Assisted reproductive technology induces different secondary sex ratio: parental and embryonic impacts.

BACKGROUND: Assisted reproduction technology (ART) has advanced significantly, raising concerns regarding its impact on the secondary sex ratio (SSR), which is the sex ratio at birth in offspring. This study aimed to explore factors affecting SSR in singletons, singletons from twin gestation, and twins from twin gestation within the context of ART. METHODS: A retrospective analysis was conducted on data from 8335 births involving 6,223 couples undergoing ART. Binary logistic regression assessed relationships between parental and embryonic factors and SSR in singletons and singletons from twin gestation. Multinomial logistic regression models were utilized to identify factors influencing SSR in twins from twin gestation. RESULTS: Secondary infertility (OR = 1.164, 95% CI: 1.009-1.342), advanced paternal age (OR = 1.261, 95% CI: 1.038-1.534), and blastocyst embryo transfer (OR = 1.339, 95% CI: 1.030-1.742) were associated with an increased SSR, while frozen embryo transfer (FET) showed a negative association with SSR (OR = 0.738, 95% CI: 0.597-0.912) in singletons. A longer duration of gonadotropin (Gn) usage reduced SSR in singletons (OR = 0.961, 95% CI: 0.932-0.990) and singletons from twin gestation (OR = 0.906, 95% CI: 0.838-0.980). In singletons from twin gestation, male-induced infertility (OR = 2.208, 95% CI: 1.120-4.348) and higher Gn dosage (OR = 1.250, 95% CI: 1.010-1.548) were significantly associated with an increased SSR. Women aged > 35 years and intracytoplasmic sperm injection (ICSI) were associated with lower SSR (OR = 0.539, 95% CI: 0.293-0.990 and OR = 0.331, 95% CI: 0.158-0.690, respectively). In twins from twin gestation, paternal age exceeded maternal age (OR = 0.682, 95% CI: 0.492-0.945) and higher Gn dosage (OR = 0.837, 95% CI: 0.715-0.980) were associated with a higher proportion of male twins. Cleavage stage transfer (OR = 1.754, 95% CI: 1.133-2.716) resulted in a higher percentage of boy-girl twins compared to blastocyst transfer. CONCLUSION: This study demonstrates the complex interplay of various factors in determining the SSR in ART, highlighting the importance of considering infertility type, paternal age, fertilization method, embryo transfer stage, and Gn use duration when assessing SSR. Nevertheless, further research with a large sample size is necessary to confirm and expand upon the findings of this study.

Characterization of a Hyaluronidase-Producing Bacillus sp. CQMU-D Isolated from Soil.

Hyaluronidase (HAase) can depolymerize mucopolysaccharide hyaluronic acid (HA) to increase the efficacy of drug diffusion in the case of pathogenic bacteria. Due to its widespread medical applications, HAase originating from microorganisms has attracted significant attention. In this study, the HAase-producing bacterium Bacillus sp. CQMU-D was isolated from soil and identified based on its morphology and 16S rRNA gene sequencing. Enzyme activity was detected by measuring the content of reducing sugar in HA degradation products with 3,5-dinitrosalicylic acid (DNSA) or p-dimethylaminobenzaldehyde (DMAB) as the detection reagent. The results revealed that HAase reached maximum activity after 48 h of cultivation. Gene function annotation after full-length sequencing showed increased transport and metabolic activities associated with HAase. Additionally, the HAase gene of Bacillus sp. CQMU-D was different from the existing microbial HAase, and its protein was predicted to be a stable secretory protein with a conserved GAG_Lyase domain. These results characterized a new HAase-producing Bacillus from the soil via enzyme activity and bioinformatic analysis, expanding the knowledge on Bacillus HAase for potential industrial applications.

Molecular Detection and Genotyping of Toxoplasma gondii from Pigs for Human Consumption in Zhejiang and Jiangsu Provinces, Eastern China.

