The microRNA miR-202 prevents precocious spermatogonial differentiation and meiotic initiation during mouse spermatogenesis.

Spermatogonial differentiation and meiotic initiation during spermatogenesis are tightly regulated by a number of genes, including those encoding enzymes for miRNA biogenesis. However, whether and how single miRNAs regulate these processes remain unclear. Here, we report that miR-202, a member of the let-7 family, prevents precocious spermatogonial differentiation and meiotic initiation in spermatogenesis by regulating the timely expression of many genes, including those for key regulators such as STRA8 and DMRT6. In miR-202 knockout (KO) mice, the undifferentiated spermatogonial pool is reduced, accompanied by age-dependent decline of fertility. In KO mice, SYCP3, STRA8 and DMRT6 are expressed earlier than in wild-type littermates, and Dmrt6 mRNA is a direct target of miR-202-5p. Moreover, the precocious spermatogonial differentiation and meiotic initiation were also observed in KO spermatogonial stem cells when cultured and induced in vitro, and could be partially rescued by the knockdown of Dmrt6. Therefore, we have not only shown that miR-202 is a regulator of meiotic initiation but also identified a previously unknown module in the underlying regulatory network.

MicroRNA-202 safeguards meiotic progression by preventing premature SEPARASE-mediated REC8 cleavage.

MicroRNAs (miRNAs) are believed to play important roles in mammalian spermatogenesis but the in vivo functions of single miRNAs in this highly complex developmental process remain unclear. Here, we report that miR-202, a member of the let-7 family, plays an important role in spermatogenesis by phenotypic evaluation of miR-202 knockout (KO) mice. Loss of miR-202 results in spermatocyte apoptosis and perturbation of the zygonema-to-pachynema transition. Multiple processes during meiosis prophase I including synapsis and crossover formation are disrupted, and inter-sister chromatid synapses are detected. Moreover, we demonstrate that Separase mRNA is a miR-202 direct target and provides evidence that miR-202 upregulates REC8 by repressing Separase expression. Therefore, we have identified miR-202 as a new regulating noncoding gene that acts on the established SEPARASE-REC8 axis in meiosis.

Effects of aluminum (Al) stress on the isoprenoid metabolism of two Citrus species differing in Al-tolerance.

Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, ß-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.

The transcription factor SOX30 is a key regulator of mouse spermiogenesis.

The postmeiotic development of male germ cells, also known as spermiogenesis, features the coordinated expression of a large number of spermatid-specific genes. However, only a limited number of key transcription factors have been identified and the underlying regulatory mechanisms remain largely unknown. Here, we report that SOX30, the most-divergent member of the Sry-related high-motility group box (SOX) family of transcription factors, is essential for mouse spermiogenesis. The SOX30 protein was predominantly expressed in spermatids, while its transcription was regulated by retinoic acid and by MYBL1 before and during meiosis. Sox30 knockout mice arrested spermiogenesis at step 3 round spermatids, which underwent apoptosis and abnormal chromocenter formation. We also determined that SOX30 regulated the expression of hundreds of spermatid-specific protein-coding and long non-coding RNA genes. SOX30 bound to the proximal promoter of its own gene and activated its transcription. These results reveal SOX30 as a novel key regulator of spermiogenesis that regulates its own transcription to enforce and activate this meiotic regulatory pathway.

Wt1 directs the lineage specification of sertoli and granulosa cells by repressing Sf1 expression.

Supporting cells (Sertoli and granulosa) and steroidogenic cells (Leydig and theca-interstitium) are two major somatic cell types in mammalian gonads, but the mechanisms that control their differentiation during gonad development remain elusive. In this study, we found that deletion of Wt1 in the ovary after sex determination caused ectopic development of steroidogenic cells at the embryonic stage. Furthermore, differentiation of both Sertoli and granulosa cells was blocked when Wt1 was deleted before sex determination and most genital ridge somatic cells differentiated into steroidogenic cells in both male and female gonads. Further studies revealed that WT1 repressed Sf1 expression by directly binding to the Sf1 promoter region, and the repressive function was completely abolished when WT1 binding sites were mutated. This study demonstrates that Wt1 is required for the lineage specification of both Sertoli and granulosa cells by repressing Sf1 expression. Without Wt1, the expression of Sf1 was upregulated and the somatic cells differentiated into steroidogenic cells instead of supporting cells. Our study uncovers a novel mechanism of somatic cell differentiation during gonad development.

