Effect of genotyping errors on linkage map construction based on repeated chip analysis of two recombinant inbred line populations in wheat (Triticum aestivum L.).

Linkage maps are essential for genetic mapping of phenotypic traits, gene map-based cloning, and marker-assisted selection in breeding applications. Construction of a high-quality saturated map requires high-quality genotypic data on a large number of molecular markers. Errors in genotyping cannot be completely avoided, no matter what platform is used. When genotyping error reaches a threshold level, it will seriously affect the accuracy of the constructed map and the reliability of consequent genetic studies. In this study, repeated genotyping of two recombinant inbred line (RIL) populations derived from crosses Yangxiaomai × Zhongyou 9507 and Jingshuang 16 × Bainong 64 was used to investigate the effect of genotyping errors on linkage map construction. Inconsistent data points between the two replications were regarded as genotyping errors, which were classified into three types. Genotyping errors were treated as missing values, and therefore the non-erroneous data set was generated. Firstly, linkage maps were constructed using the two replicates as well as the non-erroneous data set. Secondly, error correction methods implemented in software packages QTL IciMapping (EC) and Genotype-Corrector (GC) were applied to the two replicates. Linkage maps were therefore constructed based on the corrected genotypes and then compared with those from the non-erroneous data set. Simulation study was performed by considering different levels of genotyping errors to investigate the impact of errors and the accuracy of error correction methods. Results indicated that map length and marker order differed among the two replicates and the non-erroneous data sets in both RIL populations. For both actual and simulated populations, map length was expanded as the increase in error rate, and the correlation coefficient between linkage and physical maps became lower. Map quality can be improved by repeated genotyping and error correction algorithm. When it is impossible to genotype the whole mapping population repeatedly, 30% would be recommended in repeated genotyping. The EC method had a much lower false positive rate than did the GC method under different error rates. This study systematically expounded the impact of genotyping errors on linkage analysis, providing potential guidelines for improving the accuracy of linkage maps in the presence of genotyping errors.

Identification of reliable QTLs and designed QTL breeding for grain shape and milling quality in the reciprocal introgression lines in rice.

Milling quality (MQ) and grain shape (GS) of rice (Oryza sativa L.) are correlated traits, both determine farmers' final profit. More than one population under multiple environments may provide valuable information for breeding selection on these MQ-GS correlations. However, suitable analytical methods for reciprocal introgression lines with linkage map for this kind of correlation remains unclear. In this study, our major tasks were (1) to provide a set of reciprocal introgression lines (composed of two BC2RIL populations) suitable for mapping by linkage mapping using markers/bins with physical positions; (2) to test the mapping effects of different methods by using MQ-GS correlation dissection as sample case; (3) to perform genetic and breeding simulation on pyramiding favorite alleles of QTLs for representative MQ-GS traits. Finally, with four analysis methods and data collected under five environments, we identified about 28.4 loci on average for MQ-GS traits. Notably, 52.3% of these loci were commonly detected by different methods and eight loci were novel. There were also nine regions harboring loci for different MQ-GS traits which may be underlying the MQ-GS correlations. Background independent (BI) loci were also found for each MQ and GS trait. All these information may provide useful resources for rice molecular breeding.

Molecular-Weight-Dependent Degradation of Plastics: Deciphering Host-Microbiome Synergy Biodegradation of High-Purity Polypropylene Microplastics by Mealworms.

