Isoniazide modified Ag nanoparticles triggered photothermal immunoassay for carcinoembryonic antigen detection.

As the most well-known analytical tool, the thermometer has been extended to the field of biological analysis based on the photothermal effect. Herein, isoniazide modified Ag nanoparticles were prepared as nanolabels to build an immunoassay. The nanoparticles were characterized by transmission electron microscope (TEM), dynamic laser scattering (DLS), X-ray powder diffraction (XRD), and Fourier transform infrared (FT-IR). When the target protein was present, the sandwich immunoassay was developed and the photothermal reaction was triggered by isoniazide modified Ag nanoparticles. As a reducing agent, isoniazide is used to transform phosphomolybdic acid hydrate into molybdenum blue solution. And molybdenum blue had good photothermal stability and high photothermal conversion efficiency. The temperature variation of molybdenum blue solution showed a positive correlation with the concentration of carcinoembryonic antigen (CEA). Thus, the target protein of CEA was quantitative detection by thermometer. The linear response range is 0.1 ng mL-1 to 40 ng mL-1, and the detection limit is 0.08 ng mL-1. Moreover, the proposed protocol had satisfactory selectivity, accuracy, and reproducibility.

Molybdenum blue mediated photothermal immunoassay for CEA detection based on Ag4P2O7@Ag nanocomposites.

A photothermal immunoassay was built for tumor marker detection based on Ag4P2O7@Ag nanocomposites. Ag4P2O7@Ag nanomaterials were synthesized by precipitation-photoreduction reaction, and characterized by transmission electron microscope (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectra (XPS) and X-ray powder diffraction (XRD). Come about PO43- derived from Ag4P2O7@Ag under acidic conditions react with ammonium molybdate in the action of reductant generating molybdenum blue. The photothermal change is due to molybdenum blue solution depending on the concentration of carcinoembryonic antigen (CEA) in immunoassay. Under optimal conditions, there is a linear relation between ΔT and CEA concentration in the range of 1 pg mL-1-40 ng mL-1 with the detection limit of 0.33 pg mL-1. Meanwhile, the developed photothermal immunoassay displays preferable selectivity, repeatability, and stability.

A Colorimetric Immunoassay Based on g-C3N4@Fe3O4 Nanocomposite for Detection of Carcinoembryonic Antigen.

We proposed a colorimetric immunosensor based on g-C3N4@Fe3O4 nanocomposite-mediated transformation strategy for sensitive detection of carcinoembryonic antigen (CEA). The g-C3N4@Fe3O4 nanocomposite was synthesized and characterized by the scanning electron microscope (SEM), energy dispersive X-ray spectra (EDX), X-ray powder diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Fe3+ derived from g-C3N4@Fe3O4 nanocomposite could combine with sodium salicylate to form purple complex products. Based on this color development, the sandwich colorimetric immunoassay was built by utilizing g-C3N4@Fe3O4 nanocomposite as nanolabels on the microplate. With the increasement of CEA concentration, the purple color showed a gradient change. Under optimal conditions, the linearity range is 0.001-50 ng/mL with the detection limit of 0.35 pg/mL for CEA. More importantly, the colorimetric immunoassay has good selectivity, specificity, repeatability, and stability.

Transposon-induced epigenetic silencing in the X chromosome as a novel form of dmrt1 expression regulation during sex determination in the fighting fish.

BACKGROUND: Fishes are the one of the most diverse groups of animals with respect to their modes of sex determination, providing unique models for uncovering the evolutionary and molecular mechanisms underlying sex determination and reversal. Here, we have investigated how sex is determined in a species of both commercial and ecological importance, the Siamese fighting fish Betta splendens. RESULTS: We conducted association mapping on four commercial and two wild populations of B. splendens. In three of the four commercial populations, the master sex determining (MSD) locus was found to be located in a region of ~ 80 kb on LG2 which harbours five protein coding genes, including dmrt1, a gene involved in male sex determination in different animal taxa. In these fish, dmrt1 shows a male-biased gonadal expression from undifferentiated stages to adult organs and the knockout of this gene resulted in ovarian development in XY genotypes. Genome sequencing of XX and YY genotypes identified a transposon, drbx1, inserted into the fourth intron of the X-linked dmrt1 allele. Methylation assays revealed that epigenetic changes induced by drbx1 spread out to the promoter region of dmrt1. In addition, drbx1 being inserted between two closely linked cis-regulatory elements reduced their enhancer activities. Thus, epigenetic changes, induced by drbx1, contribute to the reduced expression of the X-linked dmrt1 allele, leading to female development. This represents a previously undescribed solution in animals relying on dmrt1 function for sex determination. Differentiation between the X and Y chromosomes is limited to a small region of ~ 200 kb surrounding the MSD gene. Recombination suppression spread slightly out of the SD locus. However, this mechanism was not found in the fourth commercial stock we studied, or in the two wild populations analysed, suggesting that it originated recently during domestication. CONCLUSIONS: Taken together, our data provide novel insights into the role of epigenetic regulation of dmrt1 in sex determination and turnover of SD systems and suggest that fighting fish are a suitable model to study the initial stages of sex chromosome evolution.

