A white-light-emitting sensor array based on cucurbit[8]uril for quantitative detection of multicomponent nitroaniline isomers.

A white-light-emitting supramolecular complex through supramolecular interactions has been assembled; the white luminescent supramolecular complex exhibits two emission spectra. Based on this, a dual-channel white-light array sensor was constructed. The results show that it can quickly identify and detect nitroaniline isomer pollutants (p-nitroaniline, m-nitroaniline, o-nitroaniline). When these three nitroaniline isomers were added to the supramolecular white-light array sensor, the fluorescence intensity of the white-light complex decreased to varying degrees. Linear discriminant analysis (LDA) showed that the supramolecular white-light array sensor could recognize and distinguish three nitroaniline isomers and could classify mixtures containing different concentrations. Factor 1 of the array had a good linear relationship with the concentration of pollutants, and the detection limit (LOD) was as low as 0.7 µM. The method has good reproducibility and stability. In addition, it can also qualitatively detect the nitroaniline isomers in river water and contaminated rice seedling extract. It provides an ideal platform for constructing multiresponse sensors.

Supramolecular fluorescent probe based on a host-guest complex for amino acids recognition and detection in aqueous solution.

A supramolecular fluorescent probe based on a host-guest complex has been developed for amino acid recognition and detection in aqueous solution. Cucurbit[7]uril (Q[7]) with 4-(4-dimethylamino-styrene) quinoline (DSQ) formed a fluorescent probe (DSQ@Q[7]). The DSQ@Q[7] fluorescent probe nearly generated changes in fluorescence in response to four amino acids (arginine, histidine, phenylalanine and tryptophan). These changes were attributed to the host-guest interaction between DSQ@Q[7] and amino acids, which occurred as a consequence of the subtle cooperation of ionic dipole and hydrogen bonding. Linear discriminant analysis showed that the fluorescent probe could recognize and distinguish four amino acids, and a mixture with different concentration ratios could be well categorized in ultrapure water and tap water.

Supramolecular Fluorescence Sensor Array Based on Cucurbit[8]uril Complexes Used for the Detection of Multiplex Quaternary Ammonium Pesticides.

The simultaneous detection of multiple quaternary ammonium pesticides (QAPs) in water is a challenge due to their high solubility in water and similar structures. In this paper, we have developed a quadruple-channel supramolecular fluorescence sensor array for the simultaneous analysis of five QAPs, including paraquat (PQ), diquat (DQ), difenzoquat (DFQ), mepiquat (MQ), and chlormequat (CQ). Not only were QAP samples of different concentrations (10, 50, and 300 µM) in water distinguished with 100% accuracy but also single QAP and binary QAP mixed samples (DFQ-DQ) were sensitively quantified. Our experimental interference study confirmed that the developed array has good anti-interference ability. The array can quickly identify five QAPs in river and tap water samples. In addition, it also qualitatively detected QAP residues in Chinese cabbage and wheat seedlings extract. This array has rich output signals, low cost, easy preparation, and simple technology, demonstrating great potential in environmental analysis.

A supramolecular fluorescence array sensor for toxic heavy metal ion detection in environmental water and rice seedling extracts.

A supramolecular fluorescence array sensor based on cucurbituril-dye host-guest complexes (6-QAA@Q[7], PyY@Q[7], and TO@Q[8]) was constructed. The results showed that it can quickly identify and detect toxic heavy metal ions, such as Ag+, Cr3+, Hg2+, Ni2+, and Pb2+. When these five toxic heavy metal ions were added to the supramolecular fluorescence array sensor, different fluorescence responses were produced due to the different binding capacities of the metal ions to the cucurbituril-dye complex. Linear discriminant analysis (LDA) showed that the supramolecular fluorescence array sensor could identify and distinguish these five toxic heavy metal ions and a mixture containing different concentration ratios could be classified. The linear correlation between the metal ion concentration and factor 1 (F1) was strong, and the detection limit (LOD) was as low as 10-6-10-7 mol L-1. These five toxic heavy metal ions in environmental water and rice seedling extracts were identified using the supramolecular fluorescence array sensor. This sensor provides a quick and convenient method for monitoring toxic heavy metal ions in sewage.