The biodegradation of polypropylene (PP), a highly persistent nonhydrolyzable polymer, by Tenebrio molitor has been confirmed using commercial PP microplastics (MPs) (Mn 26.59 and Mw 187.12 kDa). This confirmation was based on the reduction of the PP mass, change in molecular weight (MW), and a positive Δδ13C in the residual PP. A MW-dependent biodegradation mechanism was investigated using five high-purity PP MPs, classified into low (0.83 and 6.20 kDa), medium (50.40 and 108.0 kDa), and high (575.0 kDa) MW categories to access the impact of MW on the depolymerization pattern and associated gene expression of gut bacteria and the larval host. The larvae can depolymerize/biodegrade PP polymers with high MW although the consumption rate and weight losses increased, and survival rates declined with increasing PP MW. This pattern is similar to observations with polystyrene (PS) and polyethylene (PE), i.e., both Mn and Mw decreased after being fed low MW PP, while Mn and/or Mw increased after high MW PP was fed. The gut microbiota exhibited specific bacteria associations, such as Kluyvera sp. and Pediococcus sp. for high MW PP degradation, Acinetobacter sp. for medium MW PP, and Bacillus sp. alongside three other bacteria for low MW PP metabolism. In the host transcriptome, digestive enzymes and plastic degradation-related bacterial enzymes were up-regulated after feeding on PP depending on different MWs. The T. molitor host exhibited both defensive function and degradation capability during the biodegradation of plastics, with high MW PP showing a relatively negative impact on the larvae.

Diagnostic yield and novel candidate genes for neurodevelopmental disorders by exome sequencing in an unselected cohort with microcephaly.

OBJECTIVES: Microcephaly is caused by reduced brain volume and most usually associated with a variety of neurodevelopmental disorders (NDDs). To provide an overview of the diagnostic yield of whole exome sequencing (WES) and promote novel candidates in genetically unsolved families, we studied the clinical and genetic landscape of an unselected Chinese cohort of patients with microcephaly. METHODS: We performed WES in an unselected cohort of 103 NDDs patients with microcephaly as one of the features. Full evaluation of potential novel candidate genes was applied in genetically undiagnosed families. Functional validations of selected variants were conducted in cultured cells. To augment the discovery of novel candidates, we queried our genomic sequencing data repository for additional likely disease-causing variants in the identified candidate genes. RESULTS: In 65 families (63.1%), causative sequence variants (SVs) and clinically relevant copy number variants (CNVs) with a pathogenic or likely pathogenic (P/LP) level were identified. By incorporating coverage analysis to WES, a pathogenic or likely pathogenic CNV was detected in 15 families (16/103, 15.5%). In another eight families (8/103, 7.8%), we identified variants in newly reported gene (CCND2) and potential novel neurodevelopmental disorders /microcephaly candidate genes, which involved in cell cycle and division (PWP2, CCND2), CDC42/RAC signaling related actin cytoskeletal organization (DOCK9, RHOF), neurogenesis (ELAVL3, PPP1R9B, KCNH3) and transcription regulation (IRF2BP1). By looking into our data repository of 5066 families with NDDs, we identified additional two cases with variants in DOCK9 and PPP1R9B, respectively. CONCLUSION: Our results expand the morbid genome of monogenic neurodevelopmental disorders and support the adoption of WES as a first-tier test for individuals with microcephaly.

Molecular identification and validation of four stable QTL for adult-plant resistance to powdery mildew in Chinese wheat cultivar Bainong 64.

KEY MESSAGE: Four stable QTL for adult-plant resistance (APR) to powdery mildew were identified on chromosome arms 1DL, 2BS, 2DL, and 6BL in the widely grown Chinese wheat cultivar Bainong 64. These QTL had no effect on response to stripe rust or leaf rust. Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a devastating fungal disease. Seedlings of Chinese wheat Bainong 64 are susceptible to Bgt, but adult plants have maintained resistance since it was released in 1996. A population of 171 recombinant inbred lines (RILs) developed from cross Jingshuang 16/Bainong 64 (JS16/BN64) was used to dissect genetic components of powdery mildew resistance. A genetic map comprising 5383 polymorphic markers was constructed using the 15 K SNP chip and kompetitive allele-specific PCR (KASP) markers. Composite interval mapping identified four stable QTL with favorable alleles all from BN64 on chromosome arms 1DL, 2BS, 2DL, and 6BL in at least four environments. They accounted for 8.3%, 13.8%, 14.4%, and 9.0% of the total phenotypic variation explained (PVE) in maximum, respectively. QPmjbr.caas-1DL, situated about 22 Mb from centromere, is probably a new QTL. QPmjbr.caas-2DL located near the end of arm 2DL and explained the largest PVE. Using genetic maps populated with KASP markers, QPmjbr.caas-2BS and QPmjbr.caas-6BL were fine mapped to a 1.8 cM genetic intervals spanning 13.6 Mb (76.0-89.6 Mb) and 1.7 cM and 4.9 Mb (659.9-664.8 Mb), respectively. The four QTL independent of stripe rust and leaf rust resistance were validated for powdery mildew resistance in another RIL population related to BN64 and a cultivar panel using representative KASP markers. Since BN64 has been a leading cultivar and an important breeding parent in China, the QTL and markers reported in this study will be useful for marker-assisted selection of APR.

