Rapid and Sensitive Detection of Verticillium dahliae from Soil Using LAMP-CRISPR/Cas12a Technology.

Cotton Verticillium wilt is mainly caused by the fungus Verticillium dahliae, which threatens the production of cotton. Its pathogen can survive in the soil for several years in the form of microsclerotia, making it a destructive soil-borne disease. The accurate, sensitive, and rapid detection of V. dahliae from complex soil samples is of great significance for the early warning and management of cotton Verticillium wilt. In this study, we combined the loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12a technology to develop an accurate, sensitive, and rapid detection method for V. dahliae. Initially, LAMP primers and CRISPR RNA (crRNA) were designed based on a specific DNA sequence of V. dahliae, which was validated using several closely related Verticillium spp. The lower detection limit of the LAMP-CRISPR/Cas12a combined with the fluorescent visualization detection system is approximately ~10 fg/µL genomic DNA per reaction. When combined with crude DNA-extraction methods, it is possible to detect as few as two microsclerotia per gram of soil, with the total detection process taking less than 90 min. Furthermore, to improve the method's user and field friendliness, the field detection results were visualized using lateral flow strips (LFS). The LAMP-CRISPR/Cas12a-LFS system has a lower detection limit of ~1 fg/µL genomic DNA of the V. dahliae, and when combined with the field crude DNA-extraction method, it can detect as few as six microsclerotia per gram of soil, with the total detection process taking less than 2 h. In summary, this study expands the application of LAMP-CRISPR/Cas12a nucleic acid detection in V. dahliae and will contribute to the development of field-deployable diagnostic productions.

The transcript NR 134251.1 of lncRNA APTR with an opposite function to all transcripts inhibits proliferation and induces apoptosis by regulating proliferation and apoptosis-related genes.

Arsenic (As) exposure has been a global public health concern for hundreds of millions worldwide. LncRNA APTR (Alu-mediated p21 transcriptional regulator) plays an essential role in tumor growth and development. However, its function in arsenic-induced toxicological responses is still unknown. In this study, we found that the expressions of all transcripts and the transcript NR 134251.1 of APTR were increased in a dose-dependent manner in 16HBE cells treated with sodium arsenite (NaAsO2). Silencing the transcript NR 134251.1 of APTR inhibited cell proliferation and induced apoptosis. However, silencing all transcripts of APTR had the opposite function to the transcript NR 134251.1. Then we examined the protein level of the proliferation and apoptosis-related genes after silencing the transcript NR 134251.1 of APTR. The results showed that silencing the transcript NR 134251.1 of APTR up-regulated the expression of transcription factor E2F1 and regulated its downstream genes involved in proliferation and apoptosis, including p53, phospho-p53-S392, phospho-p53-T55, p21, Cyclin D1, PUMA, Fas, Bim, BIK, Caspase-3, Caspase-7, and Cyt-c. In conclusion, arsenic induced APTR expression and the transcript NR 134251.1 of APTR have an opposite function to all transcripts, providing a theoretical basis for the prevention and treatment of arsenic exposure.

The Rubber Tree (Heveae brasiliensis) MLO Protein HbMLO12 Promotes Plant Susceptibility to Sustain Infection by a Powdery Mildew Fungus.

Powdery mildew severely affects several important crops and cash plants. Disruption of mildew resistance locus O (MLO) genes elevates resistance against powdery mildew in several plants. However, whether rubber tree (Heveae brasiliensis) MLO proteins are linked to susceptibility remains unknown, owing to technical limitations in the genetic manipulation of this woody plant. A previous study showed that the H. brasiliensis MLO-like protein HbMLO12 demonstrates high amino acid sequence similarity with the known Arabidopsis MLO protein AtMLO12. In this study, we investigated whether HbMLO12 regulates susceptibility to powdery mildew. H. brasiliensis leaves take up exogenously synthesized double-stranded RNAs (dsRNAs), and foliar application of dsRNA homologous to HbMLO12 gene specifically induces HbMLO12 silencing in H. brasiliensis leaf tissues. Notably, HbMLO12 silencing inhibited fungal infection and elevated the immune response during interaction with the rubber tree powdery mildew fungus. Furthermore, the heterologous expression of HbMLO12 suppressed bacterial flg22- and fungal chitin-induced immune responses and enhanced bacterial infection in Arabidopsis. Our study provides evidence that HbMLO12 contributes to susceptibility to powdery mildew. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Characterization of conidial autofluorescence in powdery mildew.