Effects of Ocean Acidification on Transcriptomes in Asian Seabass Juveniles.

Ocean acidification is changing the fate of marine organisms. It is essential to predict the biological responses and evolutionary processes driven by ocean acidification, to maintain the equilibrium of the marine ecosystem and to facilitate aquaculture. However, how marine organisms, particularly the marine fish species, respond to ocean acidification, is still poorly understood. Consequences of ocean acidification on finfish aquaculture are largely not well known. We studied the effects of ocean acidification for 7 days on growth, behaviour and gene expression profiles in the brain, gill and kidney of Asian seabass juveniles. Results showed that growth and behaviour were not affected by short-term ocean acidification. We found tissue-specific differentially expressed genes (DEGs) involving many molecular processes, such as organ development, growth, muscle development, ion homeostasis and neurogenesis and development, as well as behaviours. Most of the DEGs, which were functionally enriched in ion homeostasis, were related to calcium transport, followed by sodium/potassium channels. We found that genes associated with neurogenesis and development were significantly enriched, implying that ocean acidification has also adversely affected the neural regulatory mechanism. Our results indicate that although the short-term ocean acidification does not cause obvious phenotypic and behavioural changes, it causes substantial changes of gene expressions in all three analysed tissues. All these changes of gene expressions may eventually affect physiological fitness. The DEGs identified here should be further investigated to discover DNA markers associated with adaptability to ocean acidification to improve fish's capability to adapt to ocean acidification.

Genomic Basis of Striking Fin Shapes and Colors in the Fighting Fish.

Resolving the genomic basis underlying phenotypic variations is a question of great importance in evolutionary biology. However, understanding how genotypes determine the phenotypes is still challenging. Centuries of artificial selective breeding for beauty and aggression resulted in a plethora of colors, long-fin varieties, and hyper-aggressive behavior in the air-breathing Siamese fighting fish (Betta splendens), supplying an excellent system for studying the genomic basis of phenotypic variations. Combining whole-genome sequencing, quantitative trait loci mapping, genome-wide association studies, and genome editing, we investigated the genomic basis of huge morphological variation in fins and striking differences in coloration in the fighting fish. Results revealed that the double tail, elephant ear, albino, and fin spot mutants each were determined by single major-effect loci. The elephant ear phenotype was likely related to differential expression of a potassium ion channel gene, kcnh8. The albinotic phenotype was likely linked to a cis-regulatory element acting on the mitfa gene and the double-tail mutant was suggested to be caused by a deletion in a zic1/zic4 coenhancer. Our data highlight that major loci and cis-regulatory elements play important roles in bringing about phenotypic innovations and establish Bettas as new powerful model to study the genomic basis of evolved changes.

Mapping and Validating QTL for Fatty Acid Compositions and Growth Traits in Asian Seabass.

Asian seabass is an important food fish species. While improving growth, increasing the nutritional value is important, omega-3 fatty acids are indispensable to human health. Identifying and validating DNA markers associated with traits is the first step towards marker-assisted selection (MAS). We quantified 13 different fatty acids and three growth traits in 213 F2 Asian seabass from a family at the age 270 days post hatch, and screened QTL for these traits. The content of total fatty acids in 100 g flesh was 2.57 ± 0.80 g, while the proportions of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were 16.96 ± 2.20% and 5.42 ± 0.90%, respectively. A linkage map with 2424 SNPs was constructed and used for QTL mapping. For fatty acid compositions, 14 significant QTL were identified on three linkage groups (LG5, LG11 and LG14), with phenotypic variance explained (PVE) from 12.8 to 24.6%. Thirty-nine suggestive QTL were detected on 16 LGs. Two significant QTL for EPA were identified on LG5 and LG14, with PVE of 15.2% and 15.1%, respectively. No significant QTL was identified for DHA. For growth traits, six significant and 13 suggestive QTL were identified on two and seven LGs, respectively. Only a few significant QTL for fatty acids overlapped with previously mapped QTL for these traits, suggesting that most QTL detected in a family are family-specific and could only be used in MAS in the family per se. To facilitate population-wide molecular breeding, more powerful methods (e.g. GWAS) should be used to identify SNPs for genomic selection.

A Universal Approach to Correct Various HBB Gene Mutations in Human Stem Cells for Gene Therapy of Beta-Thalassemia and Sickle Cell Disease.

