Massively Parallel CRISPR-Cas9 Knockout Screening in Sheep Granulosa Cells for FSH Response Genes.

Follicle-stimulating hormone (FSH) regulates ovarian follicle development through specific gene expression programs. Granulosa cells (GCs) are somatic cells surrounding the oocytes, secreting gonadotropins to regulate ovulation and promote follicular development. By analyzing the effects of different doses of FSH on the proliferation of GCs, we found that adding 10 ng/mL of FSH, as the optimal concentration, could promote the growth of GCs. Furthermore, we have successfully constructed the first CRISPR-Cas9 knockout library targeting the genes on chromosomes 2 and 3 and the X chromosomes of the sheep massively parallel coding gene, as well as an ovarian GCs knockout cell library. For the first time, we have exposed the knockout cell library to a concentration of 10 ng/mL FSH to explore the underlying mechanisms. Through this screening, we have identified 836 positive-negative screening genes that are responsive to FSH, thereby revealing the regulatory mechanisms and screening the functionality of candidate genes. Next, RNA-Seq of control (0 ng/mL), low (10 ng/mL), and high (100 ng/mL) doses of FSH revealed 1708 differentially expressed genes, and combined with 836 genes, we obtained 129 FSH dose-dependent genes with extremely significant differences. This enables us to delve deeper into investigating and identifying the mechanisms by which FSH regulates GCs. More generally, we have discovered new regulatory factors and identified reproductivity-associated major effectors. These findings provide novel research directions for further studies on sheep reproduction.

LRRC75A-AS1 drives the epithelial-mesenchymal transition in cervical cancer by binding IGF2BP1 and inhibiting SYVN1-mediated NLRP3 ubiquitination.

Cervical cancer severely affects women's health with increased incidence and poor survival for patients with metastasis. Our study aims to investigate the mechanism by which lncRNA LRRC75A-AS1 regulates the epithelial-mesenchymal transition (EMT) of cervical cancer through modulating m6A and ubiquitination modification. In this study, tumor tissues were collected from patients to analyze the expression of LRRC75A-AS1 and SYVN1. Migratory and invasive capacities of HeLa and CaSki cells were evaluated with wound healing and transwell assays. CCK-8 and EdU incorporation assays were employed to examine cell proliferation. The interaction between LRRC75A-AS1, IGF2BP1, SYVN1, and NLRP3 was evaluated through RNA immunoprecipitation, RNA pull-down, FISH, and Co-IP assays, respectively. MeRIP-qPCR was applied to analyze the m6A modification of SYVN1 mRNA. A subcutaneous tumor model of cervical cancer was established. We showed LRRC75A-AS1 was upregulated in tumor tissues, and LRRC75A-AS1 enhanced EMT through activating NLRP3/IL-1ß/Smad2/3 signaling in cervical cancer. Furthermore, LRRC75A-AS1 inhibited SYVN1-mediated NLRP3 ubiquitination by destabilizing SYVN1 mRNA. LRRC75A-AS1 competitively bound to IGF2BP1 protein and subsequently impaired the m6A modification of SYVN1 mRNA and its stability. Knockdown of LRRC75A-AS1 repressed EMT and tumor growth via inhibiting NLRP3/IL-1ß/Smad2/3 signaling in mice. In conclusion, LRRC75A-AS1 competitively binds to IGF2BP1 protein to destabilize SYVN1 mRNA, subsequently suppresses SYVN1-mediated NLRP3 ubiquitination degradation and activates IL-1ß/Smad2/3 signaling, thus promoting EMT in cervical cancer. Implications: LRRC75A-AS1 promotes cervical cancer progression, and this study suggests LRRC75A-AS1 as a new therapeutic target for cervical cancer.

Integrated Multi-Tissue Transcriptome Profiling Characterizes the Genetic Basis and Biomarkers Affecting Reproduction in Sheep (Ovis aries).