Autofluorescence is produced by endogenous fluorophores, such as NAD(P)H, lipofuscin, melanin, and riboflavin, indicating the accumulation of substances and the state of energy metabolism in organisms. As an obligate parasite, powdery mildew is wildly spread by air and parasitic crops. However, most identification studies have been based on morphology and molecular biology which were far too time- and labor-consuming, thus lacking characteristic, simple, and effective means. Using microscopy under the blue and cyan channels, we elaborated visible conidial autofluorescence in three powdery mildew species, Erysiphe quercicola, E. cichoracearum, and Podosphaera hibiscicola, with a sharp increase during the conidia senescence in E. quercicola. Additionally, the main spectral excitation detected by fluorescence spectrometery was 375 nm for these species, with a common emission peak at approximately 458-463 nm, and an additional trend at 487 nm for P. hibiscicola. Because NAD(P)H has a similar spectral feature, we further investigated the relation between NAD(P)H and conidial autofluorescence by fluorescence spectra. We observed that the reduced coenzymes prominently contributed to conidial autofluorescence; however, the conidial autofluorescence in P. hibiscicola displayed a different trend that may be affected by the oxidized coenzyme -NAD. Finally, the normalized average spectra of these three powdery mildew species and standard samples showed that the spectral trend of each species was similar but that the features in detail were specific and distinct based on principal component analysis. In conclusion, we showed and characterized conidial autofluorescence in three powdery mildew species for the first time. The specific conidial autofluorescence in these species provides a new idea for the development of field spore capture and identification devices for the discrimination of powdery mildew at the species level.

Hsa_circ_0005050 interacts with ILF3 and affects cell apoptosis and proliferation by disrupting the balance between p53 and p65.

The regulatory network between arsenic, genes and signaling pathways has been reported in arsenic carcinogenesis. Studies on circRNA represent a growing field, but the extent to circRNA potential mechanisms remains poorly understood. So this study we explore the systematic function of hsa_circ_0005050 in mediating the cell apoptosis and proliferation. We demonstrated that hsa_circ_0005050 was highly expressed in subjects who are long-term exposed to arsenic, and could be induced by NaAs2O3 in A549 and 16HBE. Knockdown of hsa_circ_0005050 promotes A549 cell viability, whereas exerts the opposite effects in 16HBE. Mechanistically, hsa_circ_0005050 regulates the p53 and NF-κB signaling pathway involved in the apoptosis and proliferation. And we found that hsa_circ_0005050 could directly bind to the RNA binding protein ILF3 and mutually influence each other's formation. Upon si-hsa_circ_0005050, ILF3 export to the cytoplasm resulting the formation of a ternary complex ILF3-p65-IκBA, breaks the balance of p53 and NF-κB pathway and induces A549 apoptosis and leads to 16HBE proliferation. As a result of these investigations, suggestions were identified for future research.

Inorganic arsenic induces MDM2, p53, and their phosphorylation and affects the MDM2/p53 complex in vitro.

Arsenic, as a human carcinogen, has posed a certain threat to environmental health globally. However, the underlying mechanism of the arsenic carcinogenic effect remains largely undetermined. The up-regulation of MDM2 seems to play a crucial part in tumors in especial carcinomas of the diffuse type. The interaction of MDM2 and p53 is closely relevant to the pathogenesis of tumors. In this study, we aimed to investigate the effect on MDM2, p53, and their phosphorylation after As(III). In the epidemiological study, we investigated that MDM2 expression was up-regulation and was positively linked to methylated metabolites (monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)) after As(III)-exposure. In vitro studies employing A549 and 16HBE cells confirmed the epidemiological data. Studies on MDM2 phosphorylation sites consisting of Ser166, Ser260, and Ser394 in response to arsenic exposure, which have not been studied presently, indicated that As(III) could induce the expression of MDM2 phosphorylation. Moreover, we studied the alterations of p53 and its N-terminus phosphorylation sites of Ser9, Ser15, and Ser33, which demonstrated that p53 and its phosphorylation were highly expressed after As(III) exposure. Subsequently, Co-immunoprecipitation assays validated our hypothesis that the bonding of MDM2 and p53 was altered by arsenic exposure. What's more, outcomes coming from different cell types of A549, 16HBE, and 60 T-16HBE revealed that MDM2 and its phosphorylation expression existed a significant difference. The study provides evidence that As(III) and its methylated metabolites modulate the expression of MDM2, p53, and their phosphorylation and then affect the interaction between MDM2 and p53.

CircP50 functions through the phosphorylation- and acetylation-activated p53 pathway to mediate inorganic arsenic-induced apoptosis in A549 cells.