Non-canonical RNA polyadenylation polymerase FAM46C is essential for fastening sperm head and flagellum in mice†.

Family with sequence similarity 46, member C (FAM46C) is a highly conserved non-canonical RNA polyadenylation polymerase that is abundantly expressed in human and mouse testes and is frequently mutated in patients with multiple myeloma. However, its physiological role remains largely unknown. In this study, we found that FAM46C is specifically localized to the manchette of spermatids in mouse testes, a transient microtubule-based structure mainly involved in nuclear shaping and intra-flagellar protein traffic. Gene knockout of FAM46C in mice resulted in male sterility, characterized by the production of headless spermatozoa in testes. Sperm heads were intermittently found in the epididymides of FAM46C knockout mice, but their fertilization ability was severely compromised based on the results of intracytoplasmic sperm injection assays. Interestingly, our RNA-sequencing analyses of FAM46C knockout testes revealed that mRNA levels of only nine genes were significantly altered compared to wild-type ones (q < 0.05). When considering alternate activities for FAM46C, in vitro assays demonstrated that FAM46C does not exhibit protein kinase or AMPylation activity against general substrates. Together, our data show that FAM46C in spermatids is a novel component in fastening the sperm head and flagellum.

MicroRNA-202 maintains spermatogonial stem cells by inhibiting cell cycle regulators and RNA binding proteins.

miRNAs play important roles during mammalian spermatogenesis. However, the function of most miRNAs in spermatogenesis and the underlying mechanisms remain unknown. Here, we report that miR-202 is highly expressed in mouse spermatogonial stem cells (SSCs), and is oppositely regulated by Glial cell-Derived Neurotrophic Factor (GDNF) and retinoic acid (RA), two key factors for SSC self-renewal and differentiation. We used inducible CRISPR-Cas9 to knockout miR-202 in cultured SSCs, and found that the knockout SSCs initiated premature differentiation accompanied by reduced stem cell activity and increased mitosis and apoptosis. Target genes were identified with iTRAQ-based proteomic analysis and RNA sequencing, and are enriched with cell cycle regulators and RNA-binding proteins. Rbfox2 and Cpeb1 were found to be direct targets of miR-202 and Rbfox2 but not Cpeb1, is essential for the differentiation of SSCs into meiotic cells. Accordingly, an SSC fate-regulatory network composed of signaling molecules of GDNF and RA, miR-202 and diverse downstream effectors has been identified.

Magnetic nanoparticles modified with hyperbranched polyamidoamine for the extraction of benzoylurea insecticides prior to their quantitation by HPLC.

A magnetic sorbent was fabricated by covalently grafting hyperbranched polyamidoamine onto the surface of Fe3O4 nanoparticles. The sorbent was used in the magnetic solid-phase extraction (MSPE) of benzoylurea insecticides (BUs) from environmental water samples. It is perceived that hydrogen bonding interactions and hydrophobic interactions are the main mechanisms for the adsorption of BUs. The sorbent was characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, elemental analysis, Brunauer-Emmett-Teller measurement, X-ray photoelectron spectroscopy, vibrating sample magnetometry and X-ray diffraction. Various experimental parameters affecting the MSPE were optimized. Following elution with acetonitrile, the BUs were quantified by HPLC with diode array detection. The method based has a wide linear response range (2.5-500.0 ng mL-1), satisfactory coefficient of determination (0.9922-0.9976), high enrichment factors (62.8-74.4), acceptable limits of quantitation (2.5 ng mL-1) and low limits of detection (0.39-0.72 ng mL-1). The intra-day precision at concentration levels of 5.0, 50.0 and 250.0 ng mL-1 ranged from 2.3-6.4% and the inter-day precision was between 1.0 and 5.5%. The sorbent can be re-used at least 15 times. It was applied to the extraction of BUs from genuine water samples where it showed satisfactory relative recoveries (75.1-111.4%) and precision (1.0-9.1%). Graphical abstract Schematic presentation of magnetic nanoparticles modified with hyperbranched polyamidoamine and their application for extraction of benzoylurea insecticides.