Association between serum 25- hydroxyvitamin D and albuminuiria in middle-aged and older Chinese patients with type 2 diabetes.

OBJECTIVE: The objective was to analyze the relationship between serum 25-hydroxy-vitamin D (25(OH)D) level and albuminuiria in middle-aged and older patients with type 2 diabetes of Gansu Province. METHODS: Data pertaining to 380 in-patients with type 2 diabetes were collected. Subjects were classified groups based on gender,age,25(OH)D,BMI and UACR.Serum 25(OH)D and other clinical characteristics among various UACR groups were compared.The relationship between albuminuiria and 25(OH)D was analyzed. RESULTS: Out of the 380 subjects, 83.4%were classified as vitamin D deficiency, 14.5%were classified as vitamin D insufficiency, while 2.1% were classified as vitamin D sufficiency. Among the participants,41% had albuminuria (microalbuminuria,28.7%;macroalbuminuria,12.3%).The prevalence of 25(OH)D deficiency in the albuminuria group(84.6%) was significantly higher than that in the normoalbuminuria group(82.6%)(Mann-Whitney U test:Z = -3.86,P = 0.000); patients with macroalbuminuria had the highest prevalence of 25(OH)D deficiency (91.5%; P < 0.01 versus normoalbuminuria).A binary logistic analysis demonstrated that 25(OH)D were protective factors for albuminuria. CONCLUSIONS: The prevalence of vitamin D deficiency in patients with albuminuria was overtly higher than that in patients without albuminuria among middle-aged and older adults with type 2 diabetes.

Ameliorative effect of rosiglitazone, a peroxisome proliferator gamma agonist on adriamycin-induced cardio toxicity via suppressing oxidative stress and apoptosis.

We investigated the rosiglitazone (RSG) effect on adriamycin (ADM)-induced cardio toxicity in experimental animals. Forty adult Wistar male rats were separated into four groups as follows: normal control; RSG (10 mg/kg)-treated; ADM (10 mg/kg)-administered; and ADM (10 mg/kg) + RSG (10 mg/kg)-treated. Serum lipid level, different biochemical biomarkers, histological analysis, and nuclear factor erythroid 2-related factor/heme oxygenase-1 (Nrf2/HO-1), Caspase 3, B-cell lymphoma 2 (Bcl-2), and Bax gene expression were assessed in serum and cardiac tissue samples. Our results show that RSG treatment in ADM-administered animals significantly diminished low-density lipoprotein cholesterol, triglyceride, and total cholesterol, and increases high-density lipoprotein cholesterol (HDL-c) in comparison with the ADM group. RSG treatment reduced the effect of ADM administration on cardiac dysfunction markers such as cardiac troponin T Creatine Kinase-MB, aspartate aminotransferase, and lactate dehydrogenase, showing the amelioration of cardio toxicity in ADM-administered rats. Additionally, RSG treatment significantly decreased the level of malondialdehyde and nitric oxide in cardiovascular tissue. RSG-treated rats in combination with ADM likewise showed a significant increase in reduced glutathione, superoxide dismutase, catalase content, and the activity of glutathione peroxidase (GPx) as compared with ADM group. Moreover, RSG treatment in ADM rats significantly increased an Nrf2 and HO-1 expression in comparison with ADM group. While in apoptosis parameters, RSG treatment in ADM rats significantly diminished a cleaved caspase-3 and Bax expression as well as expanded Bcl-2 expression when contrasted with ADM group of rats. In conclusion, RSG is capable of protecting heart toxicity in ADM-treated animals through defensive effects on oxidative stress and biochemical markers.