The heritability of litter size in sheep is low and controlled by multiple genes, but the research on its related genes is not sufficient. Here, to explore the expression pattern of multi-tissue genes in Chinese native sheep, we selected 10 tissues of the three adult ewes with the highest estimated breeding value in the early study of the prolific Xinggao sheep population. The global gene expression analysis showed that the ovary, uterus, and hypothalamus expressed the most genes. Using the Uniform Manifold Approximation and Projection (UMAP) cluster analysis, these samples were clustered into eight clusters. The functional enrichment analysis showed that the genes expressed in the spleen, uterus, and ovary were significantly enriched in the Ataxia Telangiectasia Mutated Protein (ATM) signaling pathway, and most genes in the liver, spleen, and ovary were enriched in the immune response pathway. Moreover, we focus on the expression genes of the hypothalamic-pituitary-ovarian axis (HPO) and found that 11,016 genes were co-expressed in the three tissues, and different tissues have different functions, but the oxytocin signaling pathway was widely enriched. To further explore the differences in the expression genes (DEGs) of HPO in different sheep breeds, we downloaded the transcriptome data in the public data, and the analysis of DEGs (Xinggao sheep vs. Sunite sheep in Hypothalamus, Xinggao sheep vs. Sunite sheep in Pituitary, and Xinggao sheep vs. Suffolk sheep in Ovary) revealed the neuroactive ligand-receptor interactions. In addition, the gene subsets of the transcription factors (TFs) of DEGs were identified. The results suggest that 51 TF genes and the homeobox TF may play an important role in transcriptional variation across the HPO. Altogether, our study provided the first fundamental resource to investigate the physiological functions and regulation mechanisms in sheep. This important data contributes to improving our understanding of the reproductive biology of sheep and isolating effecting molecular markers that can be used for genetic selection in sheep.

Effects of adding Sophora alopecuroides to high concentrate diet on rumen fermentation parameters and microbial diversity of sheep.

Objective: The purpose of this study was to investigate the effects of different doses of Sophora alopecuroides (SA) on the rumen fermentation and microbial diversity of sheep. Methods: A total of 32 healthy Dumont crossbred male lambs weighing 25.73 ± 2.17 kg were randomly assigned to 4 treatment groups with 8 replicates each: a control group (CG) fed a basal diet with a concentrate-to-forage ratio of 7:3 and three experimental groups - the 0.1% group(TG1), 0.3% group (TG2), and 0.5% group (TG3), which were fed the same basal diet but supplemented with increasing doses of SA. Results: (1) Increasing the SA dose led to a significant linear increase (p-< 0.05) in acetate, propionate, butyrate, and total volatile fatty acid (TVFA) concentrations in the rumen, as well as a significant quadratic effect (p-< 0.05) on the propionate concentration. In contrast, there was a significant linear decrease (p-< 0.05) in the NH3-N concentration in the rumen. (2) At the level of rumen bacterial phyla, the abundance of Bacteroidetes in the rumen increased, and that of Firmicutes decreased (p = 0.08). At the genus level, the rumen abundances of Ruminococcus and Phocaeicola of sheep in the three experimental groups were significantly higher than in the control group (p-< 0.05), and the abundances of Clostridiales and Candidatus-Hepatincola were significantly increased in the 0.1% and 0.3% groups (p < 0.05). (3) Regarding rumen anaerobic fungi, the differences between the control group and experimental groups at the phylum level and genus level were not significant (p > 0.05), but the relative abundances of Neocallimastigomycota and Piromyces in the 0.1% group were significantly higher than that in the control group. Conclusion: SA addition to a high grain diet could increase the VFA concentration and pH in the sheep rumen, reduce the NH3-N concentration in the rumen and improve rumen fermentation function. Although there was no significant change in rumen bacterial or fungal diversity, SA addition increased the rumen abundances of Bacteroidetes, Ruminococcus, Phocaeicola, Clostridiales, Neocallimastigomycota and Piromyces, decreased the rumen abundance of Firmicutes, and had a positive effect on the rumen microbiota to improve sheep health.