Expression dynamics, relationships, and transcriptional regulations of diverse transcripts in mouse spermatogenic cells.

Among all tissues of the metazoa, the transcritpome of testis displays the highest diversity and specificity. However, its composition and dynamics during spermatogenesis have not been fully understood. Here, we have identified 20,639 message RNAs (mRNAs), 7,168 long non-coding RNAs (lncRNAs) and 15,101 circular RNAs (circRNAs) in mouse spermatogenic cells, and found many of them were specifically expressed in testes. lncRNAs are significantly more testis-specific than mRNAs. At all stages, mRNAs are generally more abundant than lncRNAs, and linear transcripts are more abundant than circRNAs. We showed that the productions of circRNAs and piRNAs were highly regulated instead of random processes. Based on the results of a small-scale functional screening experiment using cultured mouse spermatogonial stem cells, many evolutionarily conserved lncRNAs are likely to play roles in spermatogenesis. Typical classes of transcription factor binding sites are enriched in the promoters of testis-specific m/lncRNA genes. Target genes of CREM and RFX2, 2 key TFs for spermatogenesis, were further validated by using ChIP-chip assays and RNA-seq on RFX2-knockout spermatogenic cells. Our results contribute to the current understanding of the transcriptomic complexity of spermatogenic cells and provide a valuable resource from which many candidate genes may be selected for further functional studies.

Genome-wide loss of 5-hmC is a novel epigenetic feature of Huntington's disease.

5-Hydroxymethylcytosine (5-hmC) may represent a new epigenetic modification of cytosine. While the dynamics of 5-hmC during neurodevelopment have recently been reported, little is known about its genomic distribution and function(s) in neurodegenerative diseases such as Huntington's disease (HD). We here observed a marked reduction of the 5-hmC signal in YAC128 (yeast artificial chromosome transgene with 128 CAG repeats) HD mouse brain tissues when compared with age-matched wild-type (WT) mice, suggesting a deficiency of 5-hmC reconstruction in HD brains during postnatal development. Genome-wide distribution analysis of 5-hmC further confirmed the diminishment of the 5-hmC signal in striatum and cortex in YAC128 HD mice. General genomic features of 5-hmC are highly conserved, not being affected by either disease or brain regions. Intriguingly, we have identified disease-specific (YAC128 versus WT) differentially hydroxymethylated regions (DhMRs), and found that acquisition of DhmRs in gene body is a positive epigenetic regulator for gene expression. Ingenuity pathway analysis (IPA) of genotype-specific DhMR-annotated genes revealed that alternation of a number of canonical pathways involving neuronal development/differentiation (Wnt/ß-catenin/Sox pathway, axonal guidance signaling pathway) and neuronal function/survival (glutamate receptor/calcium/CREB, GABA receptor signaling, dopamine-DARPP32 feedback pathway, etc.) could be important for the onset of HD. Our results indicate that loss of the 5-hmC marker is a novel epigenetic feature in HD, and that this aberrant epigenetic regulation may impair the neurogenesis, neuronal function and survival in HD brain. Our study also opens a new avenue for HD treatment; re-establishing the native 5-hmC landscape may have the potential to slow/halt the progression of HD.

Integrative proteomic and transcriptomic analyses reveal multiple post-transcriptional regulatory mechanisms of mouse spermatogenesis.