ARMS TaqMan real-time PCR for genotyping factor V Leiden mutation in Han Chinese.

Factor V Leiden (FVLeiden ) is a missense mutation of 1691 position (G1691A) in exon 10 of FV gene, and being a genetic risk for venous thrombosis. Currently, there are several PCR-based methods for detecting FVLeiden mutation; however, these methods have disadvantages such as time-consuming, cumbersome steps and potentially hazardous gels. The aims of present study were to develop a simple, time-saving, accurate, and gel-free method, called amplification refractory mutation system (ARMS) TaqMan real-time PCR, for detecting FVLeiden mutation. We severally designed two specific reverse primers for mutant and wild-type through intentional introduction of mismatched nucleotide at the penultimate 3' position. Although target amplicon amplification efficiency is reduced, but another corresponding amplicon is almost completely inhibited. Then, specific TaqMan-probe was designed to detect target amplicon. Established method was used to detect 500 unselected samples in Han Chinese, the results showed 499 cases of wild-type and one heterozygote. Afterward, 50 randomly picked wild-type cases and one heterozygote were reexamined by bidirectional DNA sequencing, which is considered as "Gold standard method." Exhilaratingly, the results detected by the two methods were completely consistent. At last, allelic frequency of FVLeiden was calculated the in Han Chinese. Given the above results, A FVLeiden heterozygote has been found in 500 random samples in Han Chinese, and the allelic frequency was 0.1%. In conclusion, the ARMS TaqMan real-time PCR is an ideal detecting system for genotyping FVLeiden mutation in clinical application, and FVLeiden mutation exists in Han Chinese despite extremely low prevalence.

Different Decision-Making Responses Occupy Different Brain Networks for Information Processing: A Study Based on EEG and TMS.

This study used large-scale time-varying network analysis to reveal the diverse network patterns during the different decision stages and found that the responses of rejection and acceptance involved different network structures. When participants accept unfair offers, the brain recruits a more bottom-up mechanism with a much stronger information flow from the visual cortex (O2) to the frontal area, but when they reject unfair offers, it displayed a more top-down flow derived from the frontal cortex (Fz) to the parietal and occipital cortices. Furthermore, we performed 2 additional studies to validate the above network models: one was to identify the 2 responses based on the out-degree information of network hub nodes, which results in 70% accuracy, and the other utilized theta burst stimulation (TBS) of transcranial magnetic stimulation (TMS) to modulate the frontal area before the decision-making tasks. We found that the intermittent TBS group demonstrated lower acceptance rates and that the continuous TBS group showed higher acceptance rates compared with the sham group. Similar effects were not observed after TBS of a control site. These results suggest that the revealed decision-making network model can serve as a potential intervention model to alter decision responses.

The diagnosis values of serum STAT4 and sEng in preeclampsia.

OBJECTIVE: To detect the levels of signal transducer and activator of transcription 4 (STAT4) and soluble endoglin (sEng) in preeclampsia patients and analyze the diagnostic values of STAT4 and sEng in preeclampsia. METHODS: Fifty-four pregnant women with preeclampsia from October 2017 to June 2018 were included in this study. Twenty-eight matched healthy pregnant women were set as the control group. The general clinical characteristics were measured. Serum STAT4 and sEng were detected by ELISA. Correlation between STAT4 and sEng, and their diagnostic value in preeclampsia were analyzed. RESULTS: Compared with control, the prothrombin time in preeclampsia was significantly lower, while the mean arterial pressure, 24-hour urine protein, serum creatinine, fibrinogen, and ALT were significantly higher. The circulating levels of STAT4 and sEng were significantly increased in the preeclampsia. The serum levels of STAT4 and sEng in preeclampsia were positively correlated. For the diagnosis of preeclampsia by the serum STAT4, AUC is 0.902, and the sensitivity and specificity are 0.893 and 0.929. By the serum sEng, AUC is 0.873, and the sensitivity and specificity are 0.816 and 0.905. CONCLUSION: The serum levels of STAT4 and sEng were significantly increased in preeclampsia with disease severity status, which have promise as diagnostic markers in preeclampsia.

