ABSTRACT
The sturgeon is an important commercial aquaculture species in China. The measurement of sturgeon mass plays a remarkable role in aquaculture management. Furthermore, the measurement of sturgeon mass serves as a key phenotype, offering crucial information for enhancing growth traits through genetic improvement. Until now, the measurement of sturgeon mass is usually conducted by manual sampling, which is work intensive and time consuming for farmers and invasive and stressful for the fish. Therefore, a noninvasive volume reconstruction model for estimating the mass of swimming sturgeon based on RGB-D sensor was proposed in this paper. The volume of individual sturgeon was reconstructed by integrating the thickness of the upper surface of the sturgeon, where the difference in depth between the surface and the bottom was used as the thickness measurement. To verify feasibility, three experimental groups were conducted, achieving prediction accuracies of 0.897, 0.861, and 0.883, which indicated that the method can obtain the reliable, accurate mass of the sturgeon. The strategy requires no special hardware or intensive calculation, and it provides a key to uncovering noncontact, high-throughput, and highly sensitive mass evaluation of sturgeon while holding potential for evaluating the mass of other cultured fishes.
Subject(s)
Aquaculture , Fishes , Swimming , Animals , Fishes/physiology , Swimming/physiology , Aquaculture/methodsABSTRACT
BACKGROUND: Calliptamus italicus L. is a major pest in Xinjiang grassland. The diapause overwintering strategy is one of the important reasons for the large population of this pest. This study investigated the function of the genes associated with the release of diapause (DIB, JHE and CAM) in Calliptamus italicus by RNA interference (RNAi) technology to aid in its biological control. RESULTS: The expression levels of DIB and its downstream-associated genes (EcR and FTZ-F1) in the eggs injected with dsDIB for 12 h decreased by 96.6%, 55.8% and 81.8%, respectively. Diapause began to terminate on day 3, and development was almost complete on day 6. However, the head was significantly smaller. The expression levels of JHE and its downstream-associated genes (JHEH and VgR) at 48 h after dsJHE treatment decreased by 76.5%, 85.6% and 85.9%, respectively. The termination of diapause occured on day 3 of incubation. The development was basically complete on day 6, but the yolk had been incompletely absorbed. The expression of CAM and its downstream-associated genes (CAMK4 and MYL) at 24 h after dsCAM treatment decreased by 42.4%, 95.3% and 82.7%, respectively. Diapause termination was completed on day 4 for incubation, and development was abnormal on day 6. The absorption of yolk was incomplete. CONCLUSION: DIB, JHE and CAM can delay the diapause termination of Calliptamus italicus eggs to different degrees and can be developed as potential target genes for its biological control. © 2024 Society of Chemical Industry.
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OBJECTIVE: Numerous studies have uncovered a correlation between menopausal, anxiety, and depressive symptoms. How these symptoms interrelate and influence each other, however, remains unclear. This study aimed to identify the associations between menopausal, anxiety, and depressive symptoms using network analysis. METHODS: The participants comprised 423 women (Mage = 49.21 ± 4.01 y; range, 40-60 y) recruited from a menopause clinic at a tertiary hospital in Beijing, China. Demographic characteristics and menopausal, anxiety, and depressive symptoms were obtained through self-report questionnaires. Two networks were established: a partial correlation network and a Bayesian network. RESULTS: The menopausal symptom of nervousness exhibited a robust association with anxiety symptoms in both networks. Within the partial correlation network, the depressive symptom of tiredness emerged as a pivotal symptom, facilitating the co-occurrence of menopausal and depressive symptoms. Bayesian network analysis exhibited that the depressive symptom of a loss of interest was related to certain menopausal symptoms through depressive symptoms of tiredness and motor problems, both serving as critical links between menopausal symptoms and depression. Notably, four menopausal symptoms-arthralgia/myalgia, formication, sexual complaints, and urinary tract infection-appeared independent of other menopausal, anxiety, and depressive symptoms. CONCLUSIONS: Both psychological (eg, fatigue) and somatic (eg, hot flashes, headaches, and dizziness) menopausal symptoms demonstrate strong associations with depression. In providing optimal support for women's health during menopause, psychological interventions aimed at depression, particularly among those experiencing a loss of interest or pleasure in activities, should complement conventional therapies.
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Low-coverage whole-genome sequencing (LCS) offers a cost-effective alternative for sturgeon breeding, especially given the lack of SNP chips and the high costs associated with whole-genome sequencing. In this study, the efficiency of LCS for genotype imputation and genomic prediction was assessed in 643 sequenced Russian sturgeons (â¼13.68×). The results showed that using BaseVar+STITCH at a sequencing depth of 2× with a sample size larger than 300 resulted in the highest genotyping accuracy. In addition, when the sequencing depth reached 0.5× and SNP density was reduced to 50 K through linkage disequilibrium pruning, the prediction accuracy was comparable to that of whole sequencing depth. Furthermore, an incremental feature selection method has the potential to improve prediction accuracy. This study suggests that the combination of LCS and imputation can be a cost-effective strategy, contributing to the genetic improvement of economic traits and promoting genetic gains in aquaculture species.
