Rapid Response of Daphnia magna Motor Behavior to Mercury Chloride Toxicity Based on Target Tracking.

A rapid and timely response to the impacts of mercury chloride, which is indispensable to the chemical industry, on aquatic organisms is of great significance. Here, we investigated whether the YOLOX (improvements to the YOLO series, forming a new high-performance detector) observation system can be used for the rapid detection of the response of Daphnia magna targets to mercury chloride stress. Thus, we used this system for the real-time tracking and observation of the multidimensional motional behavior of D. magna. The results obtained showed that the average velocity (v¯), average acceleration (a¯), and cumulative travel (L) values of D. magna exposed to mercury chloride stress changed significantly under different exposure times and concentrations. Further, we observed that v¯, a¯ and L values of D. magna could be used as indexes of toxicity response. Analysis also showed evident D. magna inhibition at exposure concentrations of 0.08 and 0.02 mg/L after exposure for 10 and 25 min, respectively. However, under 0.06 and 0.04 mg/L toxic stress, v¯ and L showed faster toxic response than a¯, and overall, v¯ was identified as the most sensitive index for the rapid detection of D. magna response to toxicity stress. Therefore, we provide a strategy for tracking the motile behavior of D. magna in response to toxic stress and lay the foundations for the comprehensive screening of toxicity in water based on motile behavior.

Restoring T and B cell generation in X-linked severe combined immunodeficiency mice through hematopoietic stem cells adenine base editing.

Base editing of hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematologic diseases. However, the feasibility of using adenine-base-edited HSPCs for treating X-linked severe combined immunodeficiency (SCID-X1), the influence of dose-response relationships on immune cell generation, and the potential risks have not been demonstrated in vivo. Here, a humanized SCID-X1 mouse model was established, and 86.67% ± 2.52% (n = 3) of mouse hematopoietic stem cell (HSC) pathogenic mutations were corrected, with no single-guide-RNA (sgRNA)-dependent off-target effects detected. Analysis of peripheral blood over 16 weeks post-transplantation in mice with different immunodeficiency backgrounds revealed efficient immune cell generation following transplantation of different amounts of modified HSCs. Therefore, a large-scale infusion of gene-corrected HSCs within a safe range can achieve rapid, stable, and durable immune cell regeneration. Tissue-section staining further demonstrated the restoration of immune organ tissue structures, with no tumor formation in multiple organs. Collectively, these data suggest that base-edited HSCs are a potential therapeutic approach for SCID-X1 and that a threshold infusion dose of gene-corrected cells is required for immune cell regeneration. This study lays a theoretical foundation for the clinical application of base-edited HSCs in treating SCID-X1.

Engineering of near-PAMless adenine base editor with enhanced editing activity and reduced off-target.

About 47% of pathogenic point mutations could be corrected by ABE-induced A·T-to-G·C conversions. However, the applications of ABEs are still hindered by undesired editing efficiency, limited editing scopes, and off-targeting effects. Here, we develop a new adenine base editor, by embedding TadA-8e monomer into SpRY-nCas9, named as CE-8e-SpRY, which exhibits higher activity at NRN than NYN PAMs favored by SpRY nuclease. CE-8e-SpRY could target nearly all genomic sites in principle and induces the highest targeting efficiency among tested SpRY-based ABEs. In addition, CE-8e-SpRY also shows reduced RNA and DNA off-targeting activities. With optimized sgRNAs, CE-8e-SpRY induces efficient or desired target editing at some disease-relevant loci where conventional ABEs were unable to induce precise and satisfied editing. Taken together, our CE-8e-SpRY could broaden the applicability of ABEs in correcting or introducing pathogenic point mutations.

Variant analysis of 92 Chinese Han families with hearing loss.

BACKGROUND: Hearing loss (HL) is the most frequent sensory deficit in humans, HL has strong genetic heterogeneity. The genetic diagnosis of HL is very important to aid treatment decisions and to provide prognostic information and genetic counseling for the patient's family. METHODS: We undertook pedigree analysis in 92 Chinese non-syndromic HL patients by targeted next-generation sequencing and Sanger sequencing. RESULTS: Among the 92 HL patients, 18 were assigned a molecular diagnosis with 33 different variants in 14 deafness genes. Eighteen of the variants in 12 deafness genes were novel. Variants in TMC1, CDH23, LOXHD1 and USH2A were each detected in two probands, and variants in POU3F4, OTOA, GPR98, GJB6, TRIOBP, SLC26A4, MYO15A, TNC, STRC and TMPRSS3 were each detected in one proband. CONCLUSION: Our findings expand the spectrum of deafness gene variation, which will inform genetic diagnosis of deafness and add to the theoretical basis for the prevention of deafness.