Toxoplasma gondii infections are common in humans and animals worldwide. Ingestion of raw or undercooked meat containing tissue cysts of T. gondii is one major source of transmission of this parasite. It is important to guarantee the meat quality of China since our pork industry produces about half of the world's pork. In this study, a total of 746 pig samples were collected from Zhejiang and Jiangsu provinces in eastern China, and examined for T. gondii infection by PCR amplification targeting B1 gene. In this study, we found that 57 of 746 (7.6%) pigs were positive for B1 gene, with 8.5% (48/562) in Zhejiang province and 4.9% (9/184) in Jiangsu province, respectively. The positive DNA samples were further genotyped at 11 genetic markers, including SAG1, 5'-and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico through PCR-restriction fragment length polymorphism (PCR-RFLP) technology. Two genotypes (ToxoDB 9 and ToxoDB 10) of T. gondii were identified by PCR-RFLP in Zhejiang province. However, both genotypes were not determined from Jiangsu province, which is speculated on the low DNA concentration and the small number of samples. These results indicate that T. gondii infection is endemic in pigs in eastern China and may raise public food safety concerns, suggesting more interventions for T. gondii-related risks are needed in the future.

Identification of suitable reference genes for quantitative reverse transcription PCR in Luffa (Luffa cylindrica).

Reverse transcription real-time quantitative PCR is widely used to quantify gene expression. Reference genes are usually used as internal controls to measure the target gene expression level. To date, there is no consensus on the use of systematically validated reference genes in different tissues of Luffa. This study evaluated the expression stability of 11 candidate reference genes in different tissues using five algorithms (BestKeeper, comparative delta-Ct method, GeNorm, NormFinder, and RefFinder). Protein phosphatase 2A was the most stable gene, while alpha Tubulin was the least stable. The relative expression of ethylene-related genes in different tissues was also analyzed to reveal their role in sex determination. This study provides the basis for using suitable reference genes to evaluate targeted gene expression. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01182-8.

[Pregnancy Outcomes and Prognosis of Fetal Ventriculomegaly: A Retrospective Cohort Study].

Objective: To evaluate the pregnancy outcomes and neurodevelopment prognosis of subjects prenatally diagnosed with fetal ventriculomegaly (VM). Methods: All the subjects with VM diagnosed by ultrasound and were admitted and treated at West China Second Hospital, Sichuan University between March 2011 and September 2020 were retrospectively enrolled for a chohort study, while non-VM subjects of the same period were selected with a random number table to form the control group. Pregnancy outcomes of the two groups were compared, and the fetuses of both groups were followed up after birth for further assessment and comparison of their neurodevelopmental prognosis. Results: The live birth rate of the VM group was lower than that of the control group (77.63% [229/295] vs. 94.31% [265/281], P<0.001). Furthermore, the proportion of subjects that were transferred to NICU for monitoring and observation after birth was higher in the VM group than that of the control group (20.96% [48/229] vs. 4.53% [12/265], P<0.001). During the follow-up, it was found that the rate of neurodevelopmental abnormalities of the VM group was significantly higher than that of the control group (11.79% [27/229] vs. 1.90% [5/265], P<0.001). Moreover, neurodevelopmental abnormalities of VM fetuses were correlated to the following factors, the degree of VM ( P=0.010), intrauterine progression of VM ( P=0.024), and whether the postnatal cranial ultrasound result was suggestive of VM ( P=0.001). In addition, postnatal cranial ultrasound suggestive of VM was found to be an independent risk factor for neurodevelopmental abnormalities ( OR=9.434, 95% CI: 1.791-49.688, P=0.008). Conclusion: VM reduces the fetal live birth rate and may increase the risks of neurodevelopmental abnormalities after birth. All VM fetuses should be closely followed up for neurodevelopment status after birth, especially those with severe VM, intrauterine progression, and postnatal cranial ultrasound indicative of VM.

A mutation in LacDWARF1 results in a GA-deficient dwarf phenotype in sponge gourd (Luffa acutangula).