A colorimetric supramolecular sensor array based on charge-transfer complexes for multiplex aniline and phenolic pollutants detection.

In order to effectively monitor toxic and harmful substances in sewage discharge, a rapid, highly sensitive detection of complex pollutants with similar structures has become an urgent problem to be solved. In this paper, a supramolecular colorimetric array sensor based on charge-transfer complex was constructed, which can quickly detect aniline and phenol pollutants (such as p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, m-aminophenol, hydroquinone, and resorcinol) with similar structures. When six anilines and phenol isomers with similar structures were added to the supramolecular colorimetric array sensor, different color changes were produced under natural light. Linear discriminant analysis (LDA) showed that the supramolecular colorimetric array sensor could recognize and distinguish these isomers, and a mixture with different concentration ratios could be well categorized. The total Euclidean distance (TED) of an array with pollutant concentrations had a good linear correlation, and the detection limit (LOD) was as low as 10-5-10-6 mol L-1. Six anilines and phenol isomers in real samples were identified by supramolecular colorimetric array sensor. 1H NMR results showed that the formation of charge transfer complexes in Q[8] cavity may be the cause of color change. This work provides a fast and convenient experimental basis for monitoring the complex structure pollutants in sewage discharge.

Lab-on-a-Molecule Probe: Multitarget Detection of Five Aromatic Pesticides Using a Supramolecular Probe under Single Wavelength Excitation.

In order to prevent and control the effects of pesticide residues on human health and the ecological environment, the rapid, highly sensitive, and selective detection of multiple pesticide residues has become an urgent problem to be solved. Herein, a lab-on-a-molecule probe based on a host-guest complex (ThT@Q[8] probe) has been developed to simultaneously analyze multiple aromatic pesticides under single wavelength excitation, such as fuberidazole, thiabendazole, carbendazim, thidiazuron, and tricyclazole. The fluorescence titration spectra of the ThT@Q[8] probe with the five pesticides mentioned above showed that the fluorescence intensity exhibited a good linear correlation with the pesticide concentration and the limit of detection was as low as 10-7 M. Because the ThT@Q[8] probe exhibits diverse fluorescence color changes to the five pesticides studied under a 365 nm ultraviolet lamp, we fabricated a single probe used to detect multiple analytes in the RGB triple channel by extracting the RGB variations. Principal component analysis and linear discriminant analysis proved that the ThT@Q[8] probe can recognize and distinguish five pesticides and can be applied at different concentrations. In real samples, the ThT@Q[8] probe recognized and distinguished five pesticides in tap water and Huaxi River water. The 1H NMR spectra results proved that a charge-transfer complex of ThT and pesticides in the Q[8] cavity may be formed. Moreover, we selected a test strip as a carrier to detect pesticides. The results indicate it can be used to quickly and conveniently detect different pesticides due to the rapid color change. Besides, the ThT@Q[8] probe has good cell permeability and can be used to detect pesticide residues in living cells. This work has laid the foundation for the qualitative and quantitative multitarget detection of pesticide residues.

Association Between Eating Speed and Metabolic Syndrome: A Systematic Review and Meta-Analysis.