Identification of novel deep intronic PAH gene variants in patients diagnosed with phenylketonuria.

Phenylketonuria (PKU) is caused by phenylalanine hydroxylase (PAH) gene variants. Previously, 94.21% of variants were identified using Sanger sequencing and multiplex ligation-dependent probe amplification. To investigate the remaining variants, we performed whole-genome sequencing for four patients with PKU and unknown genotypes to identify deep intronic or structural variants. We identified three novel heterozygous variants (c.706+368T>C, c.1065+241C>A, and c.1199+502A>T) in a deep PAH gene intron. We detected a c.1199+502A>T variant in 60% (6/10) of PKU patients with genetically undetermined PKU. In silico predictions indicated that the three deep variants may impact splice site selection and result in the inclusion of a pseudo-exon. A c.1199+502A>T PAH minigene and reverse transcription PCR (RT-PCR) on blood RNA from a PKU patient with biallelic variants c.1199+502A>T and c.1199G>A confirmed that the c.1199+502A>T variant may strengthen the predicted branch point and leads to the inclusion of a 25-nt pseudo-exon in the PAH mRNA. Reverse transcription polymerase chain reaction (RT-PCR) on the minigene revealed that c.706+368T>C may create an SRSF2 (SC35) binding site via a 313-nt pseudo-exon, whereas c.1065+241C>A may produce an 81-nt pseudo-exon that strengthens the predicted SRSF1 (SF2/ASF) binding site. These results augment current knowledge of PAH genotypes and show that deep intronic analysis of PAH can genetically diagnose PKU.

Novel CRISPR/Cas12a-based genetic diagnostic approach for SLC26A4 mutation-related hereditary hearing loss.

Hereditary hearing loss is a common defect of the auditory nervous system with high-incidence, seriously affecting the quality of life of the patients. The clinical manifestations of SLC26A4 mutation-related hearing loss are congenital sensorineural or mixed deafness. Sensitive and specific SLC26A4 mutation detection in the early clinical stage is key for the early indication of potential hearing loss in the lack of effective treatment. Using clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection technology, we designed a fast and sensitive detection system for SLC26A4 pathogenic mutations (c.919-2A > G, c.2168A > G and c.1229C > T). This recombinase-aided amplification-based detection system allows rapid target gene amplification and, in combination with the CRISPR-based nucleic acid testing (NAT) system, mutation site detection. Moreover, mismatches were introduced in CRISPR-derived RNA (crRNA) to increase signal differences between the wild-type genes and mutant genes. A total of 64 samples were examined using this approach and all results were verified using Sanger sequencing. The detection results were consistent with the polymerase chain reaction-Sanger sequencing results. Overall, this CRISPR-based NAT technology provides a sensitive and fast new approach for the detection of hereditary deafness and provides a crRNA optimization strategy for single-nucleotide polymorphism detection, which could be helpful for the clinical diagnosis of SLC26A4 mutation-related hereditary hearing loss.

Mutation analysis of TCOF1 gene in Chinese Treacher Collins syndrome patients.

BACKGROUND: Treacher Collins syndrome (TCS) is a rare autosomal dominant or recessive disorder, that involves unique bilateral craniofacial malformations. The phenotypes of TCS are extremely diverse. Interventional surgery can improve hearing loss and facial deformity in TCS patients. METHOD: We recruited seven TCS families. Variant screening in probands was performed by targeted next-generation sequencing (NGS). The variants identified were confirmed by Sanger sequencing. The pathogenicity of all the mutations was evaluated using the guidelines of the American College of Medical Genetics and Genomics (ACMG) and InterVar software. RESULTS: Three frameshift variants, two nonsense variants, one missense variant, and one splicing variant of TCOF1 were identified in the seven TCS probands. Five variants including c.1393C > T, c.4111 + 5G>C, c.1142delC, c.2285_2286delCT, and c.1719delG had not been previously reported. Furthermore, we report the c.149A > G variant for the first time in a Chinese TCS patient. We provided prenatal diagnosis for family 4. Proband 7 chose interventional surgery. CONCLUSION: We identified five novel variants in TCOF1 in Chinese patients with TCS, which expands the mutation spectrum of TCOF1 in TCS. Bone conduction hearing rehabilitation can improve hearing for TCS patients and prenatal diagnosis can provide fertility guidance for TCS families.

