Mitochondrial phylogenomics provides conclusive evidence that the family Ancyrocephalidae is deeply paraphyletic.

BACKGROUND: Unresolved taxonomic classification and paraphyly pervade the flatworm class Monogenea: the class itself may be paraphyletic and split into Polyopisthocotylea and Monopisthocotylea; there are some indications that the monopisthocotylean order Dactylogyridea may also be paraphyletic; single-gene markers and some morphological traits indicate that the family Ancyrocephalidae is paraphyletic and intertwined with the family Dactylogyridae. METHODS: To attempt to study the relationships of Ancyrocephalidae and Monopisthocotylea using a phylogenetic marker with high resolution, we sequenced mitochondrial genomes of two fish ectoparasites from the family Dactylogyridae: Dactylogyrus simplex and Dactylogyrus tuba. We conducted phylogenetic analyses using three datasets and three methods. Datasets were ITS1 (nuclear) and nucleotide and amino acid sequences of almost complete mitogenomes of almost all available Monopisthocotylea mitogenomes. Methods were maximum likelihood (IQ-TREE), Bayesian inference (MrBayes) and CAT-GTR (PhyloBayes). RESULTS: Both mitogenomes exhibited the ancestral gene order for Neodermata, and both were compact, with few and small intergenic regions and many and large overlaps. Gene sequences were remarkably divergent for nominally congeneric species, with only trnI exhibiting an identity value > 80%. Both mitogenomes had exceptionally low A + T base content and AT skews. We found evidence of pervasive compositional heterogeneity in the dataset and indications that base composition biases cause phylogenetic artefacts. All six mitogenomic analyses produced unique topologies, but all nine analyses produced topologies that rendered Ancyrocephalidae deeply paraphyletic. Mitogenomic data consistently resolved the order Capsalidea as nested within the Dactylogyridea. CONCLUSIONS: The analyses indicate that taxonomic revisions are needed for multiple Polyopisthocotylea lineages, from genera to orders. In combination with previous findings, these results offer conclusive evidence that Ancyrocephalidae is a paraphyletic taxon. The most parsimonious solution to resolve this is to create a catch-all Dactylogyridae sensu lato clade comprising the current Ancyrocephalidae, Ancylodiscoididae, Pseudodactylogyridae and Dactylogyridae families, but the revision needs to be confirmed by another marker with a sufficient resolution.

LRRC75A-AS1 delivered by M2 macrophage exosomes promotes cervical cancer progression via enhancing SIX1 expression.

We aimed to investigate potential roles of LRRC75A-AS1 delivered by M2 macrophage exosomes in inducing cervical cancer progression. We demonstrated LRRC75A-AS1 was highly expressed in exosomes from M2 macrophages which could be absorbed by Hela cells. M2 macrophage-derived exosomes promoted Hela cell proliferation, migration, invasion, and EMT process by delivering LRRC75A-AS1. LRRC75A-AS1 directly targeted and suppressed miR-429 in Hela cells. The regulation of cell functions by exosomes from LRRC75A-AS1-overexpressing M2 macrophages was abrogated by miR-429 mimics. miR-429 directly targeted and repressed SIX1 expression. SIX1 overexpression alleviated the modulation of cellular functions and STAT3/MMP-9 signaling by miR-429 mimics. Also, miR-429 overexpression or SIX1 silence repressed tumor formation and metastasis in nude mice, which was mitigated by exosomes from LRRC75A-AS1-overexpressing M2 macrophages. In conclusion, LRRC75A-AS1 delivered by M2 macrophage exosomes repressed miR-429 to elevate SIX1 expression and promote cervical cancer progression through activating the STAT3/MMP-9 axis.

Label-free microRNA detection through analyzing the length distribution pattern of the residual fragments of probe DNA produced during exonuclease III assisted signal amplification by mass spectrometry.

Biosensors based on various spectroscopic techniques discriminate the target microRNA (miRNA) from non-target ones with single nucleotide polymorphisms (SNPs) according to the differences in signal intensities which can be caused by other factors besides SNPs. As a result, they are liable to produce false positive results. Herein, we report an attempt to develop a false-positive resistance, sensitive and reliable mass spectrometric platform for miRNA detection. In the proposed platform, the qualitative and quantitative information of the target miRNA was obtained through analyzing mass spectral responses of the multiply charged ions of the residual fragments of the probe DNA produced during exonuclease III assisted signal amplification reaction using an advanced data analysis method. The proposed platform could achieve sensitive and accurate quantitative results for the target miRNA (e.g., miRNA-141) in complex medium with a detection limit of about 1 pM, and unambiguously identify non-target miRNAs with SNPs based on the length distribution patterns of residual fragments of probe DNA. The findings obtained in this study might open an avenue for the design of new miRNA detection methods based on mass spectrometry in combination with various nuclease assisted signal amplification strategies.

Highly specific and sensitive detection of microRNAs by tandem signal amplification based on duplex-specific nuclease and strand displacement.