Genomic Prediction for Grain Yield and Yield-Related Traits in Chinese Winter Wheat.

Genomic selection (GS) is a strategy to predict the genetic merits of individuals using genome-wide markers. However, GS prediction accuracy is affected by many factors, including missing rate and minor allele frequency (MAF) of genotypic data, GS models, trait features, etc. In this study, we used one wheat population to investigate prediction accuracies of various GS models on yield and yield-related traits from various quality control (QC) scenarios, missing genotype imputation, and genome-wide association studies (GWAS)-derived markers. Missing rate and MAF of single nucleotide polymorphism (SNP) markers were two major factors in QC. Five missing rate levels (0%, 20%, 40%, 60%, and 80%) and three MAF levels (0%, 5%, and 10%) were considered and the five-fold cross validation was used to estimate the prediction accuracy. The results indicated that a moderate missing rate level (20% to 40%) and MAF (5%) threshold provided better prediction accuracy. Under this QC scenario, prediction accuracies were further calculated for imputed and GWAS-derived markers. It was observed that the accuracies of the six traits were related to their heritability and genetic architecture, as well as the GS prediction model. Moore-Penrose generalized inverse (GenInv), ridge regression (RidgeReg), and random forest (RForest) resulted in higher prediction accuracies than other GS models across traits. Imputation of missing genotypic data had marginal effect on prediction accuracy, while GWAS-derived markers improved the prediction accuracy in most cases. These results demonstrate that QC on missing rate and MAF had positive impact on the predictability of GS models. We failed to identify one single combination of QC scenarios that could outperform the others for all traits and GS models. However, the balance between marker number and marker quality is important for the deployment of GS in wheat breeding. GWAS is able to select markers which are mostly related to traits, and therefore can be used to improve the prediction accuracy of GS.

Prioritization of pharmaceuticals in water environment in China based on environmental criteria and risk analysis of top-priority pharmaceuticals.

Numerous studies have shown that a wide range of pharmaceuticals are present in the environment and many of their adverse biological effects on the aquatic ecosystem and human health are unknown. Due to the high population density and large number of pharmaceuticals produced and consumed in China, a systematic approach is needed to identify pharmaceuticals that require greater attention. The present study provides a ranking of pharmaceuticals in China in terms of their occurrence (O), persistence, bioaccumulation, and toxicity (PBT) based on the predicted environmental concentration (PEC). The total and partial ranking method implemented in the decision analysis by ranking techniques (DART) tool was used, which is an easy-to-use tool for the analysis of datasets. Using the DART approach, 10 pharmaceuticals were selected as priority compounds. These pharmaceuticals included antibiotics, anti-inflammatory and antilipidemic. In order to identify the characteristics of the priority pharmaceuticals, ecotoxicological endpoints were considered. The results of this study and the priority list facilitate the selection of candidate pollutants in future monitoring studies.

Genetic Mapping with Background Control for Quantitative Trait Locus (QTL) in 8-Parental Pure-Line Populations.

Multiparental advanced generation intercross (MAGIC) populations provide abundant genetic variation for use in plant genetics and breeding. In this study, we developed a method for quantitative trait locus (QTL) detection in pure-line populations derived from 8-way crosses, based on the principles of inclusive composite interval mapping (ICIM). We considered 8 parents carrying different alleles with different effects. To estimate the 8 genotypic effects, 1-locus genetic model was first built. Then, an orthogonal linear model of phenotypes against marker variables was established to explain genetic effects of the locus. The linear model was estimated by stepwise regression and finally used for phenotype adjustment and background genetic variation control in QTL mapping. Simulation studies using 3 genetic models demonstrated that the proposed method had higher detection power, lower false discovery rate (FDR), and unbiased estimation of QTL locations compared with other methods. Marginal bias was observed in the estimation of QTL effects. An 8-parental recombinant inbred line (RIL) population previously reported in cowpea and analyzed by interval mapping (IM) was reanalyzed by ICIM and genome-wide association mapping implemented in software FarmCPU. The results indicated that ICIM identified more QTLs explaining more phenotypic variation than did IM; ICIM provided more information on the detected QTL than did FarmCPU; and most QTLs identified by IM and FarmCPU were also detected by ICIM.