Compound heterozygous variants of the FBXO7 gene resulting in infantile-onset Parkinsonian-pyramidal syndrome in siblings of a Chinese family.

BACKGROUND: Mutations in the FBXO7 gene can cause a rare chromosomal recessive neurodegenerative disease, Parkinsonian-pyramidal syndrome (PPS). Patients with this syndrome mainly show early-onset Parkinson's syndrome. Here, we present a Chinese family with infantile-onset PPS caused by FBXO7 mutations. METHODS: The clinical phenotypes and medical records of the proband and his family members were collected. The proband, his sibling, and his parents underwent whole-exome sequencing (WES) by next-generation sequencing. RESULTS: The proband and his sibling had a typical PPS phenotype with onset during infancy. WES identified compound heterozygous variants in the FBXO7 gene, including a nonsense mutation, p. Trp134*, and a splicing mutation, IVS5-1G > A, which were shared by both siblings and inherited from each of the parents. These variants have not been reported in literatures or databases. According to the American College of Medical Genetics and Genomics guidelines, the p. Trp134* and IVS5-1G > A mutations were classified as pathogenic variants. CONCLUSIONS: We report a case of siblings in a Chinese family with infantile-onset PPS caused by FBXO7 gene mutations determined by WES. These findings will contribute to the in-depth study of the pathogenesis of PPS among patients with FBXO7 gene mutations.

Deep targeted sequencing reveals the diversity of TRB-CDR3 repertoire in patients with preeclampsia.

Preeclampsia (PE) is one of the major causes of maternal and perinatal mortality worldwide. This study aimed to determine the immunological characteristics of PE patients and normal pregnancy at the T cell receptor beta-chain (TRB) level by using high-throughput sequencing. High-throughput sequencing was performed to analyze the expression of TRB-CDR3 in circulating T cells. T cells were isolated from 36 healthy pregnant women, 24 patients with severe PE, and 18 patients with moderate PE. Rearranged mRNA sequences were assigned to their germline V, D, and J counterparts, and translated into proper amino acids by the IMGT database. In general, PE samples had more TRB-CDR3 reads and types than those of normal pregnant woman in the circulation, but the mean number of TRB-CDR3 reads and unique TRB-CDR3 reads in severe group was lower than that in the moderate group. In PE patients, the V7_9 and V20_1 gene loci were more prevalent than in healthy pregnant women. In addition, 4 kinds of TRB-CDR3 peptides were found to be highly relevant to the pathogenesis of PE. Of them, peptides matched to herpes simplex virus antigen-specific T cells were much lower in PE samples.

Exome sequencing reveals novel IRXI mutation in congenital heart disease.

Genetic variation in specific transcription factors during heart formation may lead to congenital heart disease (CHD) or even miscarriage. The aim of the present study was to identify CHD­associated genes using next generation sequencing (NGS). The whole exome DNA sequence was obtained from a stillborn fetus diagnosed with tricuspid atresia and complete transposition of the great arteries using high­throughput sequencing methods. Subsequently, genetic variants of CHD­associated genes were selected and verified in 215 non­syndromic CHD patients and 249 healthy control subjects using polymerase chain reaction combined with Sanger sequencing. Genetic variants of previously reported CHD­inducing genes, such as cysteine rich with EGF like domains 1 and cbp/p300­interacting transactivator with Glu/Asp rich carboxy­terminal domain 2, were discovered through the NGS analysis. In addition, a novel non­synonymous mutation of the iroquois homeobox 1 (IRX1) gene (p.Gln240Glu) was identified. A total of three non­synonymous mutations (p.Gln240Glu, p.Ser298Asn and p.Ala381Glu) of the IRX1 gene were verified in 215 non­syndromic CHD patients, but not in 249 healthy volunteers. The results demonstrated that NGS is a powerful tool to study the etiology of CHD. In addition, the results suggest that genetic variants of the IRX1 gene may contribute to the pathogenesis of CHD.

High throughput sequencing reveals the diversity of TRB-CDR3 repertoire in patients with psoriasis vulgaris.