The complete mitochondrial genomes of the Leuciscus baicalensis and Rutilus rutilus: A detailed genomic comparison among closely related species of the Leuciscinae subfamily.

Cyprinidae is the largest family in the order of freshwater fish Cypriniformes. Increased subfamily members of Cyprinidae have been suggested to be re-classified for decades. In this study, we sequenced the mitochondrial genomes (mitogenomes) of Leuciscus baicalensis and Rutilus rutilus collected from northwest China and compared with other closely related species to determine their associated family or subfamily. We used Illumina NovaSeq to sequence the entire mitochondrial genomes of Leuciscus baicalensis and Rutilus rutilus and characterized the mitogenomes by the gene structure, gene order, and the secondary structures of the 22 tRNA genes. We compared mitogenome features of Leuciscinae with other subfamilies in Cyprinidae. We used the analytic Bayesian Information and Maximum Likelihood methods to determine phylogenetic trees of 13 PCGs. The mitogenomes of Leuciscus baicalensis and Rutilus rutilus were 16,607 bp and 16,606 bp, respectively. Organization and location of these genes were consistent with already studied Leuciscinae fishes. Synonymous codon usage was conservative in Leuciscinae as compared with other subfamilies in Cyprinidae. Phylogenetic analysis indicated that Leuciscinae was a monophyletic group, and genus Leuciscus was a paraphyletic group. Our approach, for the first time, of studying comparative mitochondrial genomics and phylogenetics together provided a supportive platform to the analysis of population genetics and phylogeny for Leuciscinae. Our results indicated a promising potential of comparative mitochondrial genomics in the manifestation of phylogenetic relationships between fishes, leading us to a suggestion that mitogenomes should be routinely considered in clarifying phylogenetics of family and subfamily members of fish.

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.

Estimates of Genomic Heritability and the Marker-Derived Gene for Re(Production) Traits in Xinggao Sheep.

Xinggao sheep are a breed of Chinese domestic sheep that are adapted to the extremely cold climatic features of the Hinggan League in China. The economically vital reproductive trait of ewes (litter size, LS) and productive traits of lambs (birth weight, BWT; weaning weight, WWT; and average daily gain, ADG) are expressed in females and later in life after most of the selection decisions have been made. This study estimated the genetic parameters for four traits to explore the genetic mechanisms underlying the variation, and we performed genome-wide association study (GWAS) tests on a small sample size to identify novel marker trait associations (MTAs) associated with prolificacy and growth. We detected two suggestive significant single-nucleotide polymorphisms (SNPs) associated with LS and eight significant SNPs for BWT, WWT, and ADG. These candidate loci and genes also provide valuable information for further fine-mapping of QTLs and improvement of reproductive and productive traits in sheep.

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.

Multi-Tissue Transcriptome Study of Innate Immune Gene Expression Profiling Reveals Negative Energy Balance Altered the Defense and Promoted System Inflammation of Dairy Cows.

Negative energy balance (NEB) during the perinatal period leads to metabolic and immunological disorders in dairy cows, resulting in systemic responses and inflammation. The innate immune system is crucial for the host's protection and inflammatory response. However, systematic research is still lacking on how NEB affects the innate immune system to alter the 'host defense capability and inflammatory response. In this investigation, raw transcriptome data of adipose, blood, endometrial, hypothalamus, and liver tissues were downloaded from a public database, cleaned, aligned, quantified, and batch-corrected. The innate immune gene list was retrieved from innateDB, followed by the expression matrix of innate immune genes in various tissues for differential expression analysis, principle component analysis (PCA), and gene set enrichment analysis (GSEA). Under the effect of NEB, adipose tissue had the most differentially expressed genes, which were predominantly up-regulated, whereas blood GSEA had the most enriched biological processes, which were predominantly down-regulated. The gene sets shared by different tissues, which are predominantly involved in biological processes associated with defense responses and inflammation, were dramatically down-regulated in endometrial tissues and highly up-regulated in other tissues. Under the impact of NEB, LBP, PTX3, S100A12, and LCN2 play essential roles in metabolism and immunological control. In conclusion, NEB can downregulate the defensive response of innate immune genes in endometrial, upregulate the immune and inflammatory response of other tissues, activate the host defense response, and increase the systemic inflammatory response. The analysis of the effects of NEB on innate immune genes from the multiple tissues analysis provides new insights into the crosstalk between metabolism and immunity and also provides potential molecular targets for disease diagnosis and disease resistance breeding in dairy cows.

