Sensing platform for the highly sensitive detection of catechol based on composite coupling with conductive Ni3(HITP)2 and nanosilvers.

Catechol, which has a high toxicity and low degradability, poses significant risks to both human health and the environment. Tracking of catechol residues is essential to protect human health and to assess the safety of the environment. We constructed sensing platforms to detect catechol based on the conductive metal-organic frameworks [Ni3(HITP)2] and their nanosilver composites. The reduction process of catechol at the Ni3(HITP)2/AgNP electrode is chemically irreversible as a result of the difference in compatibility of the oxidation stability and conductivity between the Ni3(HITP)2/AgNS and Ni3(HITP)2/AgNP electrodes. The electrochemical results show that the Ni3(HITP)2/AgNS electrode presents a lower detection limit of 0.053 µM and better sensitivity, reproducibility and repeatability than the Ni3(HITP)2/AgNP electrode. The kinetic mechanism of the catechol electrooxidation at the surface of the electrode is controlled by diffusion through a 2H+/2e- process. The transfer coefficient is the key factor used to illustrate this process. During the electrochemical conversion of phenol to ketone, more than half of ΔG is used to change the activation energy. We also studied the stability, anti-interference and reproducibility of these electrode systems.

Monitoring Corynespora cassiicola Resistance to Boscalid, Trifloxystrobin, and Carbendazim in China.

Cucumber leaf spot (CLS), caused by Corynespora cassiicola, is a serious disease of greenhouse cucumbers. With frequent use of existing fungicides, C. cassiicola has developed resistance to some of them, with serious implications for the control of CLS in the field. With a lack of new fungicides, it is necessary to use existing fungicides for effective control. Therefore, this study monitored the resistance of C. cassiicola to three commonly used and effective fungicides, boscalid, trifloxystrobin, and carbendazim, from 2017 to 2021. The frequency of resistance to boscalid showed an increasing trend, and the highest frequency was 85.85% in 2020. The frequency of resistance to trifloxystrobin was greater than 85%, and resistance to carbendazim was maintained at 100%. Among these fungicides, strains with multiple resistance to boscalid, trifloxystrobin, and carbendazim were found, accounting for 32.00, 25.25, 33.33, 43.06, and 37.24%, respectively. Of the strains that were resistant to boscalid, 87% had CcSdh mutations, including seven genotypes: B-H278L/Y, B-I280V, C-N75S, C-S73P, D-D95E, and D-G109V. Also, six mutation patterns of the Ccß-tubulin gene were detected: E198A, F167Y, E198A&M163I, E198A&F167Y, M163I&F167Y, and E198A&F200C. Detection of mutations of the CcCytb gene in resistant strains showed that 98.8% were found to have only the G143A mutation. A total of 27 mutation combinations were found and divided into 14 groups for analysis. The resistance levels differed according to genotype. The development of genotypes showed a complex trend, increasing from 4 in 2017 to 13 in 2021 and varying by region. Multiple fungicide resistance is gradually increasing. Therefore, it is necessary to understand the types of mutations and the trend of resistance to guide the use of fungicides to achieve disease control.

The mechanisms of target and non-target resistance to QoIs in Corynespora Cassiicola.

Corynespora leaf spot, caused by Corynespora cassiicola, is a foliar disease in cucumber. While the application of quinone outside inhibitors (QoIs) is an effective measure for disease control, it carries the risk of resistance development. In our monitoring of trifloxystrobin resistance from 2008 to 2020, C. cassiicola isolates were categorized into three populations: sensitive isolates (S, 0.01 < EC50 < 0.83 µg/mL), moderately resistant isolates (MR, 1.18 < EC50 < 55.67 µg/mL), and highly resistant isolates (HR, EC50 > 56.98 µg/mL). The resistance frequency reached up to 90% during this period, with an increasing trend observed in the annual average EC50 values of all the isolates. Analysis of the CcCytb gene revealed that both MR and HR populations carried the G143A mutation. Additionally, we identified mitochondrial heterogeneity, with three isolates carrying both G143 and A143 in MR and HR populations. Interestingly, isolates with the G143A mutation (G143A-MR and G143A-HR) displayed differential sensitivity to QoIs. Further experiments involving gene knockout and complementation demonstrated that the major facilitator superfamily (MFS) transporter (CcMfs1) may contribute to the disparity in sensitivity to QoIs between the G143A-MR and G143A-HR populations. However, the difference in sensitivity caused by the CcMfs1 transporter is significantly lower than the differences observed between the two populations. This suggests additional mechanisms contributing to the variation in resistance levels among C. cassiicola isolates. Our study highlights the alarming level of trifloxystrobin resistance in C. cassiicola in China, emphasizing the need for strict prohibition of QoIs use. Furthermore, our findings shed light on the occurrence of both target and non-target resistance mechanisms associated with QoIs in C. cassiicola.