Psoriasis is a T cell-mediated chronic inflammatory skin disease with inflammatory cell infiltrates in the dermis and epidermis. Previous studies suggested that there are some expanded T-cell receptor (TCR) clones in psoriatic skin. However, the effect of psoriasis on the immunological characteristics of TCR in circulating blood has not been reported. To address this, we performed high-throughput sequencing to reveal the immunological characteristics of TCR beta chain (TRB) in both psoriasis patients and healthy controls. Our results revealed that the TRB-CDR3 region of psoriasis patients had distinctive immunological characteristics compared with that of healthy controls, including V gene usage, nt of N addition. In addition, three types of TRB-CDR3 peptides were found highly relevant to psoriasis. Our findings show the comprehensive characteristics of psoriasis on the TRB-CDR3 repertoire of circulating blood at sequence-level resolution. These findings may contribute to a better understanding of the pathogenesis of psoriasis and open opportunities to explore potential therapeutic targets.

High-Throughput Sequencing Reveals Immunological Characteristics of the TRB-/IgH-CDR3 Region of Umbilical Cord Blood.

OBJECTIVE: To compare the differences of immunological characteristics between newborn and adults, we performed high-throughput sequencing to reveal the diversity of umbilical cord blood and adult peripheral blood at both T-cell receptor beta chain (TRB) and immunoglobulin heavy chain (IGH) levels. STUDY DESIGN: High-throughput sequencing was performed to analyze the expression of TRB-CDR3 and IGH-CDR3 in circulating T and B cells isolated from 20 healthy adults, 56 pregnant women, and 40 newborns. RESULTS: Our results revealed different immunological characteristics between newborn and adults, such as distinctive complementarity determining region 3 (CDR3) lengths, usage bias of variable and joining segments, random nucleotide addition, a large number of unique CDR3 peptides, and a greater repertoire diversity. Moreover, each newborn had a distinctive TRB-/IGH-CDR3 repertoire that was independent of the maternal immune status. CONCLUSIONS: This study presents comprehensive, unrestricted profiles of the TRB/IGH-CDR3 repertoire of newborns, pregnant women, and healthy adults at a sequence-level resolution. Our data may contribute to a better understanding of the immune system of newborns and benefit the efficient application of umbilical cord blood transplantation in future.

Contribution of TIMP3 polymorphisms to the development of preeclampsia in Han Chinese women.

PURPOSE: The aim of this study was to investigate whether polymorphisms in the tissue inhibitor of metalloproteinase 3 gene (TIMP3) are associated with the risk of preeclampsia (PE) in Han Chinese women. METHODS: Nine single TIMP3 tag-single nucleotide polymorphisms were selected by Haploview and genotyped using the Sequenom method in 181 preeclamptic and 203 healthy pregnant women from eastern China. RESULTS: The allele frequencies of the tag-single nucleotide polymorphisms were not significantly different between groups (P > 0.05). However, the genotype distribution of rs135025 was shown to differ between the multigravidity PE subgroup (>3) and controls under additive (P = 0.018) and recessive models (P = 0.008), while the genotype distribution of rs80272 differed significantly between the severe PE subgroup and controls under additive (P = 0.014) and dominant models (P = 0.041). Moreover, the H2 haplotype (A-C-G-T-A-A-G-C-G) was found to be associated with the risk of PE (P = 0.035). CONCLUSIONS: Genotypes of rs135025 and rs80272 in TIMP3 may therefore influence susceptibility to PE, and pregnant women carrying the H2 haplotype might be more prone to developing PE.

Association between CRP gene polymorphisms and the risk of preeclampsia in Han Chinese women.

BACKGROUND: As an inflammatory marker, C-reactive protein (CRP) has elevated expression in preeclampsia (PE), which is implicated in the pathogenesis of PE, but there has been a lack of information on the possible association between genetic variants of CRP and PE. In this study, we aimed to assess the genetic association between CRP polymorphisms and the risk of PE in Han Chinese Women. METHODS: Five single-nucleotide polymorphisms of CRP, rs2794521 (T>C), rs3091244 (C>T>A), rs3093068 (C>G), rs876538 (C>T), and rs1205 (C>T) were genotyped using the Sequenom method in 181 PE patients and 203 controls. RESULTS: The T allele frequency for rs2794521 was significantly higher in PE patients than in controls (odds ratios [OR]=4.091; 95% confidence interval [CI]: 1.533-10.917; p=0.002). The TT genotype of rs2794521 conferred a risk for PE (TT vs. TC+CC: OR=4.062; 95% CI: 1.499-11.008; p=0.003) and severe PE (TT vs. TC+CC: OR=9.577; 95% CI: 1.267-72.397; p=0.006). The other four polymorphic loci were not different between the groups. The CRP H2 haplotype (T-C-C-G-C) was associated with PE (OR=2.129; 95% CI: 1.47-3.085; p<0.001), whereas the H1 haplotype (C-C-C-G-C) offered protection (OR=0.23; 95% CI: 0.066-0.8; p=0.01). CONCLUSIONS: The CRP variant rs2794521 shows a strong association with PE in Han Chinese women. Pregnant women with the TT genotype of rs2794521 have higher odds of having PE, which further supports a possible role for CRP in PE.