Far infrared-assisted encapsulation of filter paper strips in poly(methyl methacrylate) for proteolysis.

Filter paper strips were enclosed between two poly(methyl methacrylate) plates to fabricate paper-packed channel microchips under pressure in the presence of far infrared irradiation. After the enclosed paper strip was oxidized by periodate, trypsin was covalently immobilized in them to fabricate microfluidic proteolysis bioreactor. The feasibility and performance of the unique bioreactor were demonstrated by digesting BSA and lysozyme. The results were comparable to those of conventional in-solution proteolysis while the digestion time was significantly reduced to ∼18 s. The suitability of the microfluidic paper-based bioreactors to complex proteins was demonstrated by digesting human serum.

Effects of Long-term Soft Contact Lenses on Tear Menisci and Corneal Nerve Density.

PURPOSE: We investigated the relationship between tear menisci and corneal subbasal nerve density (SND) in long-term soft contact lens (CL) wearers. METHODS: Three groups were enrolled for this study: asymptomatic non-CL controls (N-CL group), asymptomatic soft CL wearers (A-CL group), and symptomatic soft CL wearers with self-reported moderate to severe dryness (S-CL group). Upper and lower tear menisci height (UTMH, LTMH) and area (UTMA, LTMA) were measured by optical coherence tomography. This was followed by measurements of fluorescein tear breakup time, fluorescein staining, and the Schirmer I test. In vivo confocal microscopy measured the SND in the nasal, central, and temporal regions of the cornea. RESULTS: The UTMH, UTMA, LTMH, and LTMA were significantly lower in the S-CL group compared with the N-CL group (P<0.05). The combined corneal SND of the temporal and nasal regions of the S-CL group was lower than for the N-CL group (P<0.05). The LTMH was correlated with the SND of the temporal (r=0.410), nasal (r=0.423), combined temporal and nasal (r=0.516), and combined temporal, nasal, and central regions (r=0.430, all P<0.01). The LTMA was also correlated with the SND of the temporal (r=0.379), nasal (r=0.292), combined temporal and nasal (r=0.422), and combined temporal, nasal, and central regions (r=0.367, all P<0.05). The temporal and nasal corneal SNDs were more strongly correlated with the LTMH and LTMA than with the UTMH and UTMA. CONCLUSIONS: Soft CL wearers with dry eye symptoms have reduced tear menisci. The alteration of midperipheral corneal SND may contribute to dry eye symptoms.

[The correlations between corneal sensation, tear meniscus volume, and tear film osmolarity after femtosecond laser-assisted LASIK].