A Rapid Molecular Detection System for Sdh Mutations Conferring Differential Succinate Dehydrogenase Inhibitor Resistance in Corynespora cassiicola.

Cucumber leaf spot, caused by Corynespora cassiicola, is a serious disease of cucumbers in greenhouses. Due to the frequent application of succinate dehydrogenase inhibitors (SDHIs), resistance caused by point mutations in the SDHB/C/D gene has been reported. Different mutations lead to different resistance levels, and mutations vary over time and regions. This means that it is necessary to know the type of mutation in the field to select the appropriate SDHIs. Here, the sensitivity of mutations to SDHIs was determined, and eight resistance patterns were obtained: pattern I (BosVHR, FluoMR, PenHR, CarR); pattern II (BosMR, FluoSS, PenS, CarS); pattern III (BosVHR, FluoSS, PenLR, CarS); pattern IV (BosLR, FluoLR, PenS, CarR); pattern V (BosMR, FluoLR, PenS, CarS); pattern VI (BosMR, FluoLR, PenLR, CarS); pattern VII (BosVHR, FluoHR, PenHR, CarS); and pattern VIII (BosLR, FluoLR, PenLR, CarS). We successfully established nine allele-specific PCR (AS-PCR) assays that can detect mutation types. The sensitivity and specificity of AS-PCR were also determined. The sensitivity results showed that most of the detection thresholds of the AS-PCR assays were 100 pg/µl, while the AS-PCR assay of the B-H278R and D-G109V mutations exhibited high sensitivity, with 10 pg/µl. To validate the use of the developed AS-PCR assay, DNA from leaves inoculated with known mutations was extracted, detected by AS-PCR, and sequenced. The results showed good similarity between the two methods. Additionally, to rapidly detect mutations in the CcSdhD gene, we developed a single-tube multiplex allele-specific PCR (MAS-PCR) assay. In conclusion, AS-PCR and MAS-PCR were established for mutation detection and targeted control of CLS.

Srag Regulates Autophagy via Integrating into a Preexisting Autophagy Pathway in Testis.

Spermatogenesis is an essential process for producing sperm cells. Reproductive strategy is successfully evolved for a species to adapt to a certain ecological system. However, roles of newly evolved genes in testis autophagy remain unclear. In this study, we found that a newly evolved gene srag (Sox9-regulated autophagy gene) plays an important role in promoting autophagy in testis in the lineage of the teleost Monopterus albus. The gene integrated into an interaction network through a two-way strategy of evolution, via Sox9-binding in its promoter and interaction with Becn1 in the coding region. Its promoter region evolved a cis element for binding of Sox9, a transcription factor for male sex determination. Both in vitro and in vivo analyses demonstrated that transcription factor Sox9 could bind to and activate the srag promoter. Its coding region acquired ability to interact with key autophagy initiation factor Becn1 via the conserved C-terminal, indicating that srag integrated into preexisting autophagy network. Moreover, we determined that Srag enhanced autophagy by interacting with Becn1. Notably, srag transgenic zebrafish revealed that Srag exerted the same function by enhancing autophagy through the Srag-Becn1 pathway. Thus, the new gene srag regulated autophagy in testis by integrated into preexisting autophagy network.

An optimized base editor with efficient C-to-T base editing in zebrafish.