Mammalian spermatogenesis consists of many cell types and biological processes and serves as an excellent model for studying gene regulation at transcriptional and post-transcriptional levels. Many key proteins, miRNAs, and perhaps piRNAs have been shown to be involved in post-transcriptional regulation of spermatogenesis. However, a systematic method for assessing the relationship between protein and mRNA expression has not been available for studying mechanisms of post-transcriptional regulation. In the present study, we used the iTRAQ-based quantitative proteomic approach to identify 2008 proteins in mouse type A spermatogonia, pachytene spermatocytes, round spermatids, and elongative spermatids with high confidence. Of these proteins, 1194 made up four dynamically changing clusters, which reflect the mitotic amplification, meiosis, and post-meiotic development of germ cells. We identified five major regulatory mechanisms termed "transcript only," "transcript degradation," "translation repression," "translation de-repression," and "protein degradation" based on changes in protein level relative to changes in mRNA level at the mitosis/meiosis transition and the meiosis/post-meiotic development transition. We found that post-transcriptional regulatory mechanisms are related to the generation of piRNAs and antisense transcripts. Our results provide a valuable inventory of proteins produced during mouse spermatogenesis and contribute to elucidating the mechanisms of the post-transcriptional regulation of gene expression in mammalian spermatogenesis.

Evolution of the eye transcriptome under constant darkness in Sinocyclocheilus cavefish.

In adaptating to perpetual darkness, cave species gradually lose eyes and body pigmentation and evolve alternatives for exploring their environments. Although troglodyte features evolved independently many times in cavefish, we do not yet know whether independent evolution of these characters involves common genetic mechanisms. Surface-dwelling and many cave-dwelling species make the freshwater teleost genus Sinocyclocheilus an excellent model for studying the evolution of adaptations to life in constant darkness. We compared the mature retinal histology of surface and cave species in Sinocyclocheilus and found that adult cavefish showed a reduction in the number and length of photoreceptor cells. To identify genes and genetic pathways that evolved in constant darkness, we used RNA-seq to compare eyes of surface and cave species. De novo transcriptome assemblies were developed for both species, and contigs were annotated with gene ontology. Results from cave-dwelling Sinocyclocheilus revealed reduced transcription of phototransduction and other genes important for retinal function. In contrast to the blind Mexican tetra cavefish Astyanax mexicanus, our results on morphologies and gene expression suggest that evolved retinal reduction in cave-dwelling Sinocyclocheilus occurs in a lens-independent fashion by the reduced proliferation and downregulation of transcriptional factors shown to have direct roles in retinal development and maintenance, including cone-rod homeobox (crx) and Wnt pathway members. These results show that the independent evolution of retinal degeneration in cavefish can occur by different developmental genetic mechanisms.

Transcription factor PBX4 regulates limb development and haematopoiesis in mice.

The mammalian Pre-B cell leukaemia transcription factors 1-4 (PBX1-4) constitutes the PBC class of the homeodomain (HD)-containing proteins, which play important roles in diverse developmental processes. The functions and the underlying molecular mechanisms of PBX1-3 but not PBX4 have been extensively studied, and they have been reported to direct essential morphogenetic processes and organogenesis. In the present study, we generated knockin mice of FLAG-tagged PBX4 and the Pbx4 knockout (KO) mice and carried out in-depth characterisation of PBX4 expression and function. PBX4 was initially detected only in the testis among several organs of the adult mice and was expressed in spermatocytes and spermatids. However, no abnormality in spermatogenesis, but growth retardation and premature death after birth were observed in most adult Pbx4 KO mice. These animals were inactive and had shorter hindlimbs and lower numbers of reticulocytes and lymphocytes, probably caused by abnormalities at earlier developmental stages. Pbx4 mRNAs were indeed detected in several embryonic cell types related to limb development by in situ hybridisation and single-cell RNA-sequencing analysis. Pbx4 protein was also detected in the bone marrow of adult mice with a lower level compared with that in the testis. PBX4 preferentially binds to the promoters of a large number of genes including those for other HD-containing proteins and ribosomal proteins whose mutations are related to anaemia. PBX4-binding sites are enriched in motifs similar to those of other HD-containing proteins such as PKNOX1 indicating that PBX4 may also act as a co-transcription factor like other PBC proteins. Together, these results show that PBX4 participates in limb development and haematopoiesis while its function in spermatogenesis has not been revealed by gene KO probably due to the complementary effects of other genes.

piRNA profiling during specific stages of mouse spermatogenesis.

