive value of energy spectral CT parameters for Ki-67 expression in lung cancer. METHODS: In this retrospective analysis, 27 primary lung cancer patients confirmed by pathological examination were enrolled between December 2018 and February 2019. All patients underwent baseline arterial phase (AP) and venous phase (VP) energy spectral CT scanning followed by surgery in our institution. The iodine concentration (IC), normalized iodine concentration (NIC) and the slope of 40-80 keV energy spectrum curve (λHU) were derived from dual-energy virtual imaging on a Siemens postprocessed workstation. Immunohistochemical examination was performed to analyze Ki-67 expression. The ROC curves were used for predicting the performance of energy spectral parameters for Ki-67 expression. RESULTS: The tumors appeared larger in Ki-67 high expression group than the low expression group (P=0.046). The energy spectral parameters were higher in venous phase when compared to arterial phase, but only the venous phase NIC (vpNIC) was significantly different from that of the arterial phase NIC (apNIC) (P<0.01). There are significant differences in high and low Ki-67 expression groups for vpNIC and venous λHU (vpλHU), (P=0.033 and 0.037 for vpNIC and vpλHU, respectively). vpNIC ROC analysis showed borderline P value (P=0.056) with an AUC, sensitivity (SE), specificity (SP) and cut-off value (0.717, 92.86, 61.54 and ≤0.347), respectively. The AUC, SE, SP and cut-off value of vpλHU were 0.698, 92.86, 53.85 and ≤2.407, respectively. CONCLUSIONS: The energy spectral parameters (NIC and λHU) of venous phase might be used for predicting Ki-67 stratification. The venous phase energy spectral parameters were higher than the arterial phase. Furthermore, low expression Ki-67 group showed association with higher IC, NIC and λHU than high expression group.

NAD-independent L-lactate dehydrogenase is required for L-lactate utilization in Pseudomonas stutzeri SDM.

BACKGROUND: Various Pseudomonas strains can use L-lactate as their sole carbon source for growth. However, the L-lactate-utilizing enzymes in Pseudomonas have never been identified and further studied. METHODOLOGY/PRINCIPAL FINDINGS: An NAD-independent L-lactate dehydrogenase (L-iLDH) was purified from the membrane fraction of Pseudomonas stutzeri SDM. The enzyme catalyzes the oxidation of L-lactate to pyruvate by using FMN as cofactor. After cloning its encoding gene (lldD), L-iLDH was successfully expressed, purified from a recombinant Escherichia coli strain, and characterized. An lldD mutant of P. stutzeri SDM was constructed by gene knockout technology. This mutant was unable to grow on L-lactate, but retained the ability to grow on pyruvate. CONCLUSIONS/SIGNIFICANCE: It is proposed that L-iLDH plays an indispensable function in Pseudomonas L-lactate utilization by catalyzing the conversion of L-lactate into pyruvate.

Gellan gel beads containing magnetic nanoparticles: an effective biosorbent for the removal of heavy metals from aqueous system.

This study describes an efficient adsorbent consisting of magnetic Fe(3)O(4) and gellan gum, which couples magnetic separation with ionic exchange for heavy metal removal. Adsorption kinetics analysis showed that the adsorption capacities were in an order of Pb(2+)>Cr(3+)>Mn(2+). Different experimental parameters studies indicated that adsorbent dosage, initial metal concentration, temperature and initial pH played important roles in adsorption process. Additionally, the Freundlich model gave a better fit to the experimental data than the Langmuir model. Chemical analysis of calcium ions released into the bulk solutions demonstrated that carboxyl group is critical for binding Pb(2+), Mn(2+) and Cr(3+). Furthermore, a high desorption efficiency was obtained by sodium citrate.