BACKGROUND: Zebrafish is a model organism widely used for the understanding of gene function, including the fundamental basis of human disease, enabled by the presence in its genome of a high number of orthologs to human genes. CRISPR/Cas9 and next-generation gene-editing techniques using cytidine deaminase fused with Cas9 nickase provide fast and efficient tools able to induce sequence-specific single base mutations in various organisms and have also been used to generate genetically modified zebrafish for modeling pathogenic mutations. However, the editing efficiency in zebrafish of currently available base editors is lower than other model organisms, frequently inducing indel formation, which limits the applicability of these tools and calls for the search of more accurate and efficient editors. RESULTS: Here, we generated a new base editor (zAncBE4max) with a length of 5560 bp following a strategy based on the optimization of codon preference in zebrafish. Our new editor effectively created C-to-T base substitution while maintaining a high product purity at multiple target sites. Moreover, zAncBE4max successfully generated the Twist2 p.E78K mutation in zebrafish, recapitulating pathological features of human ablepharon macrostomia syndrome (AMS). CONCLUSIONS: Overall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.

Teaching practice in human genetics integrated into general education.

General education is an important part in higher education, which emphasizes the educational idea of integration of generality with specialty, and practices people-oriented education concept. However, there are some difficulties and puzzles in general education. Now the general education system with Chinese characteristics is needed to be established through practice and development. In this paper, we enumerate how to integrate knowledge of human genetics in practice of general education, teaching cases, and relevant analysis with concepts of general education. Using questions as "what are human beings?" as a leverage, we introduce teaching contents closely related to daily life. For example, we explain the past, present and future of human beings through contemporary evolutionary genomics teaching. In addition, we introduce problem-based deep thinking for students, thus integrating classical attributes of human beings into general education.

Population genomics identifies patterns of genetic diversity and selection in chicken.

BACKGROUND: There are hundreds of phenotypically distinguishable domestic chicken breeds or lines with highly specialized traits worldwide, which provide a unique opportunity to illustrate how selection shapes patterns of genetic variation. There are many local chicken breeds in China. RESULTS: Here, we provide a population genome landscape of genetic variations in 86 domestic chickens representing 10 phenotypically diverse breeds. Genome-wide analysis indicated that sex chromosomes have less genetic diversity and are under stronger selection than autosomes during domestication and local adaptation. We found an evidence of admixture between Tibetan chickens and other domestic population. We further identified strong signatures of selection affecting genomic regions that harbor genes underlying economic traits (typically related to feathers, skin color, growth, reproduction and aggressiveness) and local adaptation (to high altitude). By comparing the genomes of the Tibetan and lowland fowls, we identified genes associated with high-altitude adaptation in Tibetan chickens were mainly involved in energy metabolism, body size maintenance and available food sources. CONCLUSIONS: The work provides crucial insights into the distinct evolutionary scenarios occurring under artificial selection for agricultural production and under natural selection for success at high altitudes in chicken. Several genes were identified as candidates for chicken economic traits and other phenotypic traits.

The long non-coding RNA OLC8 enhances gastric cancer by interaction with IL-11.

BACKGROUND: The gastric cancer (GC) represents a common malignancy especially in China. Long non-coding RNAs (lncRNAs) are critically involved in various types of cancer. However, the underlying mechanisms of OLC8 in gastric cancer are still largely unknown. METHODS: The lncRNA profiling was used to identify novel lncRNAs associated with GC. The expression of OLC8 was quantified using qRT-PCR. Migration and viability assays were performed to evaluate the in vitro effects. Xenograft tumor models were conducted to investigate the in vivo oncogenic potential. RNA-seq was used to identify IL-11 as OLC8 binding partner. RESULTS: In current study, we have identified a novel lncRNA termed OLC8. OLC8 was significantly overexpressed in gastric cancer specimens and cell lines. In vitro experiments showed that OLC8 facilitated migration and viability of MKN1 and AGS cells. As expected, in vivo experiments also confirmed an oncogenic role for OLC8. Mechanistic study indicated that OLC8 associated with IL-11 transcripts. The OLC8-IL-11 binding greatly impaired the degradation of IL-11 mRNAs. Not surprisingly, enhanced expression of IL-11 could increase STAT3 activation to favor gastric cancer development. CONCLUSIONS: Our current research has identified OLC8 as a novel oncogenic lncRNA in IL-11/STAT3 signaling, and OLC8 may constitute a potential target for gastric cancer intervention.