KEY MESSAGE: A dwarfism gene LacDWARF1 was mapped by combined BSA-Seq and comparative genomics analyses to a 65.4 kb physical genomic region on chromosome 05. Dwarf architecture is one of the most important traits utilized in Cucurbitaceae breeding because it saves labor and increases the harvest index. To our knowledge, there has been no prior research about dwarfism in the sponge gourd. This study reports the first dwarf mutant WJ209 with a decrease in cell size and internodes. A genetic analysis revealed that the mutant phenotype was controlled by a single recessive gene, which is designated Lacdwarf1 (Lacd1). Combined with bulked segregate analysis and next-generation sequencing, we quickly mapped a 65.4 kb region on chromosome 5 using F2 segregation population with InDel and SNP polymorphism markers. Gene annotation revealed that Lac05g019500 encodes a gibberellin 3ß-hydroxylase (GA3ox) that functions as the most likely candidate gene for Lacd1. DNA sequence analysis showed that there is an approximately 4 kb insertion in the first intron of Lac05g019500 in WJ209. Lac05g019500 is transcribed incorrectly in the dwarf mutant owing to the presence of the insertion. Moreover, the bioactive GAs decreased significantly in WJ209, and the dwarf phenotype could be restored by exogenous GA3 treatment, indicating that WJ209 is a GA-deficient mutant. All these results support the conclusion that Lac05g019500 is the Lacd1 gene. In addition, RNA-Seq revealed that many genes, including those related to plant hormones, cellular process, cell wall, membrane and response to stress, were significantly altered in WJ209 compared with the wild type. This study will aid in the use of molecular marker-assisted breeding in the dwarf sponge gourd.

Metabolic and transcriptomic analysis of two Cucurbita moschata germplasms throughout fruit development.

BACKGROUND: Pumpkins (Cucurbita moschata; Cucurbitaceae) are valued for their fruits and seeds and are rich in nutrients. Carotenoids and sugar contents, as main feature of pumpkin pulp, are used to determine the fruit quality. RESULTS: Two pumpkin germplasms, CMO-X and CMO-E, were analyzed regarding the essential quality traits such as dry weight, soluble solids, organic acids, carotenoids and sugar contents. For the comparison of fruit development in these two germplasms, fruit transcriptome was analyzed at 5 different developmental stages from 0 d to 40 d in a time course manner. Putative pathways for carotenoids biosynthesis and sucrose metabolism were developed in C. moschata fruit and homologs were identified for each key gene involved in the pathways. Gene expression data was found consistent with the accumulation of metabolites across developmental stages and also between two germplasms. PSY, PDS, ZEP, CRTISO and SUS, SPS, HK, FK were found highly correlated with the accumulation of carotenoids and sucrose metabolites, respectively, at different growth stages of C. moschata as shown by whole transcriptomic analysis. The results of qRT-PCR analysis further confirmed the association of these genes. CONCLUSION: Developmental regulation of the genes associated with the metabolite accumulation can be considered as an important factor for the determination of C. moschata fruit quality. This research will facilitate the investigation of metabolic profiles in other cultivars.

Light- and temperature-regulated BjAPY2 may have a role in stem expansion of Brassica juncea.

Tuber mustard (Brassica juncea (L.) Czern. et Coss. var. tumida Tsen et Lee) is an important vegetable crop with a characteristic of expanded stem that is edible. The underlying molecular mechanism of the stem expansion is not well understood. Here, we reported that a total of 51 differentially expressed fragments (DEFs) with three expression patterns during stem expansion of tuber mustard were identified by cDNA-AFLP analysis. Among the DEFs, DEF11 with high homology to Arabidopsis thaliana apyrase 2 (AtAPY2) that encodes an enzyme with ATPase and ADPase activity was development- and tissue-specific. DEF11 was thus renamed as BjAPY2. The expression levels of BjAPY2 increased with the stem expression and were the highest at stage IV, a developmental stage at which the stem expanded most rapidly. In contrast, the BjAPY2 expression levels in leaves were much lower and remained unchanged during leaf development and expansion, suggesting that BjAPY2 was closely associated with the expansion of stems but not of leaves in the tuber mustard. Interestingly, the expression of BjAPY2 was higher in the mustard under short-day (SD) photoperiod (8 h/16 h) than that under long-day (LD) photoperiod (16 h/8 h); similarly, the transcript levels of BjAPY2 were higher in the mustard grown at low temperature (14 °C/12 °C) than that at high temperature (26 °C /24 °C). The SD photoperiod and low temperature were two environmental conditions that favored the mustard stem expansion. Further cloning and analysis of the promoter region of BjAPY2 revealed that there were indeed several types of motifs in the promoter region, including the light and temperature responsive elements. These results suggested that BjAPY2 might play an important role during the stem expansion of the tuber mustard.