Candidate Genes and Gene Networks Change with Age in Japanese Black Cattle by Blood Transcriptome Analysis.

Age is an important physiological factor that affects the metabolism and immune function of beef cattle. While there have been many studies using the blood transcriptome to study the effects of age on gene expression, few have been reported on beef cattle. To this end, we used the blood transcriptomes of Japanese black cattle at different ages as the study subjects and screened 1055, 345, and 1058 differential expressed genes (DEGs) in the calf vs. adult, adult vs. old, and calf vs. old comparison groups, respectively. The weighted co-expression network consisted of 1731 genes. Finally, blue, brown, and yellow age-specific modules were obtained, in which genes were enriched in signaling pathways related to growth and development and immune metabolic dysfunction, respectively. Protein-protein interaction (PPI) analysis showed gene interactions in each specific module, and 20 of the highest connectivity genes were chosen as potential hub genes. Finally, we identified 495, 244, and 1007 genes by exon-wide selection signature (EWSS) analysis of different comparison groups. Combining the results of hub genes, we found that VWF, PARVB, PRKCA, and TGFB1I1 could be used as candidate genes for growth and development stages of beef cattle. CORO2B and SDK1 could be used as candidate marker genes associated with aging. In conclusion, by comparing the blood transcriptome of calves, adult cattle, and old cattle, the candidate genes related to immunity and metabolism affected by age were identified, and the gene co-expression network of different age stages was constructed. It provides a data basis for exploring the growth, development, and aging of beef cattle.

Blood Transcriptome Analysis of Beef Cow with Different Parity Revealed Candidate Genes and Gene Networks Regulating the Postpartum Diseases.

Maternal parity is an important physiological factor influencing beef cow reproductive performance. However, there are few studies on the influence of different calving periods on early growth and postpartum diseases. Here, we conducted blood transcriptomic analysis on cows of different parities for gene discovery. We used Short Time Series Expression Miner (STEM) analysis to determine gene expression levels in cows of various parities and divided multiple parities into three main periods (nulliparous, primiparous, and multiparous) for subsequent analysis. Furthermore, the top 15,000 genes with the lowest median absolute deviation (MAD) were used to build a co-expression network using weighted correlation network analysis (WGCNA), and six independent modules were identified. Combing with Exon Wide Selection Signature (EWSS) and protein-protein interaction (PPI) analysis revealed that TPCN2, KIF22, MICAL3, RUNX2, PDE4A, TESK2, GPM6A, POLR1A, and KLHL6 involved in early growth and postpartum diseases. The GO and KEGG enrichment showed that the Parathyroid hormone synthesis, secretion, and action pathway and stem cell differentiation function-related pathways were enriched. Collectively, our study revealed candidate genes and gene networks regulating the early growth and postpartum diseases and provided new insights into the potential mechanism of reproduction advantages of different parity selection.

A Fifteen-Gene Classifier to Predict Neoadjuvant Chemotherapy Responses in Patients with Stage IB to IIB Squamous Cervical Cancer.