Objective: This review aimed to systematically summarize and meta-analyze the association between eating speed and metabolic syndrome (MetS). Methods: Following the Preferred Reporting Items for Systematic Reviews, and Meta Analyses (PRISMA) guidelines, four electronic databases (PubMed, Web of Science, MEDLINE, and EMBASE) were searched until March 2021 to identify eligible articles based on a series of inclusion and exclusion criteria. Heterogeneity was examined using I 2 statistics. Using random-effects models, the pooled odds ratios (ORs), and 95% CIs were calculated to evaluate the association between eating speed with MetS and its components, including central obesity, blood pressure (BP), high-density lipoprotein cholesterol (HDL), triglyceride (TG), and fasting plasma glucose (FPG). Results: Of the 8,500 original hits generated by the systematic search, 29 eligible studies with moderate-to-high quality were included, involving 465,155 subjects. The meta-analysis revealed that eating faster was significantly associated with higher risks of MetS (OR = 1.54, 95% CI: 1.27-1.86), central obesity (OR = 1.54, 95% CI: 1.37-1.73), elevated BP (OR = 1.26, 95% CI: 1.13-1.40), low HDL (OR = 1.23, 95% CI: 1.15-1.31), elevated TG (OR = 1.29, 95% CI: 1.18-1.42), and elevated FPG (OR = 1.16, 95% CI: 1.06-1.27) compared to eating slowly. Conclusions: The results of the review indicated that eating speed was significantly associated with MetS and its components. Interventions related to decreasing eating speed may be beneficial for the management of MetS. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021242213, identifier: CRD42021242213.

"Turn-Off" Supramolecular Fluorescence Array Sensor for Heavy Metal Ion Identification.

A "turn-off" supramolecular fluorescence array sensor based on the host-guest complexes between fluorescence dyes and cucurbit[n]urils for sensing metal ions was developed. Three fluorescent probes (RhB@Q[7], H33342@2Q[7], and BRE@Q[7]) were used as the sensing units to construct a supramolecular fluorescence array sensor. The binding ability of the metal ions and cucurbituril-dye probes varied; therefore, the probes and metal ions produced different fluorescence responses. When combined with linear discriminant analysis (LDA), the qualitative and quantitative detection of seven metal ions was achieved. In analytical samples, the supramolecular fluorescence array sensor recognized and distinguish seven metal ions. These results provided new research ideas for the rapid analysis and real-time monitoring of different heavy metal ions.

PhiC31 integrase induces efficient site-specific recombination in the Capra hircus genome.

Streptomyces phage φC31 integrase induces efficient site-specific recombination capable of integrating exogenous genes at pseudo attP sites in human, mouse, rat, rabbit, sheep, Drosophila, and bovine genomes. However, the φC31-mediated recombination between attB and the corresponding pseudo attP sites has not been investigated in Capra hircus. Here, we identified eight pseudo attP sites located in the intron or intergenic regions of the C. hircus genome, and demonstrated different levels of foreign gene expression after φC31 integrase-mediated integration. These pseudo attP sites share similar sequences with each other and with pseudo attP sites in other mammalian genomes, and these are associated with a neighboring consensus motif found in other genomes. The application of the φC31 integrase system in C. hircus provides a new option for genetic engineering of this economically important goat species.

Engraftment of genetically modified human amniotic fluid-derived progenitor cells to produce coagulation factor IX after in utero transplantation in mice.

Human amniotic fluid derived progenitor cells (hAFPCs) may be multipotent and can be considered a potential tool in the field of cell therapy for haemophilia B. Their capacity to express human coagulation factor IX (hFIX) after transduction and their fate after in utero transplantation is unknown. hAFPCs isolated from second trimester pregnancies were assessed for their phenotypic markers, multilineage capacity, and expression of hFIX after transduction. Their engraftment potential was analysed in a mouse model after in utero transplantation at embryonic day 12.5. Immunohistochemistry, fluorescence in situ, ELISA and PCR were used to assess post-transplant chimeras. hAFPCs expressed several pluripotent markers, including NANOG, SOX2, SSEA4 and TRA-1-60, and could differentiate into adipocytes and osteocytes. In vitro, after transduction with hFIX and EGFP cDNAs, constitutive hFIX protein expression and clotting activity were found. Engraftment was achieved in various foetal tissues after in utero transplantation. Safe engraftment without oncogenesis was confirmed, with low donor cell levels, but persistent engraftment, into different organs (liver, heart and lung) through to 12 weeks of age. Transgenic expression of circulating hFIX was detected in recipient mice for up to 12 weeks. hAFPCs can be engrafted long-term in immunocompetent mice after in utero transplantation. Thus, cell transplantation approaches using genetically engineered hAFPCs may prove valuable for the prenatal treatment for haemophilia B.