The role of small RNAs on phenotypes in reciprocal hybrids between Solanum lycopersicum and S. pimpinellifolium.

BACKGROUND: Reciprocal hybrids showing different phenotypes have been well documented in previous studies, and many factors accounting for different phenotypes have been extensively investigated. However, less is known about whether the profiles of small RNAs differ between reciprocal hybrids and how these small RNAs affect gene expression and phenotypes. To better understand this mechanism, the role of small RNAs on phenotypes in reciprocal hybrids was analysed. RESULTS: Reciprocal hybrids between Solanum lycopersicum cv. Micro-Tom and S. pimpinellifolium line WVa700 were generated. Significantly different phenotypes between the reciprocal hybrids were observed, including fruit shape index, single fruit weight and plant height. Then, through the high-throughput sequencing of small RNAs, we found that the expression levels of 76 known miRNAs were highly variable between the reciprocal hybrids. Subsequently, a total of 410 target genes were predicted to correspond with these differentially expressed miRNAs. Furthermore, gene ontology (GO) annotation indicated that those target genes are primarily involved in metabolic processes. Finally, differentially expressed miRNAs, such as miR156f and 171a, and their target genes were analysed by qRT-PCR, and their expression levels were well correlated with the different phenotypes. CONCLUSIONS: This study showed that the profiles of small RNAs differed between the reciprocal hybrids, and differentially expressed genes were also observed based on the different phenotypes. The qRT-PCR results of target genes showed that differentially expressed miRNAs negatively regulated their target genes. Moreover, the expression of target genes was well correlated with the observations of different phenotypes. These findings may aid in elucidating small RNAs contribute significantly to different phenotypes through epigenetic modification during reciprocal crossing.

The role of small RNAs in wide hybridisation and allopolyploidisation between Brassica rapa and Brassica nigra.

BACKGROUND: An allopolyploid formation consists of the two processes of hybridisation and chromosome doubling. Hybridisation makes a different genome combined in the same cell, and genome "shock" and instability occur during this process, whereas chromosome doubling results in doubling and reconstructing the genome dosage. Recent studies have demonstrated that small RNAs, play an important role in maintaining the genome reconstruction and stability. However, to date, little is known regarding the role of small RNAs during the process of wide hybridisation and chromosome doubling, which is essential to elucidate the mechanism of polyploidisation. Therefore, the genetic and DNA methylation alterations and changes in the siRNA and miRNA were assessed during the formation of an allodiploid and its allotetraploid between Brassica rapa and Brassica nigra in the present study. RESULTS: The phenotypic analysis exhibited that the allotetraploid had high heterosis compared with their parents and the allodiploid. The methylation-sensitive amplification polymorphism (MSAP) analysis indicated that the proportion of changes in the methylation pattern of the allodiploid was significantly higher than that found in the allotetraploid, while the DNA methylation ratio was higher in the parents than the allodiploid and allotetraploid. The small RNAs results showed that the expression levels of miRNAs increased in the allodiploid and allotetraploid compared with the parents, and the expression levels of siRNAs increased and decreased compared with the parents B. rapa and B. nigra, respectively. Moreover, the percentages of miRNAs increased with an increase in the polyploidy levels, but the percentages of siRNAs and DNA methylation alterations decreased with an increase in the polyploidy levels. Furthermore, qRT-PCR analysis showed that the expression levels of the target genes were negatively corrected with the expressed miRNAs. CONCLUSIONS: The study showed that siRNAs and DNA methylation play an important role in maintaining the genome stability in the formation of an allotetraploid. The miRNAs regulate gene expression and induce the phenotype variation, which may play an important role in the occurrence of heterosis in the allotetraploid. The findings of this study may provide new information for elucidating that the allotetraploids have a growth advantage over the parents and the allodiploids.