Based on the full understanding of the hydrolysis patterns of duplex-specific nuclease (DSN) against the probe DNAs in DNA/microRNA heteroduplexes, a simple and generic platform for highly specific and sensitive detection of microRNAs was developed by seamlessly integrating DSN-assisted target recycling amplification and strand displacement amplification in tandem.

[Lentivirus-mediated BMP-2 overexpression plasmid transfection into bone marrow mesenchymal stem cells combined with silk fibroin scaffold for osteoblast transformation].

OBJECTIVE: To explore the effect of lentivirus-mediated BMP-2 overexpression plasmid transfection into bone marrow mesenchymal stem cells and silk fibroin scaffold on osteoblast transformation. METHODS: The lentivirus BMP-2 overexpression vector was constructed, bone marrow mesenchymal stem cells were cultured, and the combined culture system of nuclear scaffolds was constructed. Alizarin red staining and alkaline phosphatase staining were used to detect the osteogenic transformation of bone marrow mesenchymal stem cells in vitro. Ten New Zealand white rabbits, weighing 3.2 to 4.5 kg(averaging 3.9 kg), aged (2.89±0.45) years old, were selected to construct the rabbit tibial defect model by drilling a conical tibial defect (5 mm in length, 2 mm in width and 3 mm in depth) with an oral drill. The repair of the tibial defect in the animal model was observed by HE staining. The experimental group was implanted with silk fibroin scaffold + BMP-2 overexpression vector bone marrow mesenchymal stem cell complex, while the negative control group was implanted with silk fibroin scaffold+non-transfected bone marrow mesenchymal stem cell complex. RESULTS: Compared with the control group(silk fibroin scaffold+non-transfected bone marrow mesenchymal stem cells), the number of adherent cells on the surface of the scaffold in the experimental group(silk fibroin scaffold+transfected BMP-2 overexpression vector BMP-2 complex) increased significantly. Compared with the control group, the ECM secretion in the experimental group increased significantly. EDX analysis showed that the content of calcium ion was 0.22% in the control group and 0.86% in the experimental group, which showed that the ability of inducing calcium ion formation in the experimental group was stronger than that in the control group. Alizarin red staining of calcium nodules showed that there was no obvious change in the naked eye of the control group, and a small amount of calcium nodules could be seen under the microscope. In the experimental group, obvious red area staining was observed by naked eye, and a large number of calcium nodules were observed by microscopy. The results of alkaline phosphatase staining showed that there was no obvious change in the naked eye of the control group, and no obvious change in the microscopic observation. In the experimental group, purple area staining was observed by naked eyes, and ALP staining was strongly positive by microscopy. The combined culture system of silk fibroin scaffold and bone marrow mesenchymal stem cells can repair cartilage defects. The repair effect of BMP-2 bone marrow mesenchymal stem cells after transfection is obviously better than that of non-transfection group. HE staining showed that inflammatory cells decreased and scaffolds disappeared slightly in the control group. In the experimental group, inflammatory cells were significantly reduced, scaffolds disappeared and angiogenesis was observed. CONCLUSIONS: Lentivirus-mediated BMP-2 overexpression plasmid can promote BMSC to differentiate into osteocytes and secrete more extracellular matrix containing Ca²âº to promote bone defect repair.

Quantification of enantiomers by mass spectrometry based on chemical derivatization and spectral shape deformation quantitative theory.

A facile mass spectrometric kinetic method for quantitative analysis of chiral compounds was developed by integrating mass spectrometry based on chemical derivatization and the spectral shape deformation quantitative theory. Chemical derivatization was employed to introduce diastereomeric environments to the chiral compounds of interest, resulting in different abundance distribution patterns of fragment ions of the derivatization products of enantiomers in mass spectrometry. The quantitative information of the chiral compounds of interest was extracted from complex mass spectral data by an advanced calibration model derived based on the spectral shape deformation quantitative theory. The performance of the proposed method was tested on the quantitative analysis of R-propranolol in propranolol tablets. Experimental results demonstrated that it could achieve accurate and precise concentration ratio predictions for R-propranolol with an average relative predictive error (ARPE) of about 4%, considerably better than the corresponding results of the mass spectrometric method based on chemical derivatization and the univariate ratiometric model (ARPE: about 12%). The limit of detection (LOD) and limit of quantification (LOQ) of the proposed method for the concentration ratio of R-propranolol were estimated to be 1.5% and 6.0%, respectively. The proposed method is complementary to the existing methods designed for the quantification of enantiomers such as the Cooks kinetic method.

Label-Free and Multiplexed Quantification of microRNAs by Mass Spectrometry Based on Duplex-Specific-Nuclease-Assisted Recycling Amplification.