Progesterone production requires activation of caspase-3 in preovulatory granulosa cells in a serum starvation model.

Granulosa cells proliferate, differentiate, and undergo apoptosis throughout follicular development. Previous studies have demonstrated that stimulation of progesterone production is accompanied by caspase-3 activation. Moreover, we previously reported that arsenic enhanced caspase-3 activity coupled with progesterone production. Inhibition of caspase-3 activity can significantly inhibit progesterone production induced by arsenic or follicle-stimulating hormone (FSH). Here, we report that serum starvation induces caspase-3 activation coupled with augmentation of progesterone production. Serum starvation also increased the levels of cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein, both of which may contribute to progesterone synthesis in preovulatory granulosa cells. Inhibition of caspase-3 activity resulted in a decrease in progesterone production. Deactivation of caspase-3 activity by caspase-3 specific inhibitor also resulted in decreases in P450scc and StAR expression, which may partly contribute to the observed decrease in progesterone production. Our study demonstrates for the first time that progesterone production in preovulatory granulosa cells is required for caspase-3 activation in a serum starvation model. Inhibition of caspase-3 activity can result in decreased expression of the steroidogenic proteins P450scc and StAR. Our work provides further details on the relationship between caspase-3 activation and steroidogenesis and indicates that caspase-3 plays a critical role in progesterone production by granulosa cells.

Arsenic induced progesterone production in a caspase-3-dependent manner and changed redox status in preovulatory granulosa cells.

Arsenic contamination is a principal environmental health threat throughout the world. However, little is known about the effect of arsenic on steroidogenesis in granulosa cells (GCs). We found that the treatment of preovulatory GCs with arsenite stimulated progesterone production. A significant increase in serum level of progesterone was observed in female Sprague-Dawley rats following arsenite treatment at a dose of 10 mg/L/rat/day for 7 days. Further experiments demonstrated that arsenite treatment did not change the level of intracellular cyclic AMP (cAMP) or phosphorylated ERK1/2 in preovulatory GCs; however, progesterone production was significantly decreased when cAMP-dependent protein kinase (PKA) or ERK1/2 pathway was inhibited. This implied that the effect of arsenite on progesterone production may require cAMP/PKA and ERK1/2 signaling but not depend on them. Furthermore, we found that arsenite decreased intracellular reactive oxygen species (ROS) but increased the antioxidant glutathione (GSH) levels and mitochondrial membrane potential (ΔΨm) in parallel to the changes in progesterone production. Progesterone antagonist blocked the arsenic-stimulated increase of GSH levels. Arsenite treatment induced caspase-3 activation, although no apoptosis was observed. Inhibition of caspase-3 activity significantly decreased progesterone production stimulated by arsenite or follicle-stimulating hormone (FSH). GSH depletion with buthionine sulfoximine led to cell apoptosis in response to arsenite treatment. Collectively, this study demonstrated for the first time that arsenite stimulates progesterone production through cleaved/active caspase-3-dependent pathway, and the increase of GSH level promoted by progesterone production may protect GCs against apoptosis and maintain the steroidogenesis of GCs in response to arsenite treatment.

Polycomb group proteins are essential for spinal cord development.

Birth defects are the leading cause of infantile mortality, followed by neural tube defects (NTD) and congenital heart defects. Spina bifida and anencephaly are among the most common forms of NTD. NTD etiologies are complex, and are associated with both genetic and environmental factors. Polycomb group proteins are essential for vertebrate development; therefore, the purpose of this study was to determine the role of PcGs in spinal cord morphogenesis in normal and all-trans-retinoic acid (RA)-treated fetal rat models of spina bifida. Pregnant rats were gavage-fed RA, resulting in fetal NTD, and embryos were obtained on day 15.5, 17.5, and 19.5. Western blot and immunohistochemistry were used to reveal PcGs expression in the normal and RA-treated E15.5-19.5 rat sacral cords. Western blot and immunohistochemistry revealed decreased EED, RNF2, SUZ12, and H3K27me3 expression in the normal, E15.5-19.5, rat sacral cords. In addition, the spinal cord of RA-treated rats during embryonic development exhibited altered PcGs protein expression. Administration of excess RA results in NTD. Our results suggest that the Polycomb proteins may be involved in spinal cord development.