Construction of a lentiviral T/A vector for direct analysis of PCR-amplified promoters.

The promoter plays an important role in the regulation of gene expression. To analyze a promoter's activity, we developed a novel lentiviral T/A vector that contains two reporter genes, a luciferase (Luc2) gene and a green fluorescent protein (Venus) gene, that are linked via an internal ribosome entry site (IRES2). To test the performance of this vector, phosphoglycerate kinase-1 (PGK) and elongation factor-1α (EF1α) promoters were amplified by PCR and inserted into this lentiviral T/A vector using T4 DNA ligase, yielding two promoter-reporter vectors: pLent-T-PGK and pLent-T-EF1α. When these vectors were transfected into 293T cells, we observed a higher level of Venus expression under a fluorescence microscopy in the case of pLent-T-EF1α as compared to pLent-T-PGK. The results of the luciferase reporter assay showed that the ratio of the promoter activities of EF1α and PGK was approximately 9:1. The two promoter-reporter vectors were also packaged as lentiviral particles to conduct promoter activity assay in cultured cells. The ratio of the promoter activities of EF1α and PGK was 4.23:1 when they were infected into 293T cells at a multiplicity of infection of 1. This value is comparable to that of a parallel experiment using the commercial luciferase reporter vector pGL4.10 with an activity ratio of 5.99:1 for EF1α and PGK. These results indicate that lentiviral T/A vector will be a useful tool for analysis of promoter activity and specificity.

Occurrence Mechanism of the Abnormal Gelation Phenomenon of High Temperature Cementing Slurry Induced by a Polycarboxylic Retarder.

The class G oil well cement is a type of special cement that can be subjected to a high temperature formation environment. It was found that the class G cement tail slurry with a low polycarboxylic retarder dosage (usually ≤1% by weight of cement) was more prone to cause the abnormal gelation phenomenon (AGP) than the lead slurry with a high retarder dosage at a high temperature (usually when T ≥ 120 °C). This study aimed at the occurrence mechanism of this unfavorable phenomenon that seriously endangers the cementing security. Results showed that the abnormal gelatinous region underwent premature hydration; namely, the calcium hydroxide and calcium silicate hydrate (C-S-H) content were all higher than the nongelatinous region, while the copolymer content was the opposite. Correspondingly, the theory of "premature hydration and crystal nucleation" was proposed to explain the abnormal gelation mechanism of a cementing tail slurry with an insufficient retarder dosage. Furthermore, a novel functionalized copolymer retarder "PAIANS" was synthesized to alleviate the AGP.

Association between strenuous sports or other exercises and lung cancer risk: a mendelian randomization study.