Correction of ß654-thalassaemia mice using direct intravenous injection of siRNA and antisense RNA vectors.

Although the therapeutic efficacy of ß(654)-thalassaemia treatment using a combination of RNAi and antisense RNA to balance the synthesis of α- and ß-globin chains has been demonstrated previously, and the safety of lentiviral delivery remains unclear. Herein, we used the same ß(654)-thalassaemia mouse model to develop a therapy involving direct delivery of siRNA and antisense RNA plasmids via intravenous injection to simultaneously knock down α-globin transcript levels and restore correct ß-globin splicing. The amount of α-globin mRNAs in siRNA-treated MEL cells decreased significantly, and the properly spliced ß-globin mRNA was restored in HeLaß(654) cells transfected with pcDNA-antisense plasmid. Furthermore, treatment of ß(654)-thalassaemic mice with siRNA and antisense RNA plasmids resulted in significant reduction of poikilocytosis and reticulocyte counts in blood samples, decreased nucleated cell populations in bone marrow, and reduced intrasinusoidal extramedullary haematopoiesis loci and iron accumulation in liver. RT-PCR analysis revealed that treatment resulted in down-regulation of α-globin mRNA synthesis by ~50% along with an increase in the presence of normally spliced ß-globin transcripts, indicating that the phenotypic changes observed in ß(654)-thalassaemic mice following treatment resulted from restoration of the balance of α/ß-globin biosynthesis.

Donor-host mitochondrial compatibility improves efficiency of bovine somatic cell nuclear transfer.

BACKGROUND: The interaction between the karyoplast and cytoplast plays an important role in the efficiency of somatic cell nuclear transfer (SCNT), but the underlying mechanism remains unclear. It is generally accepted that in nuclear transfer embryos, the reprogramming of gene expression is induced by epigenetic mechanisms and does not involve modifications of DNA sequences. In cattle, oocytes with various mitochondrial DNA (mtDNA) haplotypes usually have different ATP content and can further affect the efficiency of in vitro production of embryos. As mtDNA comes from the recipient oocyte during SCNT and is regulated by genes in the donor nucleus, it is a perfect model to investigate the interaction between donor nuclei and host oocytes in SCNT. RESULTS: We investigated whether the in vitro development of reconstructed bovine embryos produced by SCNT would be influenced by mtDNA haplotype compatibility between the oocytes and donor cells. Embryos from homotype A-A or B-B showed significantly higher developmental ability at blastocyst stages than the heterotype A-B or B-A combinations. Post-implantation development ability, pregnancy rate up to day 90 of gestation, as well as percent of term births were higher in the homotype SCNT groups than in the heterotype groups. In addition, homotype and heterotype SCNT embryos showed different methylation patterns of histone 3-lysine 9 (H3K9) genome-wide and at pluripotency-related genes (Oct-4, Sox-2, Nanog). CONCLUSION: Both histone and DNA methylation show that homotype SCNT blastocysts have a more successful epigenetic asymmetry pattern than heterotype SCNT blastocysts, which indicates more complete nuclear reprogramming. This may result from variability in their epigenetic patterns and responses to nuclear reprogramming. This suggests that the compatibility of mtDNA haplotypes between donor cells and host oocytes can significantly affect the developmental competence of reconstructed embryos in SCNT, and may include an epigenetic mechanism.