Neoadjuvant chemotherapy (NACT) remains an attractive alternative for controlling locally advanced cervical cancer. However, approximately 15-34% of women do not respond to induction therapy. To develop a risk stratification tool, 56 patients with stage IB-IIB cervical cancer are included in 2 research centers from the discovery cohort. Patient-specific somatic mutations led to NACT non-responsiveness are identified by whole-exome sequencing. Next, CRISPR/Cas9-based library screenings are performed based on these genes to confirm their biological contribution to drug resistance. A 15-gene classifier is developed by generalized linear regression analysis combined with the logistic regression model. In an independent validation cohort of 102 patients, the classifier showed good predictive ability with an area under the curve of 0.80 (95% confidence interval (CI), 0.69-0.91). Furthermore, the 15-gene classifier is significantly associated with patient responsiveness to NACT in both univariate (odds ratio, 10.8; 95% CI, 3.55-32.86; p = 2.8 × 10-5) and multivariate analysis (odds ratio, 17.34; 95% CI, 4.04-74.40; p = 1.23 × 10-4) in the validation set. In conclusion, the 15-gene classifier can accurately predict the clinical response to NACT before treatment, representing a promising approach for guiding the selection of appropriate treatment strategies for locally advanced cervical cancer.

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%.

Effect of oxidative stress from nanoscale TiO2 particles on a Physarum polycephalum macroplasmodium under dark conditions.

Information regarding the effect of nanoscale titanium dioxide particles (nTiO2) on the environment under dark conditions is scarce, and the effect of nTiO2 on fungi is largely unknown. Due to its huge size and high sensitivity to external stimuli, the slime mold fungi cell, Physarum polycephalum macroplasmodium, was utilized as a novel subject for the toxicity investigations in the present study, and oxidative stress from nTiO2 on the macroplasmodium was assessed under dark conditions. Short exposure (2-3 h) caused an intracellular reactive oxygen species (ROS) imbalance, and an anti-oxidative mechanism was activated from intermediate doses of nTiO2 (5-18 mg/mL). At long exposure times (~3 days), relatively low doses of nTiO2 (≤9 mg/mL) stimulated the growth of macroplasmodium and oxidative stress without DNA damage, whereas higher doses of nTiO2 (≥15 mg/mL) led to growth inhibition, significant DNA oxidative damage, and activation of the DNA single-strand repairing system. Although DNA oxidative damage was decreased to the same level as the control group by the supplementation of the anti-oxidant vitamin C, growth of the macroplasmodium failed to be completely restored. We inferred that nTiO2 induced a complicated toxicity effect on P. polycephalum in addition to DNA oxidative damage. Taken as a whole, the present study implied the probability of using P. polycephalum macroplasmodium for toxicity studies at the single-cell level, indicating that nTiO2 could induce oxidative stress or damage in P. polycephalum even under dark conditions and suggesting that the release of nTiO2 could lead to a growth imbalance of slime molds in the environment.

Overexpression of Cathepsin L is associated with chemoresistance and invasion of epithelial ovarian cancer.

Paclitaxel is recommended as a first-line chemotherapeutic agent against, ovarian cancer, however, the development of chemoresistance is a major obstacle in patients with aggressive ovarian cancer and results in recurrence after conventional therapy. The key molecule or mechanism associated with paclitaxel resistance in ovarian cancer still remains unclear. Cathepsin L (CTSL) is overexpressed in various cancers, however, the association between CTSL expression and paclitaxel resistance remains unclear. In the present study, we investigated the role of CTSL in paclitaxel-resistant SKOV3/TAX cells by CTSL silencing. Expression of CTSL was examined by immunohistochemistry and qRT-PCR in 58 clinical samples, and in SKOV3 cells and SKOV3/TAX cells. Effects of CTSL knockdown on ovarian cancer cell proliferation, apoptosis, migration, and invasion were also studied. The IHC and real-time PCR results showed that the difference of CTSL expression between ovarian cancer and the adjacent non-tumourous ovarian tissues was statistically significant. Western blot analysis showed that the CTSL was overexpressed in SKOV3/TAX cells and weakly detectable in paclitaxel-sensitive SKOV3 cells. Knocking-down of CTSL in ovarian cancer cells could decrease cell proliferation, migration, and invasion, and potentiate apoptosis induced by paclitaxel, suggesting CTSL may contribute to Paclitaxel resistance in ovarian cancer.