Isolation and characterization of string-forming female germline stem cells from ovaries of neonatal mice.

Germline stem cells are essential in the generation of both male and female gametes. In mammals, the male testis produces sperm throughout the entire lifetime, facilitated by testicular germline stem cells. Oocyte renewal ceases in postnatal or adult life in mammalian females, suggesting that germline stem cells are absent from the mammalian ovary. However, studies in mice, rats, and humans have recently provided evidence for ovarian female germline stem cells (FGSCs). A better understanding of the role of FGSCs in ovaries could help improve fertility treatments. Here, we developed a rapid and efficient method for isolating FGSCs from ovaries of neonatal mice. Notably, our FGSC isolation method could efficiently isolate on average 15 cell "strings" per ovary from mice at 1-3 days postpartum. FGSCs isolated from neonatal mice displayed the string-forming cell configuration at mitosis (i.e. a "stringing" FGSC (sFGSC) phenotype) and a disperse phenotype in postnatal mice. We also found that sFGSCs undergo vigorous mitosis especially at 1-3 days postpartum. After cell division, the sFGSC membranes tended to be connected to form sFGSCs. Moreover, F-actin filaments exhibited a cell-cortex distribution in sFGSCs, and E-cadherin converged in cell-cell connection regions, resulting in the string-forming morphology. Our new method provides a platform for isolating FGSCs from the neonatal ovary, and our findings indicate that FGCSs exhibit string-forming features in neonatal mice. The sFGSCs represent a valuable resource for analysis of ovary function and an in vitro model for future clinical use to address ovarian dysfunction.

Biased Duplications and Loss of Members in Tdrd Family in Teleost Fish.

Gene expansion and contraction are important evolution events. Some tdrd genes, especially multi-Tudor members, participate in Piwi-interacting RNA pathway and spermatogenesis. However, tdrd evolution and their functions in teleost fish are poorly understood. Here, we identified 14 tdrds in the teleost fish, swamp eel, which were clustered into 12 tdrd branches. Comparative synteny showed biased duplications and loss of members in the tdrd family. Both tdrd6 and tdrd7 were duplicated in the teleost fish, whereas tdrd8 was lost from the original locus. Expression analysis at both RNA and protein levels showed that tdrd6l, a duplicated multi-Tudor member, was gonad enriched. Expression pattern of tdrd6l in follicular epithelium and seminiferous epithelium during sex reversal supports its potential role in genome defense in germline.

Long-term artificial selection reveals a role of TCTP in autophagy in mammalian cells.

Understanding genomic variation and detecting selection signatures in a genome under selection have been great challenges for a century. Activation, development/exhaustion of primordial follicles in mammalian ovary determines reproductive success, menopause/end of female reproductive life. However, molecular mechanisms underlying oogenesis, particularly under artificial selection, are largely unknown. We report that a proteome-wide scan for selection signatures in the genome over 9,000 years of artificial pressure on the ovary revealed a general picture of selection signatures in the genome, especially genomic variations through artificial selection were detected in promoter and intron regions. Crossbreeding between domestic and wild species results in more than half of the protein spots exhibiting heterosis. Translationally controlled tumor protein (TCTP) is upregulated by artificial selection and positively regulates autophagy through the AMP-activated protein kinase pathway. Notably, TCTP interacts with ATG16 complex. In addition to cytoplasmic autophagy, nucleophagy occurs in the nuclei of granulosa and cumulus cells in ovaries, indicating an importance of the nuclear material for degradation by nucleophagy. Our findings provide insight into cellular and molecular mechanisms relevant for improvement of ovary functions, and identify selection signatures in the genome for ovary function over long-term artificial selection pressure.

Loss-of-function mutation in the X-linked TBX22 promoter disrupts an ETS-1 binding site and leads to cleft palate.