Rapid and reliable detection of glucose-6-phosphate dehydrogenase (G6PD) gene mutations in Han Chinese using high-resolution melting analysis.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked inherited disease, is one of the most common enzymopathies and affects over 400 million people worldwide. In China at least 21 distinct point mutations have been identified so far. In this study high-resolution melting (HRM) analysis was used to screen for G6PD mutations in 260 unrelated Han Chinese individuals, and the rapidity and reliability of this method was investigated. The mutants were readily differentiated by using HRM analysis, which produced distinct melting curves for each tested mutation. Interestingly, G1388A and G1376T, the two most common variants accounting for 50% to 60% of G6PD deficiency mutations in the Chinese population, could be differentiated in a single reaction. Further, two G6PD mutations not previously reported in the Chinese population were identified in this study. One of these mutations, designated "G6PD Jiangxi G1340T," involved a G1340T substitution in exon 11, predicting a Gly447Val change in the protein. The other mutation involved a C406T substitution in exon 5. The frequencies of the common polymorphism site C1311T/IVS (intervening sequence) XI t93c between patients with G6PD and healthy volunteers were not significantly different. Thus, HRM analysis will be a useful alternative for screening G6PD mutations.

Hematopoietic stem cell engraftment by early-stage in utero transplantation in a mouse model.

A novel intrauterine transplantation (IUT) approach was developed to improve the efficiency of engraftment of hematopoietic stem cells (HSCs). HSCs with a green fluorescent protein (GFP) reporter gene were transplanted in utero on days 12.5, 13.5 and 14.5 post coitum (p.c.). The degree of chimerism of donor cells in recipient newborn mice was examined using fluorescent microscopy, polymerase chain reaction (PCR), fluorescence-activated cell sorting (FACS), and fluorescence in situ hybridization (FISH) analyses. Microscopic examination revealed the presence of green fluorescent signal in the peripheral blood of the chimeric mice. The highest survival rate (47%) as well as the highest chimerism rate (73%) were achieved by our new approach in the newborn mice that were subjected to in utero transplantation (IUT) on day 12.5 p.c. (E12.5) compared to the conventional IUT method. FACS analysis indicated that 1.55+/-1.10% of peripheral blood cells from the newborn mice were GFP-positive donor cells. FISH showed that cells containing the donor-specific GFP sequence were present in the bone marrow (BM) of the chimeric mice. Thus, the efficiency of chimera production with this new method of IUT was significantly improved over the existing IUT techniques and instruments.

Improved efficiency of bovine somatic cell nuclear transfer by optimizing operational procedures.

To improve bovine somatic cell nuclear transfer (SCNT) efficiency, we studied various aspects to optimize the experimental procedures. Firstly, donor cells were treated with pronase, which resulted in a higher fusion rate than that of cells without the pronase treatment (78.3 vs. 53.9%). Secondly, when fused embryos were activated either by chemical (ionomycin + cyclohemixide (CHX)) or electrical + CHX stimulation, the cleavage and blastocyst formation rates were comparable amongst these treatment groups (P>0.05); however, mortality following electrical + CHX activation was significantly higher than that observed with the chemical activation, regardless of the pronase treatment (P<0.05). Finally, we compared the culture conditions of the reconstructed embryos using ACM medium plus mouse embryonic fibroblasts (MEF) vs. B2 medium plus granulose cells (GC), and the results clearly demonstrated that the former culture conditions led to a higher blastocyst rate, 90-day pregnancy rate, and newborn rate, than that observed for culture in B2 medium plus GC (46.7 vs. 34.7%, 36.1 vs. 9.6% and 25.9 vs. 5.8% for the blastocyst, pregnancy and newborn rates, respectively). In summary, the efficiency of bovine SCNT can be greatly improved using optimized operational procedures, including treating the donor cells with pronase, activation of fused embryos by ionomycin + CHX and the culture of the reconstructed embryos in ACM + MEF media.