Beta-thalassemia is one of the most common recessive genetic diseases, caused by mutations in the HBB gene. Over 200 different types of mutations in the HBB gene containing three exons have been identified in patients with ß-thalassemia (ß-thal) whereas a homozygous mutation in exon 1 causes sickle cell disease (SCD). Novel therapeutic strategies to permanently correct the HBB mutation in stem cells that are able to expand and differentiate into erythrocytes producing corrected HBB proteins are highly desirable. Genome editing aided by CRISPR/Cas9 and other site-specific engineered nucleases offers promise to precisely correct a genetic mutation in the native genome without alterations in other parts of the human genome. Although making a sequence-specific nuclease to enhance correction of a specific HBB mutation by homology-directed repair (HDR) is becoming straightforward, targeting various HBB mutations of ß-thal is still challenging because individual guide RNA as well as a donor DNA template for HDR of each type of HBB gene mutation have to be selected and validated. Using human induced pluripotent stem cells (iPSCs) from two ß-thal patients with different HBB gene mutations, we devised and tested a universal strategy to achieve targeted insertion of the HBB cDNA in exon 1 of HBB gene using Cas9 and two validated guide RNAs. We observed that HBB protein production was restored in erythrocytes derived from iPSCs of two patients. This strategy of restoring functional HBB gene expression will be able to correct most types of HBB gene mutations in ß-thal and SCD. Stem Cells Translational Medicine 2018;7:87-97.

The impact of luteinizing hormone supplementation in gonadotropin-releasing hormone antagonist cycles: a retrospective cohort study.

The impact of exogenous luteinizing hormone (LH) supplementation to patients undergoing controlled ovarian stimulation with gonadotropin-releasing hormone (GnRH) antagonists on cycle outcomes is controversial. Here, we present a retrospective cohort study including cycles from December 2015 to December 2016. Totally 320 cycles were divided into two groups according to with or without exogenous LH supplementation. No significant differences regarding the number of retrieved oocytes, the number of good-quality embryos, and clinical pregnancy rate between the two groups were found. The logistic regression analysis revealed that LH supplementation was not independently associated with clinical pregnancy rate (OR = 0.577, 95% CI: 0.272-1.222, p = .58) or a biochemical pregnancy rate (OR = 0.922, 95% CI: 0.444-1.916, p = .83). When patients were divided into subgroups based on age, more retrieved oocytes (5.60 vs. 3.97, p = .04) and good-quality embryos (3.07 vs. 1.93, p = .01) were achieved in cycles with exogenous LH supplementation for 40 years and over group. We conclude that for aged women (40 years old and over), LH supplementation has a positive impact on the number of retrieved oocytes and good-quality embryos in GnRH antagonist cycles.

Tim-1+ B cells suppress T cell interferon-gamma production and promote Foxp3 expression, but have impaired regulatory function in coronary artery disease.

Atherosclerosis and its associated coronary artery disease (CAD) represent another chronic low-grade inflammatory disorder. Regulatory B cells (Bregs) possess essential functions in maintaining peripheral tolerance and inhibiting pathogenic inflammation through IL-10. Here, we investigated one subset of Bregs, Tim-1+ B cell, and its role in atherosclerosis and CAD patients. In healthy individuals, IL-10-producing B cells were predominantly found in the Tim-1+ B cells. Upon stimulation of the B cell receptor (BCR) and Toll-like receptor 9 (TLR-9) by anti-BCR antibodies and CpG, respectively, the Tim-1+ B cells could further upregulate IL-10 expression. In contrast, the Tim-1+ B cells were present at normal frequency in CAD patients, but showed impaired capacity to upregulate IL-10 with or without BCR + CpG stimulation. The stimulated Tim-1+ B cells from healthy individuals also suppressed expression of interferon gamma (IFN-γ), an atherogenic cytokine in T cells, in an IL-10-dependent fashion, and strongly promoted the expression of Foxp3 in naive CD4+ CD45RO- T cells. In contrast, the Tim-1+ B cells from CAD patients were unable to suppress IFN-γ secretion, and only minimally increased the expression of Foxp3 in naive CD4+ CD45RO- T cells. Despite this, the frequency of Tim-1+ B cells in the atherosclerotic lesions from CAD patients was inversely correlated with the frequency of IFN-γ-expressing T cells. Together, these results demonstrated that CAD patients presented an inflammatory disorder in regulatory B cells, which could be used as a therapeutic target.

Deriving cells expressing markers of female germ cells from premature ovarian failure patient-specific induced pluripotent stem cells.