OBJECTIVE: To investigate the correlations between corneal sensation, tear meniscus volume, and tear film osmolarity after femtosecond laser-assisted LASIK (FS-LASIK) surgery. METHODS: In this prospective clinical study, 31 patients undergoing FS-LASIK for myopia were recruited. The upper and lower tear meniscus volumes (UTMV and LTMV) were measured by customized anterior segment optical coherence tomography, tear film osmolarity was measured by a TearLab Osmolarity test device, central corneal sensation was measured by a Cochet-Bonner esthesiometer preoperatively, at 1 week, 1 and 3 months postoperatively. Repeated measures analysis of variance was used to evaluate whether the tear film osmolarity, tear meniscus volume, and corneal sensation were changed after surgery. The correlations between these variables were analyzed by the Pearson correlation analysis. RESULTS: The tear film osmolarity was (310.03 ± 16.48) mOsms/L preoperatively, (323.51 ± 15.92) mOsms/L at 1 week, (319.93 ± 14.27) mOsms/L at 1 month, and (314.97±12.91) mOsms/L at 3 months. The UTMV was (0.42±0.15), (0.25± 0.09), (0.30±0.11), and (0.35±0.09) µL, respectively; the LTMV was (0.60±0.21),(0.37±0.08), (0.44± 0.14), and (0.52±0.17) µL, respectively. The tear film osmolarity was significantly higher at 1 week and 1 month postoperatively compared with the baseline (P=0.001, 0.004), and reduced to the preoperative level at 3 months (P=0.573). The UTMV, LTMV, and corneal sensation values presented significant decreases at all postoperative time points (all P<0.05). The Pearson correlation analysis showed the postoperative UTMV had a weak relationship with corneal sensation at 1 week after surgery (r=0.356,P=0.005). There were significant correlations between the preoperative LTMV and corneal sensation at 1 week, 1 and 3 months (respectively, r=0.422, 0.366, 0.352;P=0.001, 0.004, 0.006). No significant correlations were found between the tear film osmolarity, tear meniscus volume, and corneal sensation after surgery (all P>0.05). CONCLUSION: The tear film osmolarity, tear meniscus volume, and corneal sensation became aggravated due to the FS-LASIK surgery procedures. There were significant correlations between the preoperative tear meniscus volume and recovery of corneal sensation early after surgery. A higher tear meniscus volume before surgery may contribute to a faster corneal sensation recovery.

[Interferon-γ Induced by Toxoplasma gondii Excreted/Secreted Antigens Promotes Apoptosis of CD4+CD25+ Regulatory T Cells].

Objective: To investigate the effect of excreted/secreted antigens (ESAs) from Toxoplasma gondii RH strain and TgCtwh3 strain on apoptosis of CD4+CD25+ regulatory T cells and interferon-γ (IFN-γ) secretion. Methods: ESAs of Toxoplasma gondii RH strain and TgCtwh3 strain were prepared. Splenic mononuclear cells were isolated from C57BL/6 mice and randomly divided into RH ESA group（2×106 cells/well with addition of 10 µg/ml RH ESA）, TgCtwh3 ESA group （2×106 cells/well with addition of 10 µg/ml TgCtwh3 ESA） and control group（2×106 cells/well with addition of 10 µg/ml ovalbumin）. Flow cytometry was performed to examine the early apoptosis of CD4+CD25+ regulatory T cells after treatment for 48 h and 72 h. ELISA was conducted to determine the level of IFN-γ in the supernatant after treatment for 72 h. In another experiment, the 3 groups of splenic mononuclear cells were added with 10 µg/ml anti-IFN-γ antibody for 72 h and flow cytometry was performed to examine the early apoptosis of CD4+CD25+ regulatory T cells. Meanwhile, splenic mononuclear cells from IFN-γ knockout and wild-type C57BL/6 mice were also divided into the above-described 3 groups, and flow cytometry was performed to examine the early apoptosis of CD4+CD25+ regulatory T cells after treatment for 72 h. Results: The concentrations of RH ESA and TgCtwh3 ESA were 0.54 mg/ml and 2.14 mg/ml, respectively. Flow cytometry showed that the early apoptosis rate of CD4+CD25+ regulatory T cells in the RH ESA group and the TgCtwh3 ESA group after 48 h treatment was （12.90±1.26）% and （9.71±1.04）%, respectively （P＜0.05）, both significantly higher than that in control group （4.48±0.48）% （P<0.01） . The early apoptosis rate of CD4+CD25+ regulatory T cells after 72 h in the RH ESA group was（15.21±1.11）%, significantly higher than that in the TgCtwh3 ESA group[（11.02±0.92）%] （P<0.05） and the control group[（10.10±1.49）%]（P<0.01）. ELISA showed that the level of interferon-γ in the RH ESA group and the TgCtwh3 ESA group after 72 h was（4 764.0±118.7） pg/ml and （3 629.0±33.6） pg/ml, respectively （P＜0.01）, both significantly higher than that in the control［（679.4±30.6） pg/ml］（P<0.01）. Flow cytometry revealed lower early apoptosis rate of CD4+CD25+ regulatory T cells in the RH ESA group added with anti-IFN-γ antibody［（10.44±1.44）%ï¼½ compared with that without the addition of the antibody［（14.96±0.83）］（P<0.05）. But this difference was not observed for the TgCtwh3 ESA group. Moreover, the RH ESA-induced apoptosis rate of regulatory T cells from IFN-γ knockout mice［（10.64±0.55）%ï¼½ was significantly lower than that from the wild-type mice ［（15.21±1.11）%］（P<0.01）. But this difference was not found for the TgCtwh3 ESA treatment. Conclusion: T. gondii RH ESA induces apoptosis of CD4+CD25+ regulatory T cells and IFN-γ secretion, and these effects are stronger than those of TgCtwh3 ESA. The T. gondii ESA-induced IFN-γ stimulates generation of anti-Toxoplasma immunity during acute Toxoplasma infection through mediation of regulatory T cell apoptosis.