A phiC31 integrase-mediated integration hotspot in favor of transgene expression exists in the bovine genome.

phiC31 integrase, a site-specific recombinase, can effectively mediate foreign genes bearing an attB sequence integrated into pseudo attP sites. We have previously identified two pseudo attP sites, BpsF1 and BpsM1 from the bovine genome. In this study, two new pseudo attP sites, BF4 and BF10, were discovered using half-nested inverse PCR from cow fibroblasts. The genomic locations of these two pseudo attP sites were identified by direct sequencing and a BLAST search, and it was confirmed that they reside at positions 4q31 and 10q35 by fluorescence in situ hybridization analysis. Subsequently, the distinct integration frequencies of the four pseudo attP sites were examined. The BF4 site was identified as a hotspot where site-specific integration occurred in most of the cell clones examined, accounting for 74% (42/57) of the integration; much more than the integration frequency for BF10 (7%; 4/57), BpsF1 (7%; 4/57) and BpsM1 (0/57). Interestingly, similar to other hotspots identified in the human and mouse genomes, in which transgenes integrated at hotspots result in high expression, the GFP gene integrated at hotspot BF4 was expressed at high levels in cow fibroblasts, as confirmed by fluorescence microscopy and FACS analysis. Furthermore, ELISA showed that the expression level of the GFP gene integrated at the BF4 site averaged approximately 328 microg x mg(-1), which is more than twofold higher than that integrated at the BF10 site. This study suggests that somatic cells carrying a desired gene integrated at the BF4 site can be used as nuclear donors to generate valuable transgenic animals by nuclear transfer.

Effect of individual heifer oocyte donors on cloned embryo development in vitro.

The aim of this study was to determine the effect of individual oocyte donors on cloned embryo development in vitro. Five Holstein heifers of varied genetic origins were subject to ovum pick up (OPU) once weekly. In total, 913 oocytes were recovered from 1304 follicles. A mean of 7.7+/-0.4 oocytes was recovered per session per animal. Individual mean oocyte production varied significantly in quantity but not in quality (morphological categories) among heifers. Oocytes from individual heifers were used as recipient cytoplasm for somatic cell nuclear transfer (SCNT). Cumulus cells, collected from a single Holstein cow genetically unrelated to the oocyte donor, were used as donor cells. Although the percentage of reconstructed embryos that started to cleave was nearly constant, the percentage of cleaved embryos that developed into blastocysts showed clear individual heifer variation (61%, 51%, 31%, 28% and 24%, respectively), with a mean of 38% showing blastocyst formation. In vitro fertilization (IVF) was also conducted with oocyte from the same heifers used in SCNT. A variation of blastocyst production among individual heifers was also shown in the IVF experiment, but the rank of oocyte donor based on the blastocyst rate was changed. In conclusion, individual oocyte donor may have an effect on cloned embryo development in vitro, which differed from the effect on IVF embryos.

Array-MLPA: comprehensive detection of deletions and duplications and its application to DMD patients.

Multiplex ligation-dependent probe amplification (MLPA) is widely used to screen genes of interest for deletions and duplications. Since MLPA is usually based on size-separation of the amplification products, the maximum number of target sequences that can be screened in parallel is usually limited to approximately 40. We report the design of a robust array-based MLPA format that uses amplification products of essentially uniform size (100-120 bp) and distinguishes between them by virtue of incorporated tag sequences. We were thus able to increase probe complexity to 124, with very uniform product yields and signals that have a low coefficient of variance. The assay designed was used to screen the largest set studied so far (249 patients) of unrelated Duchenne muscular dystrophy (DMD) cases from the Chinese population. In a blind study we correctly assigned 98% of the genotypes and detected rearrangements in 181 cases (73%); i.e., 163 deletions (65%), 13 duplications (5%), and five complex rearrangements (2%). Although this value is significantly higher for Chinese patients than previously reported, it is similar to that found for other populations. The location of the rearrangements (76% in the major deletion hotspot) is also in agreement with other findings. The 96-well flow-through microarray system used in this research provides high-throughput and speed; hybridization can be completed in 5 to 30 minutes. Since array processing and data analysis are fully automated, array-MLPA should be easy to implement in a standard diagnostic laboratory. The universal array can be used to analyze any tag-modified MLPA probe set.

Derivation of human embryonic stem cell lines from parthenogenetic blastocysts.

Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.