Background: Studying the relationship between strenuous sports or other exercises (SSOE) and lung cancer risk remains underexplored. Traditional observational studies face challenges like confounders and inverse causation. However, Mendelian randomization (MR) provides a promising approach in epidemiology and genetics, using genetic variants as instrumental variables to investigate causal relationships. By leveraging MR, we have scrutinized the causal link between SSOE and lung cancer development. Methods: Twelve single-nucleotide polymorphisms (SNPs) associated with SSOE, as identified in previously published genome-wide association studies, were utilized as instrumental variables in our investigation. Summary genetic data at the individual level were obtained from relevant studies and cancer consortia. The study encompassed a total of 11,348 cases and 15,861 controls. The statistical technique of inverse variance-weighting (IVW), commonly employed in meta-analyses and MR studies, was employed to assess the causal relationship between SSOE and lung cancer risk. Results: The MR risk analysis indicated a causal relationship between SSOE and the incidence of lung cancer, with evidence of a reduced risk for overall lung cancer [odds ratio (OR) =0.129; 95% confidence interval (CI): 0.021-0.779; P=0.03], lung adenocarcinoma (OR =0.161; 95% CI: 0.012-2.102; P=0.16) and squamous cell lung cancer (OR =0.045; 95% CI: 0.003-0.677; P=0.03). The combined OR for lung cancer from SSOE (controlling for waist circumference and smoking status) was 0.054 (95% CI: 0.010-0.302, P<0.001). Conclusions: Our MR analysis findings indicate a potential correlation between SSOE and a protective effect against lung cancer development. Further investigation is imperative to uncover the precise mechanistic link between them.

Heritable variation and small RNAs in the progeny of chimeras of Brassica juncea and Brassica oleracea.

Chimeras have been used to study the transmission of genetic material and the resulting genetic variation. In this study, two chimeras, TCC and TTC (where the origin of the outer, middle, and inner cell layers, respectively, of the shoot apical meristem is designated by a 'T' for tuber mustard and 'C' for red cabbage), as well as their asexual and sexual progeny, were used to analyse the mechanism and the inheritance of the variation induced by grafting. Asexual TCC progeny were obtained by adventitious shoot regeneration, while TTC sexual progeny were produced by self-crossing. This study observed similar morphological variations in both the asexual and sexual progeny, including changes in leaf shape and the pattern of shoot apical meristem termination. The leaf shape variation was stable, while the rate of shoot apical meristem termination in the TTC progenies decreased from 74.52% to 3.01% after three successive rounds of self-crossing. Specific red cabbage small RNAs were found in the asexually regenerated plants (rTTT) that were not present in TTT, indicating that small RNAs might be transmitted from red cabbage to tuber mustard during grafting. Moreover, in parallel with the variations in phenotype observed in the progeny, some conserved miRNAs were differentially expressed in rTTT and TTT, which correlated with changes in expression of their target genes. These results suggest that the change in small RNA expression induced by grafting may be an important factor for introducing graft-induced genetic variations, providing a basis for further investigating the mechanism of graft-induced genetic variation through epigenetics.

Integrative transcriptomic and metabolomic analysis in mice reveals the mechanism by which ginseng stem-leaf saponins enhance mucosal immunity induced by a porcine epidemic diarrhea virus vaccination.

Porcine epidemic diarrhea virus (PEDV) is a main cause of severe enteric disease in piglets, leading to millions of dollars lost annually in the global pig industry. Parenteral vaccination is limited in generating sufficient mucosal immunity, which is crucial for early defense against PEDV. Here, we orally administered ginseng stem-leaf saponins (GSLS) to mice before parenteral vaccination and found that GSLS significantly enhanced the phagocytosis of dendritic cells, promoted the activities of CD4+ T cells and increased PEDV-specific IgA antibodies in the intestinal mucosa. Transcriptomic results showed that the altered genes following GSLS treatment were mostly related to the immune response and metabolism. In addition, integrated analysis of the transcriptome and metabolome revealed that the mechanism by which GSLS enhances mucosal immunity may be associated with progesterone-related pathways. Further studies are needed to explore the detailed molecular mechanisms.

Early Oral Administration of Ginseng Stem-Leaf Saponins Enhances the Peyer's Patch-Dependent Maternal IgA Antibody Response to a PEDV Inactivated Vaccine in Mice, with Gut Microbiota Involvement.