GACD: Integrated Software for Genetic Analysis in Clonal F1 and Double Cross Populations.

Clonal species are common among plants. Clonal F1 progenies are derived from the hybridization between 2 heterozygous clones. In self- and cross-pollinated species, double crosses can be made from 4 inbred lines. A clonal F1 population can be viewed as a double cross population when the linkage phase is determined. The software package GACD (Genetic Analysis of Clonal F1 and Double cross) is freely available public software, capable of building high-density linkage maps and mapping quantitative trait loci (QTL) in clonal F1 and double cross populations. Three functionalities are integrated in GACD version 1.0: binning of redundant markers (BIN); linkage map construction (CDM); and QTL mapping (CDQ). Output of BIN can be directly used as input of CDM. After adding the phenotypic data, the output of CDM can be used as input of CDQ. Thus, GACD acts as a pipeline for genetic analysis. GACD and example datasets are freely available from www.isbreeding.net.

Quantitative trait locus mapping with background control in genetic populations of clonal F1 and double cross.

In this study, we considered five categories of molecular markers in clonal F1 and double cross populations, based on the number of distinguishable alleles and the number of distinguishable genotypes at the marker locus. Using the completed linkage maps, incomplete and missing markers were imputed as fully informative markers in order to simplify the linkage mapping approaches of quantitative trait genes. Under the condition of fully informative markers, we demonstrated that dominance effect between the female and male parents in clonal F1 and double cross populations can cause the interactions between markers. We then developed an inclusive linear model that includes marker variables and marker interactions so as to completely control additive effects of the female and male parents, as well as the dominance effect between the female and male parents. The linear model was finally used for background control in inclusive composite interval mapping (ICIM) of quantitative trait locus (QTL). The efficiency of ICIM was demonstrated by extensive simulations and by comparisons with simple interval mapping, multiple-QTL models and composite interval mapping. Finally, ICIM was applied in one actual double cross population to identify QTL on days to silking in maize.

Compound heterozygous variants of ANKFY1 in a child with infantile-onset proteinuria and movement disorder.

The ANKFY1 gene encodes a protein that belongs to double zinc finger proteins involved in endocytosis. Only one family with steroid-resistant nephrotic syndrome has been reported carrying a homozygous variant in ANKFY1 so far. Here we describe the second case where a 13-year-old boy presented with infantile-onset proteinuria and movement disorder. Whole-exome sequencing showed compound heterozygous variants (NM_001330063.2: c.2753C>G; p.Ser918Ter, and c.3287-11_3287-10del) in ANKFY1. In vitro functional study revealed the two variants led to reduced protein expression level of ANKFY1. This is the first case of co-existence of renal and nervous system phenotypes in a child with variants in ANKFY1, suggesting that bi-allelic variants in ANKFY1 might be associated with a new neuro-renal syndrome.