The cleft palate only (CPO) is a common congenital defect with complex etiology in humans. The molecular etiology of the CPO remains unknown. Here, we report a loss-of-function mutation in X-linked TBX22 gene (T-box 22) in a six-generation family of the CPO with obvious phenotypes of both cleft palate and hyper-nasal speech. We identify a functional -73G>A mutation in the promoter of TBX22, which is located at the core-binding site of transcription factor ETS-1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1). Phylogenetic analysis showed that the sequence around the -73G>A mutation site is specific in primates. The mutation was detected in all five affected male members cosegregating with the affected phenotype and heterozygote occurred only in some unaffected females of the family, suggesting an X-linked transmission of the mutation in the family. The -73G>A variant is a novel single nucleotide mutation. Cell co-transfections indicated that ETS-1 could activate the TBX22 promoter. Moreover, EMSA and ChIP assays demonstrated that the allele A disrupts the binding site of ETS-1, thus markedly decreases the activity of the TBX22 promoter, which is likely to lead to the birth defect of the CPO without ankyloglossia. These results suggest that a loss-of-function mutation in the X-linked TBX22 promoter may cause the cleft palate through disruption of TBX22-ETS-1 pathway.

DNA demethylation and USF regulate the meiosis-specific expression of the mouse Miwi.

Miwi, a member of the Argonaute family, is required for initiating spermiogenesis; however, the mechanisms that regulate the expression of the Miwi gene remain unknown. By mutation analysis and transgenic models, we identified a 303 bp proximal promoter region of the mouse Miwi gene, which controls specific expression from midpachytene spermatocytes to round spermatids during meiosis. We characterized the binding sites of transcription factors NF-Y (Nuclear Factor Y) and USF (Upstream Stimulatory Factor) within the core promoter and found that both factors specifically bind to and activate the Miwi promoter. Methylation profiling of three CpG islands within the proximal promoter reveals a markedly inverse correlation between the methylation status of the CpG islands and germ cell type-specific expression of Miwi. CpG methylation at the USF-binding site within the E2 box in the promoter inhibits the binding of USF. Transgenic Miwi-EGFP and endogenous Miwi reveal a subcellular co-localization pattern in the germ cells of the Miwi-EGFP transgenic mouse. Furthermore, the DNA methylation profile of the Miwi promoter-driven transgene is consistent with that of the endogenous Miwi promoter, indicating that Miwi transgene is epigenetically modified through methylation in vivo to ensure its spatio-temporal expression. Our findings suggest that USF controls Miwi expression from midpachytene spermatocytes to round spermatids through methylation-mediated regulation. This work identifies an epigenetic regulation mechanism for the spatio-temporal expression of mouse Miwi during spermatogenesis.

Genome-wide analysis reveals origin of transfer RNA genes from tRNA halves.

Transfer RNAs (tRNAs) play an important role linking mitochondrial RNA and amino acids during protein biogenesis. Four types of tRNA genes have been identified in living organisms. However, the evolutionary origin of tRNAs remains largely unknown. In this article, we conduct a deep sequence analysis of diverse genomes that cover all three domains of life to unveil the evolutionary history of tRNA genes from tRNA halves. tRNA half homologs were detected in diverse organisms, and some of them were expressed in mouse tissues. Continuous tRNA genes have a conserved pattern similar to indels, which is, more closely flanking regions have higher single nucleotide substitution rates, whereas tRNA half homologs do not have this pattern. In addition, tRNAs tend to break into tRNA halves when tissues are incubated in vitro, the tendency of tRNA to break into tRNA halves may be a "side-effect" of tRNA genes evolving from tRNA halves. These results suggest that modern tRNAs originated from tRNA halves through a repeat element-mediated mechanism. These findings provide insight into the evolutionary origin of tRNA genes.

Identification of Dmrt genes and their up-regulation during gonad transformation in the swamp eel (Monopterus albus).