As a class I carcinogen, arsenic has been reported to cause diseases accompanied by circRNAs regulating proliferation and apoptosis at the molecular level, but whether circP50 (circBase ID: hsa_circ_0008012) does the same has not been demonstrated. The aim of this study is to provide the basis for anti-lung cancer mechanism research, by studying the expression of circP50 under arsenic-induced conditions, and the effect and mechanism on the proliferation and apoptosis of A549 cells based on the circP50 knockdown models. To explore whether the circP50 is responsive to arsenic exposure, the qRT-PCR was applied to discover that the relative expression of circP50 in A549 cells increased only with increasing NaAsO2 dose and independent of its metabolites. We further determined the mechanism of circP50 by establishing circP50 knockdown models. The results of cell viability and EdU assays indicated the proliferation of A549 cells. According to the western blotting, phosphorylation of p53 at Ser15, Ser376, and Ser392 and acetylation of p53 at Lys370 and Lys382 were inhibited, resulting in the deficiency of p53 expression. Subsequently, the expression of genes downstream of p53 was reduced, including p21, PUMA, Caspase3, and Bcl-xS. Furthermore, the expressions of IKB-α, p65, and p50 decreased, but C-myc expression did not change significantly, referring to the NF-κB pathway was not dominant. The results suggest that circP50 mainly functions through the p53 pathway to mediate apoptosis in response to arsenic exposure.

Sodium arsenite-mediated upregulation of circDHX34 promotes apoptosis in hormone-independent breast cancer cells by regulating apoptotic genes.

Arsenic and the compounds thereof can be carcinogens or therapeutic agents for different cancer types. However, for breast cancer (BC), studies have yielded conflicted results on the role of arsenic. A previous study by the present authors indicated a potential relationship between circDHX34 and sodium arsenite-treated BC cells. As such, the expression, function, and potential mechanism of circDHX34 in sodium arsenite-treated MDA-MB-231 cells were further detected. In the present study, findings were made that sodium arsenite upregulated circDHX34 expression in MDA-MB-231 cells in a dose-dependent manner, and knockdown of circDHX34 could promote cell proliferation and inhibit apoptosis. Further investigations revealed that knockdown of circDHX34 upregulated the expression levels of antiapoptotic genes BCL2 and BCL2L1 and downregulated the expression levels of proapoptotic genes CASP8 and CASP9. To conclude, by regulating apoptotic genes, sodium arsenite-mediated upregulation of circDHX34 promotes apoptosis in hormone-independent breast cancer cells.

Up-regulation of PUMA caused the activation of p53 phosphorylation and acetylation, enhancing the interaction between PUMA and Bcl-X and mediating arsenic-induced apoptosis.

Arsenic is a toxic metalloid vastly dispersed all over the occupational environments, manifesting multiple adverse health issues related to apoptosis. PUMA (p53 up-regulated modulator of apoptosis) is a crucial member of the Bcl-2 protein family and plays a key role in pro-apoptosis. The purpose of this work was to determine whether inorganic arsenic (NaAsO2) and its metabolites influenced the expression of PUMA in vivo and vitro, followed by investigating the mechanisms. RNA was extracted from serum and used to determine the expression of PUMA in vivo. The urine samples performed arsenic speciation analysis. This trial tested three-dose proportions in two cell lines (A549: 20, 40, 60 µM/L; 16HBE: 1.5, 3.0, 4.5 µM/L), respectively. According to the results of qRT-PCR and western blotting, NaAsO2 caused the overexpression of PUMA, not its metabolites. Furthermore, NaAsO2 induced phosphorylation of p53 at Ser315, 376, 392, and Thr55, and acetylation of p53 at K370, 382 with a dose-response relationship, suggesting the contribution of PUMA up-regulation to p53 phosphorylation and acetylation. CCK-8, JC-1 (5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetramethylbenzimi-dazolylcarbocyanine iodide), Hoechst33342/PI and the caspase3 and PARP1 blots were utilized to reveal apoptosis responding to NaAsO2 exposure. The co-immunoprecipitation assay showed that the interaction between PUMA and Bcl-X enhanced in intensity responding to NaAsO2 exposure, disrupting the complexes of Bcl-X with other pro-survival Bcl-2-related proteins. To our knowledge, we first reported that NaAsO2 activated phosphorylation of p53 at Ser315, 376, and Thr55, as well as acetylation of p53 at K370.

The Relationship Between GSTT1, GSTM1, GSTO1, GSTP1 and MTHFR Gene Polymorphisms and DNA Damage of BRCA1 and BRCA2 Genes in Arsenic-Exposed Workers.

OBJECTIVE: To investigate the associations between genetic polymorphisms of GSTT1, GSTM1, GSTO1, GSTP1 and MTHFR genes and the DNA damage levels of BRCA1 and BRCA2 genes. METHODS: Peripheral blood samples were used to measure DNA damage levels and genetic polymorphisms, and urine samples were collected to analyze arsenic metabolites in 79 arsenic-exposed workers and 24 non-arsenic-exposed workers. RESULTS: The BRCA1 and BRCA2 damage levels in exposure group were significantly higher than that in control group. Significant associations were detected between GSTT1 and GSTO1 polymorphisms and DNA damage levels of BRCA1 and BRCA2 genes in subjects (P < 0.05). CONCLUSIONS: Our findings suggest that the DNA damage levels of BRCA1 and BRCA2 genes may modulate by genetic variations of GSTT1 and GSTO1 when individuals are exposed to carcinogens, such as arsenic.