PIWI-interacting RNAs (piRNAs) are a class of small RNAs abundantly expressed in animal gonads. piRNAs that map to retrotransposons are generated by a "ping-pong" amplification loop to suppress the activity of retrotransposons. However, the biogenesis and function of other categories of piRNAs have yet to be investigated. In this study, we first profiled the expression of small RNAs in type A spermatogonia, pachytene spermatocytes, and round spermatids by deep sequencing. We then focused on the computational analysis of the potential piRNAs generated in the present study as well as other published sets. piRNAs mapping to retrotransposons, mRNAs, and intergenic regions had different length distributions and were differentially regulated in spermatogenesis. piRNA-generating mRNAs (PRMRs), whose expression positively correlated with their piRNA products, constituted one-third of the protein-coding genes and were evolutionarily conserved and enriched with splicing isoforms and antisense transcripts. PRMRs with piRNAs preferentially mapped to CDSs and 3' UTRs partitioned into three clusters differentially expressed during spermatogenesis and enriched with unique sets of functional annotation terms related to housekeeping activities as well as spermatogenesis-specific processes. Intergenic piRNAs were divided into 2992 clusters probably representing novel transcriptional units that have not been reported. The transcripts of a large number of genes involved in spermatogenesis are the precursors of piRNAs, and these genes are intricately regulated by alternative splicing and antisense transcripts. piRNAs, whose regulatory role in gene expression awaits to be identified, are clearly products of a novel regulatory process that needs to be defined.

Abnormal chromatin remodeling caused by ARID1A deletion leads to malformation of the dentate gyrus.

ARID1A, an SWI/SNF chromatin-remodeling gene, is commonly mutated in cancer and hypothesized to be a tumor suppressor. Recently, loss-of-function of ARID1A gene has been shown to cause intellectual disability. Here we generate Arid1a conditional knockout mice and investigate Arid1a function in the hippocampus. Disruption of Arid1a in mouse forebrain significantly decreases neural stem/progenitor cells (NSPCs) proliferation and differentiation to neurons within the dentate gyrus (DG), increasing perinatal and postnatal apoptosis, leading to reduced hippocampus size. Moreover, we perform single-cell RNA sequencing (scRNA-seq) to investigate cellular heterogeneity and reveal that Arid1a is necessary for the maintenance of the DG progenitor pool and survival of post-mitotic neurons. Transcriptome and ChIP-seq analysis data demonstrate that ARID1A specifically regulates Prox1 by altering the levels of histone modifications. Overexpression of downstream target Prox1 can rescue proliferation and differentiation defects of NSPCs caused by Arid1a deletion. Overall, our results demonstrate a critical role for Arid1a in the development of the hippocampus and may also provide insight into the genetic basis of intellectual disabilities such as Coffin-Siris syndrome, which is caused by germ-line mutations or microduplication of Arid1a.

Global identification of SMAD2 target genes reveals a role for multiple co-regulatory factors in zebrafish early gastrulas.

Nodal and Smad2/3 signals play pivotal roles in mesendoderm induction and axis determination during late blastulation and early gastrulation in vertebrate embryos. However, Smad2/3 direct target genes during those critical developmental stages have not been systematically identified. Here, through ChIP-chip assay, we show that the promoter/enhancer regions of 679 genes are bound by Smad2 in the zebrafish early gastrulas. Expression analyses confirm that a significant proportion of Smad2 targets are indeed subjected to Nodal/Smad2 regulation at the onset of gastrulation. The co-existence of DNA-binding sites of other transcription factors in the Smad2-bound regions allows the identification of well known Smad2-binding partners, such as FoxH1 and Lef1/ß-catenin, as well as many previously unknown Smad2 partners, including Oct1 and Gata6, during embryogenesis. We demonstrate that Oct1 physically associates with and enhances the transcription and mesendodermal induction activity of Smad2, whereas Gata6 exerts an inhibitory role in Smad2 signaling and mesendodermal induction. Thus, our study systemically uncovers a large number of Smad2 targets in early gastrulas and suggests cooperative roles of Smad2 and other transcription factors in controlling target gene transcription, which will be valuable for studying regulatory cascades during germ layer formation and patterning of vertebrate embryos.