Neonatal piglets during the first week of life are highly susceptible to porcine epidemic diarrhoea virus (PEDV) infection, with mortality rates reaching 80-100%. Passive lactogenic immunity remains the most effective way to protect neonates from infection. Although safe, inactivated vaccines provide little or no passive protection. Here, we administered ginseng stem-leaf saponins (GSLS) to mice before parenteral immunization with an inactivated PEDV vaccine to investigate the effect of GSLS on the gut-mammary gland (MG)-secretory IgA axis. Early oral GSLS administration potently increased PEDV-specific IgA plasma cell generation in the intestine, facilitated intestinal IgA plasma cell migration to the MG by enhancing the chemokine receptor (CCR)10-chemokine ligand (CCL)28 interaction, and ultimately promoted specific IgA secretion into milk, which was dependent on Peyer's patches (PPs). Additionally, GSLS improved the gut microbiota composition, especially increasing probiotic abundance, and these microflora members promoted the GSLS-enhanced gut-MG-secretory IgA axis response and were regulated by PPs. In summary, our findings highlight the potential of GSLS as an oral adjuvant for PEDV-inactivated vaccines and provide an attractive vaccination strategy for lactogenic immunity induction in sows. Further studies are required to evaluate the mucosal immune enhancement efficacy of GSLS in pigs.

Sequential application of copy number variation sequencing and quantitative fluorescence polymerase chain reaction in genetic analysis of miscarriage and stillbirth.

BACKGROUND: Copy number variation sequencing (CNV-seq) could detect most chromosomal abnormalities except polyploidy, and quantitative fluorescence polymerase chain reaction (QF-PCR) is a supplementary method to CNV-seq in triploid detection. This study aimed to evaluate the feasibility of sequential application of CNV-seq and QF-PCR in genetic analysis of miscarriage and stillbirth. METHODS: A total of 261 fetal specimens were analyzed by CNV-seq, and QF-PCR was only further performed for samples with normal female karyotype identified by CNV-seq. Cost and turnaround time (TAT) was analyzed for sequential detection strategy. Subgroup analysis and logistic regression were carried out to evaluate the relationship between clinical characteristics (maternal age, gestational age, and number of pregnancy losses) and the occurrence of chromosomal abnormalities. RESULTS: Abnormal results were obtained in 120 of 261 (45.98%) cases. Aneuploidy was the most common abnormality (37.55%), followed by triploidy (4.98%) and pathogenic copy number variations (pCNVs) (3.45%). CNV-seq could detect the triploidy with male karyotype, and QF-PCR could further identify the remaining triploidy with female karyotype. In this study, we found more male triploidies than female triploidies. With the same ability in chromosomal abnormalities detection, the cost of sequential strategy decreased by 17.35% compared with combined strategy. In subgroup analysis, significant difference was found in the frequency of total chromosomal abnormalities between early abortion group and late abortion group. Results of logistic regression showed a trend that pregnant women with advanced age, first-time abortion, and abortion earlier than 12 weeks were more likely to detect chromosomal aberrations in their products of conception. CONCLUSION: Sequential application of CNV-seq and QF-PCR is an economic and practical strategy to identify chromosomal abnormalities in fetal tissue.

Enhanced in vivo antiviral activity against pseudorabies virus through transforming gallic acid into graphene quantum dots with stimulation of interferon-related immune responses.

With the urgent need for antiviral agents, antiviral materials with high biocompatibility and antiviral effects have attracted a lot of attention. In this study, gallic acid, a natural polyphenolic compound, was transformed into biocompatible graphene quantum dots (GAGQDs) which exhibit enhanced antiviral activity against pseudorabies virus (PRV). The as-prepared GAGQDs inhibit PRV proliferation with a 104-fold reduction in viral titers. Investigation of the antiviral mechanism revealed that GAGQDs inhibit the adsorption, invasion and replication of PRV infection. Treatment with GAGQDs regulates the expression levels of interferon-related antiviral proteins, including mitochondrial antiviral-signaling protein (MAVS), signal transducer and activator of transcription 1 (STAT1) and 2',5'-oligoadenylate synthetase 1 (OAS1), suggesting that GAGQDs can stimulate innate antiviral immune responses, resulting in enhanced antiviral effects. More importantly, GAGQD treatments alleviate clinical symptoms and reduce mortality in PRV-infected mice. Our results reveal the enhanced therapeutic effects of GAGQDs against PRV infection in vitro and in vivo, suggesting the potential of GAGQDs as a promising novel antiviral agent.