MicroRNAs (miRNAs) are important biomarker candidates for cancer screening and early detection research. Generally, miRNAs undergo synergistic adjustments in tumor cells. Herein, a mass-spectrometric method based on a duplex-specific-nuclease (DSN)-enzyme-assisted signal-amplification technique was proposed for label-free and multiplexed detection of multiple miRNAs, and applied to the quantification of three miRNAs (i.e., miRNA-141, miRNA-21, and let-7a) in samples of HeLa and MDA-MB231 cell extracts. Experimental results showed that the digestion modes of DSN against three different DNAs complementary to miRNA-141, miRNA-21, and let-7a in their DNA-miRNA heteroduplexes were quite different, verifying the multiplexed-detection capability of the proposed method. Moreover, an advanced calibration model was derived for the quantitative analysis of the complex mass-spectral data measured during the label-free and multiplexed detection of miRNA-141, miRNA-21, and let-7a by the proposed mass-spectrometric method. With the aid of the advanced calibration model, the proposed mass-spectrometric method achieved quite reliable quantitative results for miRNA-141, miRNA-21, and let-7a in samples of HeLa and MDA-MB231 cell extracts, with recovery rates within the range of 89.2 to 111.6%. The limits of detection (LODs) of the proposed mass-spectrometric method for miRNA-141, miRNA-21, and let-7a in standard samples were estimated to be 42, 41, and 95 pM, respectively. Therefore, it is reasonable to expect that the proposed mass-spectrometric method can be a competitive alternative for the label-free and multiplexed detection of multiple miRNAs in clinical diagnosis.

[Reducing Membrane Fouling from Micro-Flocculation in a Humic Acid Ultrafiltration Process].

This study investigated the influence of different floc morphologies produced by micro-flocculation process on filtration of a self-constructed polyvinylidene fluoride (PVDF) ultrafiltration membrane. Aluminum sulfate was used as a flocculant and humic acid (HA) and kaolin as raw water. Both the properties of flocs formed during the micro-flocculation process (floc size and distribution, fractal dimension) and the effects of floc formation on membrane flux under different conditions were investigated. The surface morphology of the contaminated membrane was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM), and adhesion between the PVDF membrane and organic pollutants was measured to analyze the membrane fouling mechanism. Results showed that the main mechanism during a micro-flocculation process using Al3+ as a flocculant is electrical neutralization to remove organic matter. With an increase in flocculant dosage, floc size increased and the fractal dimension of flocs decreased. The attenuation rate of membrane flux was negatively correlated with floc size. The larger the floc, the lower the membrane flux attenuation rate, and the looser the filter cake layer formed during the ultrafiltration process. Comparatively, membrane fouling caused by flocs with smaller fractal dimension was lighter, and the membrane flux recovery rate was also higher. The interaction force between PVDF and organic matter was positively correlated with the corresponding membrane flux attenuation rate during the initial stage of operation. When dosage of Al3+ was 5 mg·L-1 and initial pH was 7, the HA removal rate was 96.7%, the membrane flux attenuation rate was lowest, and the flux recovery rate reached 88%.

Generalized multiple internal standard method for quantitative liquid chromatography mass spectrometry.

In this contribution, a multiplicative effects model for generalized multiple-internal-standard method (MEMGMIS) was proposed to solve the signal instability problem of LC-MS over time. MEMGMIS model seamlessly integrates the multiple-internal-standard strategy with multivariate calibration method, and takes full use of all the information carried by multiple internal standards during the quantification of target analytes. Unlike the existing methods based on multiple internal standards, MEMGMIS does not require selecting an optimal internal standard for the quantification of a specific analyte from multiple internal standards used. MEMGMIS was applied to a proof-of-concept model system: the simultaneous quantitative analysis of five edible artificial colorants in two kinds of cocktail drinks. Experimental results demonstrated that MEMGMIS models established on LC-MS data of calibration samples prepared with ultrapure water could provide quite satisfactory concentration predictions for colorants in cocktail samples from their LC-MS data measured 10days after the LC-MS analysis of the calibration samples. The average relative prediction errors of MEMGMIS models did not exceed 6.0%, considerably better than the corresponding values of commonly used univariate calibration models combined with multiple internal standards. The advantages of good performance and simple implementation render MEMGMIS model a promising alternative tool in quantitative LC-MS assays.

Surface-enhanced Raman spectroscopy based on conical holed enhancing substrates.

In this contribution, surface-enhanced Raman spectroscopy (SERS) based on conical holed glass substrates deposited with silver colloids was reported for the first time. It combines the advantages of both dry SERS assays based on plane films deposited with silver colloids and wet SERS assays utilizing cuvettes or capillary tubes. Compared with plane glass substrates deposited with silver colloids, the conical holed glass substrates deposited with silver colloids exhibited five-to ten-folds of increase in the rate of signal enhancement, due to the internal multiple reflections of both the excitation laser beam and the Raman scattering photons within conical holes. The application of conical holed glass substrates could also yield significantly stronger and more reproducible SERS signals than SERS assays utilizing capillary tubes to sample the mixture of silver colloids and the solution of the analyte of interest. The conical holed glass substrates in combination with the multiplicative effects model for surface-enhanced Raman spectroscopy (MEMSERS) achieved quite sensitive and precise quantification of 6-mercaptopurine in complex plasma samples with an average relative prediction error of about 4% and a limit of detection of about 0.02 µM using a portable i-Raman 785H spectrometer. It is reasonable to expect that SERS technique based on conical holed enhancing substrates in combination with MEMSERS model can be developed and extended to other application areas such as drug detection, environmental monitoring, and clinic analysis, etc.