Mining and characterization of ubiquitin E3 ligases expressed in the mouse testis.

BACKGROUND: Ubiquitin-mediated protein modification and degradation are believed to play important roles in mammalian spermatogenesis. The catalogues of ubiquitin activating enzymes, conjugating enzymes, and ligases (E3s) have been known for mammals such as mice and humans. However, a systematic characterization of E3s expressed during spermatogenesis has not been carried out. RESULTS: In present study, we set out to mine E3s from the mouse genome and to characterize their expression pattern, subcellular localization, and enzymatic activities based on microarray data and biochemical assays. We identified 398 putative E3s belonging to the RING, U-box, and HECT subfamilies and found that most genes were conserved between mice and humans. We discovered that 73 of them were highly or specifically expressed in the testes based on the microarray expression data. We selected 10 putative E3 genes to examine their mRNA expression pattern, and several genes to study their subcellular localization and E3 ligase activity. RT-PCR results showed that all the selected genes were predominately expressed in the testis. Some putative E3s were localized in the cytoplasm while others were in both the cytoplasm and the nucleus. Moreover, all the selected proteins were enzymatically active as demonstrated by in vitro and in vivo assays. CONCLUSIONS: We have identified a large number of putative E3s that are expressed during mouse spermatogenesis. Among these, a significant portion is highly or specifically expressed in the testis. Subcellular localization and enzymatic activity assays suggested that these E3s might execute diverse functions in mammalian spermatogenesis. Our results may serve as an initial guide to the field for further functional analysis.

[Polyamidoamine dendrimer-functionalized silica nanocomposite with polydopamine coating for dispersive micro solid-phase extraction of benzoylurea insecticides in water samples].

Pesticides are used in the agricultural production process to ensure the yield and quality of agricultural products. However, in recent years, environmental pollution issues caused by pesticide residues have sparked widespread concern in society. It is important to develop convenient and efficient approaches to detect and monitor pesticide residues. In this study, targeting benzoylurea insecticides (BUs), polyamidoamine dendrimer-functionalized silica nanocomposite with polydopamine coating (SiO2-PAMAM-PDA) was designed and successfully synthesized. First, monodisperse silica nanoparticles were prepared by the hydrolysis of tetraethyl orthosilicate (TEOS) in mixed solution of ethanol, water and ammonia. The silane coupling agent (3-aminopropyl)triethoxysilane was then employed to introduce amino groups into the silica. Silica with the zeroth generation of polyamidoamine (PAMAM) modification (SiO2-PAMAM-G0) was obtained through Michael addition reaction of methyl acrylate. Ethylenediamine was added to polymerize with methyl acrylate using an amidation reaction to form the first-generation PAMAM (SiO2-PAMAM-G1). Finally, by polymerizing dopamine under alkaline conditions (pH=8.5), the SiO2-PAMAM-G1 was coated with PDA. Thus, the final product named SiO2-PAMAM-PDA was obtained. The composite was characterized using a transmission electron microscope (TEM) and an increase in surface roughness indicated the successful grafting of PDA coating. Dopamine structure contains abundant benzene rings and amino and hydroxyl groups. It could bind with BUs through multiple secondary interactions, such as hydrogen bond and π-π stacking interaction. Therefore, the introduction of PDA could effectively enhance the affinity of the material toward benzoylurea insecticides. The prepared nanocomposites were used as sorbents in a dispersive micro solid-phase extraction approach (D-µ-SPE). The established approach was employed to extract and enrich the BUs in water samples before high-performance liquid chromatography (HPLC) analysis. Diflubenzuron, triflumuron, hexaflumuron, and teflubenzuron were chosen as target analytes. The following was a typical D-µ-SPE procedure. The prepared adsorbents measuring 40 mg were first dispersed in an 8-mL sample solution containing 150 g/L NaCl. The dispersion was assisted by 120-s vortexing to ensure full contact between the SiO2-PAMAM-PDA and the targets. Next, the adsorbents were separated from the liquid phase by 4-min centrifugation (5000 r/min). Thereafter, the adsorbed benzoylureas were eluted using 1 mL acetonitrile as desorption solvent by 120-s vortexing. Separated by centrifugation, the eluate was dried under a mild nitrogen stream. The solid remains were redissolved in 0.1 mL of acetonitrile, filtered by filter membrane (0.22 µm), and then analyzed by HPLC. The experimental conditions in the D-µ-SPE process could have a great impact on the extraction efficiency. Experimental conditions were optimized using a single factor optimization approach to further enhance the extraction recoveries. The optimized conditions included adsorbent amount, extraction time, desorption solvent type, desorption solvent volume, desorption time, and NaCl addition amount. Under the optimal conditions, a linearity range of 10-500 µg/L and limits of detection (LODs, S/N=3) of 1.1-2.1 µg/L were obtained. The extraction recoveries and relative standard deviations (RSDs) of the four BUs were 82.8%-94.1% and 2.1%-8.0%, respectively. The established approach was compared with reported approaches targeting benzoylurea insecticides. It was discovered that this approach consumed less sample, material, organic solvent, and pretreatment time. It provided a more rapid and green choice for the determination of benzoylurea pesticides. To determine the applicability, the proposed approach was applied to analyze the four benzoylurea insecticides in three river water samples. The real water samples were pretreated using the developed approach ahead of instrumental analysis, and no benzoylurea pesticides residue was detected. Next, standard addition experiments were performed under three spiking levels, including 15, 50, and 200 µg/L. The established approach had good accuracy and feasibility with satisfactory recoveries (69.5%-99.4%) and RSDs (0.2%-9.5%).