Metabolic and molecular mechanisms underlying the foliar Zn application induced increase of 2-acetyl-1-pyrroline conferring the 'taro-like' aroma in pumpkin leaves.

Introduction: Fresh pumpkin leaf is popular vegetable for its rich nutrition. The pleasant taro-like odour is important aroma quality of crops, and mostly contributed by 2-acetyl-1-pyrroline in pumpkin. Element Zn can impact metabolite biosynthesis in plants, including aroma formation. However, Zn-induced biochemical responses, especially 2-acetyl-1-pyrroline formation in pumpkin, haven't been elucidated. Methods: This study integrated metabolome and transcriptome to explore molecular fluctuations in pumpkin leaves at different time intervals after foliar Zn treatment. Result and Discussion: We first identified more than one thousand metabolites from pumpkin leaves by integrating different mass spectrometry methods according to the form in which a metabolite exists. Comparative metabolomic analysis revealed there were separately 25 out of 50 and 286 out of 963 metabolites that were respectively identified by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, differentially regulated by Zn treatment. Our findings revealed that 50mg/L of Zn significantly enhanced 2-acetyl-1-pyrroline production by more than 38%, which was contributed by increased biosynthesis of its precursors, including ornithine and proline. The following transcriptome analysis discovered 30,574 genes, including 953 novel genes. Zn treatment induced the differential expression of 41.6% of identified genes which were supposed to regulate the downstream metabolite changes in a time-dependent manner. Pathway analysis indicated that alternations in primary metabolism, including carbon metabolism and biosynthesis of amino acids, were vital to the fluctuated aromatic compound generation. Phytohormones and transcription factors may regulate the expression of gene P5CS and proline biosynthesis, which, therefore, affect 2-acetyl-1-pyrroline production. This research reveals molecular mechanisms of 2-acetyl-1-pyrroline formation in pumpkin, which will provide the molecular basis for desired aroma compound production through metabolite engineering.

The role of DNA methylation in the maintenance of phenotypic variation induced by grafting chimerism in Brassica.

Grafting facilitates the interaction between heterologous cells with different genomes, resulting in abundant phenotypic variation, which provides opportunities for crop improvement. However, how grafting-induced variation occurs and is transmitted to progeny remains elusive. A graft chimera, especially a periclinal chimera, which has genetically distinct cell layers throughout the plant, is an excellent model to probe the molecular mechanisms of grafting-induced variation maintenance. Here we regenerated a plant from the T-cell layer of a periclinal chimera, TCC (where the apical meristem was artificially divided into three cell layers - from outside to inside, L1, L2, and L3; T = Tuber mustard, C = red Cabbage), named rTTT0 (r = regenerated). Compared with the control (rsTTT, s = self-grafted), rTTT0 had multiple phenotypic variations, especially leaf shape variation, which could be maintained in sexual progeny. Transcriptomes were analyzed and 58 phenotypic variation-associated genes were identified. Whole-genome bisulfite sequencing analyses revealed that the methylome of rTTT0 was changed, and the CG methylation level was significantly increased by 8.74%. In rTTT0, the coding gene bodies are hypermethylated in the CG context, while their promoter regions are hypomethylated in the non-CG context. DNA methylation changes in the leaf shape variation-associated coding genes, ARF10, IAA20, ROF1, and TPR2, were maintained for five generations of rTTT0. Interestingly, grafting chimerism also affected transcription of the microRNA gene (MIR), among which the DNA methylation levels of the promoters of three MIRs associated with leaf shape variation were changed in rTTT0, and the DNA methylation modification of MIR319 was maintained to the fifth generation of selfed progeny of rTTT0 (rTTT5). These findings demonstrate that DNA methylation of coding and non-coding genes plays an important role in heterologous cell interaction-induced variation formation and its transgenerational inheritance.