The swamp eel is a teleost fish with a characteristic of natural sex reversal and an ideal model for vertebrate sexual development. However, underlying molecular mechanisms are poorly understood. We report the identification of five DM (doublesex and mab-3) domain genes in the swamp eel that include Dmrt2, Dmrt2b, Dmrt3, Dmrt4 and Dmrt5, which encode putative proteins of 527, 373, 471, 420 and 448 amino acids, respectively. Phylogenetic tree showed that these genes are clustered into corresponding branches of the DM genes in vertebrates. Southern blot analysis indicated that the Dmrt1-Dmrt3-Dmrt2 genes are tightly linked in a conserved gene cluster. Notably, these Dmrt genes are up-regulated during gonad transformation. Furthermore, mRNA in situ hybridisation showed that Dmrt2, Dmrt3, Dmrt4 and Dmrt5 are expressed in developing germ cells. These results are evidence that the DM genes are involved in sexual differentiation in the swamp eel.

TCTP increases stability of hypoxia-inducible factor 1α by interaction with and degradation of the tumour suppressor VHL.

BACKGROUND INFORMATION: The translationally controlled tumour protein (TCTP) plays an important role in maintaining cell proliferation and its high expression is associated with many tumours. The tumour suppressor von Hippel-Lindau protein (VHL) has been shown to function as an E3 ubiquitin ligase. Although great progress has been made, biological roles of these factors and relevant molecular mechanisms remain largely unknown. RESULTS: In this study, we have shown that TCTP specifically binds to VHL through its ß domain and competes with hypoxia-inducible factor-1α (HIF1α). TCTP over-expression decreased the protein level of VHL and the inhibition of TCTP expression by miRNA resulted in an increase of the VHL protein level. Moreover, TCTP over-expression promoted the K48-linked ubiquitination of VHL, thus degradation through the ubiquitin-proteasome pathway. In addition, we showed that TCTP increased the protein level of HIF1α, which promoted both vascular endothelial growth factor-hypoxic response element-promoter-driven luciferase reporter and endogenous VEGF expression. CONCLUSIONS: These data have demonstrated that TCTP binds to the ß domain of VHL through competition with HIF1α, which promotes VHL degradation by the ubiquitin-proteasome system and HIF1α stability.

Regulation of the transcriptional activation of the androgen receptor by the UXT-binding protein VHL.

Loss and/or inactivation of the VHL (von Hippel-Lindau) tumour suppressor causes various tumours. Using a yeast two-hybrid system, we have identified the AR (androgen receptor) co-activator UXT (ubiquitously expressed transcript), as a VHL-interacting protein. GST pull-down and co-immunoprecipitation assays show that UXT interacts with VHL. In addition, UXT recruits VHL to the nucleus. VHL associates with the DBD (DNA-binding domain) and hinge domains of the AR and induces AR ubiquitination. Moreover, VHL interaction with the AR activates AR transactivation upon DHT (dihydrotestosterone) treatment. VHL knockdown inhibits AR ubiquitination and decreases transcriptional activation of the AR. Our data suggest that the VHL-UXT interaction and VHL-induced ubiquitination of AR regulate transcriptional activation of the AR.

RAB37-mediated autophagy guards ovarian homeostasis and function.

Loss of ovarian homeostasis is associated with ovary dysfunction and female diseases; however, the underlying mechanisms responsible for the establishment of homeostasis and its function in the ovary have not been fully elucidated. Here, we showed that conditional knockout of Rab37 in oocytes impaired macroautophagy/autophagy proficiency in the ovary and interfered with follicular homeostasis and ovary development in mice. Flunarizine treatment upregulated autophagy, thus rescuing the impairment of follicular homeostasis and ovarian dysfunction in rab37 knockout mice by reprogramming of homeostasis. Notably, both the E2F1 and EGR2 transcription factors synergistically activated Rab37 transcription and promoted autophagy. Thus, RAB37-mediated autophagy ensures ovary function by maintaining ovarian homeostasis.Abbreviations: AMH: anti-Mullerian hormone; ATG: autophagy related; BECN1: beclin 1; cKO: conditional knockout; Cre: cyclization recombination enzyme; dpp: days postpartum; E2: estradiol; E2F1: E2F transcription factor 1; EBF1: EBF transcription factor 1; EGR2: early growth response 2; FSH: follicle stimulating hormone; LH: luteinizing hormone; mpp: months postpartum; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; RAB37: RAB37, member RAS oncogene family; SQSTM1: sequestosome 1; TFEB: transcription factor EB; Zp3: zona pellucida glycoprotein 3.