Identification and characterization of BEND2 as a key regulator of meiosis during mouse spermatogenesis.

The chromatin state, which undergoes global changes during spermatogenesis, is critical to meiotic initiation and progression. However, the key regulators involved and the underlying molecular mechanisms remain to be uncovered. Here, we report that mouse BEND2 is specifically expressed in spermatogenic cells around meiotic initiation and that it plays an essential role in meiotic progression. Bend2 gene knockout in male mice arrested meiosis at the transition from zygonema to pachynema, disrupted synapsis and DNA double-strand break repair, and induced nonhomologous chromosomal pairing. BEND2 interacted with chromatin-associated proteins that are components of certain transcription-repressor complexes. BEND2-binding sites were identified in diverse chromatin states and enriched in simple sequence repeats. BEND2 inhibited the expression of genes involved in meiotic initiation and regulated chromatin accessibility and the modification of H3K4me3. Therefore, our study identified BEND2 as a previously unknown key regulator of meiosis, gene expression, and chromatin state during mouse spermatogenesis.

Ionic liquid-type surfactant modified attapulgite as a novel and efficient dispersive solid phase material for fast determination of pyrethroids in tea drinks.

In this study, a novel ionic liquid-type surfactant modified attapulgite named as 1-dodecyl-3-methylimidazolium bromide-attapulgite (C12MIM-ATP) is successfully prepared and applied in dispersive solid phase extraction (dSPE) for the fast determination of pyrethroid residues in tea drinks. The primary factors that influenced the extraction efficiency, including sorbent type, amount of sorbent, extraction time, desorption conditions, pH and ionic strength, are investigated. The optimized results reveal that the extraction and desorption equilibria are rapidly obtained within 1 min. Under the optimized conditions, good linearity (2-500 µg/L) is observed for four pyrethroids in tea drinks with determination coefficients (r2) ranged from 0.9992 to 1.0000. The limits of detection (LODs) are 0.6 µg/L for all pesticides. Acceptable extraction recoveries of target analytes are found from 90.28 to 107.56% with relative standard deviations (RSDs) less than 8.30% in real tea drink samples. The batch-to-bath repeatability is evaluated by recovery test on five independent synthesized C12MIM-ATP sorbents. Satisfactory batch-to-batch repeatability is obtained with the recovery factors varied in 15%. A small matrix effect is observed using C12MIM-ATP as the sorbent for detection pyrethroids in tea drinks.