AIM: We proposed a two-step protocol for deriving cells expressing markers of female germ cells (FGCs) from premature ovarian failure patient-specific induced pluripotent stem cells (POF-iPSCs). MATERIAL & METHODS: We cultured POF-iPSCs in suspension and pretreated them with TGFß-1 (1 ng/ml) for 2 days and continued with both TGFß-1 and BMP4 (50 ng/ml) for 5 more days. Then changed to media containing retinoic acid (1 µM) and 5% follicular fluid for another 7 days. Expression of markers of different stages of FGCs were detected. RESULTS: c-KIT, STELLA/DPPA3, VASA/DDX4, SCP3, GDF9 and ZP3 were positively detected and statistically significant different when compared with control groups. CONCLUSION: Our in vitro system was beneficial for POF-iPSCs differentiated cells to express STELLA, VASA and SCP3, which were the markers of meiosis initiation of FGCs.

Doctors' Preferences for Controlled Ovarian Stimulation Protocols in Intrauterine Insemination.

INTRODUCTION: Intrauterine insemination (IUI) is an important treatment for infertility. IUI combined with controlled ovarian stimulation (COS) is widely used because of the higher pregnancy rates compared to IUI cycles without COS. MATERIAL AND METHODS: We retrospectively analyzed a single center data from 458 patients underwent the first IUI cycle and had only 1 mature follicle from May 2009 to January 20144. 48 cycles were performed with Clomiphene citrate/Letrozole (CC/LE), 244 cycles with gonadotropins (Gn), 71 cycles with CC/LE+Gn, and 95 cycles in NC group. RESULTS: Results showed that doctors preferred Gn protocol (53.3%) (p<0.05). Older patients were more likely to be allocated to CC/LE or NC group. 98.95% patients in NC group had regular menstruation cycle, with only 49.3% in CC/LE+Gn group (p<0.05). Estradiol (E2) level was much higher in COS groups than in NC group (p<0.05, for one mature follicle patients), and no significant differences were found within the COS groups. Duration of reaching follicles maturation was the shortest in Gn group and the longest in NC group, and NC group has the smallest follicular diameter (p<0.05). CONCLUSION: No significances were found regarding the IUI outcomes. To sum up, doctors prefer COS for IUI. Patients' age, menstruation cycle, infertile etiology and ovary function were the main factors affecting doctors' selection of COS protocols.

Forensic Identification for Erectile Dysfunction: Experience of a Single Center.

OBJECTIVE: To analyze the characteristics of forensic identification cases and evaluate the importance of integrating penile erection length, angle, and rigidity in diagnosing erectile dysfunction (ED). METHODS: Retrospective analysis of the forensic identification cases between Jan 2009 and May 2013. Correlation between ED diagnosis and nocturnal penile tumescence (NPT) test result or the site of injury was analyzed. RESULTS: In total, 148 patients came for forensic identification of sexual function because of rape charges, divorce, medical accidents, or injury: 126 of 148 (85.1%) because of injury, of which 95 (75.4%) resulted from traffic accidents. There was a significant correlation between the site of injury and ED diagnosis; pelvic fracture with urethral or perineum injury was the most common. Our data showed that ED diagnosis was in general significantly associated with NPT results. However, we also identified three cases of diagnosed organic ED with normal NPT reactions. Our analyses showed that abnormal length and/or angle of the erectile penis were contributing factors to the diagnoses in these cases. CONCLUSION: In addition to NPT test, which measures the rigidity of the erectile penis, the length and angle of the erectile penis should also be considered in diagnosing ED, particularly in the case of forensic identification of sexual function.

Polymorphism of stromal cell-derived factor-1 selectively upregulates gene expression and is associated with increased susceptibility to coronary artery disease.

Stromal cell-derived factor-1 (SDF-1) plays critical roles in vascular development and hematopoiesis. Here, we investigated the function of SDF-1 rs1801157G/A polymorphism in various immune cells and examined its association with susceptibility to coronary artery disease (CAD). Protein and mRNA levels of SDF-1 were tested in peripheral CD4+ T cell, CD8+ T cells, monocytes, and natural killer (NK) T cells from healthy donors with different genotypes of rs1801157G/A polymorphism. Prevalence of the polymorphism was compared between CAD patients and healthy controls. Data revealed that SDF-1 mRNA and protein were detectable in CD4+ T cells, CD8+ T cells, monocytes and NK T cells. Interestingly, both protein level and mRNA level of SDF-1 were significantly increased in the monocytes with rs1801157AA genotype, whereas the same phenomenon was not observed in the other three cell types. Blockage of CD14 completely inhibited the upregulation of SDF-1 in the monocytes with rs1801157AA genotype. Association analysis showed that frequencies of the rs1801157AA genotype and A allele were significantly higher in CAD cases than in controls (odds ratio [OR]=2.28, 95% confidence interval [CI], 1.50-3.29, p<0.0001, and OR=1.46, 95% CI, 1.21-3.73, p<0.0001, respectively). Also, prevalence of rs1801157AA genotype was further increased in cases with ST-elevation myocardial infarction (OR=1.65, 95% CI, 1.04-2.56, p=0.028). Our data suggest a novel pathway for regulating SDF-1 and a new risk factor for CAD.