Subject(s)
Fishes , Polymorphism, Single Nucleotide , Fishes/genetics , Animals , Whole Genome Sequencing/economics , Whole Genome Sequencing/methods , Genomics/methods , Genomics/economics , Cost-Benefit Analysis , Linkage DisequilibriumABSTRACT
OBJECTIVE: To explore the improvement effect of probiotics combined with dietary fiber on constipation in patients with schizophrenia. METHODS: To compare the improvement scores of constipation, constipation symptoms, quality of life, neurotrophic factors-related indicators, and clinical efficacy between the two groups. RESULTS: There was no statistically significant difference in Cleveland Constipation Scoring System (CCS) scores in the control group before and after treatment (p > 0.05), while the CCS scores in the observation group decreased significantly after treatment (p < 0.05); Patient Assessment of Constipation Symptoms scores significantly decreased in the observation group compared to the control group (p < 0.05), with no significant difference in Patient Assessment of Constipation Quality of Life scores between the two groups pre- and post-treatment; Neuron-specific enolase levels decreased significantly in both groups post-treatment, while brain-derived neurotrophic factor, neuregulin-1, and nerve growth factor levels increased significantly, with a more pronounced rise in the observation group (p < 0.05). Additionally, the total effective rate of clinical treatment in the observation group was higher than that in the control group (p < 0.05). CONCLUSION: Probiotics combined with dietary fiber can improve constipation symptoms in patients with schizophrenia accompanied by constipation, effectively maintain the balance of intestinal microbiota, and improve the quality of life of patients. Additionally, levels of neurotrophic factors associated with bowel function and neurological health increased significantly, with a higher total effective rate of clinical treatment observed in the probiotics and dietary fiber group. These findings suggest the potential efficacy of probiotics and dietary fiber in managing constipation in this patient population.
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Given the universality of autopolyploid species in nature, it is crucial to develop genomic selection methods that consider different allele dosages for autopolyploid breeding. However, no method has been developed to deal with autopolyploid data regardless of the ploidy level. In this study, we developed a modified genomic best linear unbiased prediction (GBLUP) model (polyGBLUP) through constructing additive and dominant genomic relationship matrices based on different allele dosages. polyGBLUP could carry out genomic prediction for autopolyploid species regardless of the ploidy level. Through comprehensive simulations and analysis of real data of autotetraploid blueberry and guinea grass and autohexaploid sweet potato, the results showed that polyGBLUP achieved higher prediction accuracy than GBLUP and its superiority was more obvious when the ploidy level of autopolyploids is high. Furthermore, when the dominant effect was added to polyGBLUP (polyGDBLUP), the greater the dominance degree, the more obvious the advantages of polyGDBLUP over the diploid models in terms of prediction accuracy, bias, mean squared error and mean absolute error. For real data, the superiority of polyGBLUP over GBLUP appeared in blueberry and sweet potato populations and a part of the traits in guinea grass population due to the high correlation coefficients between diploid and polyploidy genomic relationship matrices. In addition, polyGDBLUP did not produce higher prediction accuracy than polyGBLUP for most traits of real data as dominant genetic variance was not captured for these traits. Our study will be a significant promising method for genomic prediction of autopolyploid species.
Subject(s)
Genome , Genomics , Humans , Genomics/methods , Phenotype , Ploidies , Polyploidy , Models, Genetic , Genotype , Polymorphism, Single NucleotideABSTRACT
BACKGROUND: Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a comprehensive syndrome with endocrine and metabolic complications. This review aims to explore the correlation between thyroid hormone levels and the severity of OSAHS in patients. METHODS: The protocol for this meta-analysis has been registered on PROSPERO. Searches were carried out from the inception of the databases to July 18, 2023, utilizing 6 databases (PubMed, CNKI, EMBASE, Web of Science, Cochrane Library, China Biology Medicine, and Wanfang). Standardized mean difference (SMD) and correlation coefficients were used as the effect size measures. Additionally, random effects or fixed effects models were used for pooled analysis. Moreover, data were statistically evaluated with the help of STATA 11.0 and R 4.1.3. RESULTS: This study included 23 articles that satisfied the pre-defined criteria. The prevalence of hypothyroidism and subclinical hypothyroidism in OSAHS patients was 6% and 8%, whereas hyperthyroidism had a prevalence of 2%. Moreover, thyroid hormone levels in OSAHS individuals exhibited no significant difference relative to healthy subjects. Subgroup analysis based on disease severity also established no significant changes in thyroid hormone levels between OSAHS individuals and controls. There was no significant correlation between the Apnea-Hypopnea Index (AHI) and free triiodothyronine (FT3), serum thyroid stimulating hormone (TSH), and free thyroxine (FT4) levels. CONCLUSION: The prevalence of thyroid dysfunction is relatively low in OSAHS individuals. Thyroid hormone levels show no significant difference between OSAHS patients and healthy subjects. Furthermore, there is no significant correlation between AHI and serum TSH, FT3, and FT4 levels. Based on existing data, the relationship between OSAHS and thyroid function remains controversial, and further in-depth research is warranted to validate the connection and elucidate the underlying mechanisms.
Subject(s)
Hypothyroidism , Sleep Apnea, Obstructive , Thyroid Diseases , Humans , Thyroid Diseases/complications , Thyroid Diseases/epidemiology , Hypothyroidism/complications , Hypothyroidism/epidemiology , Thyroid Hormones , Sleep Apnea, Obstructive/complications , Sleep Apnea, Obstructive/epidemiology , Thyrotropin , SyndromeABSTRACT
Paranosema locustae is an entomopathogenic microsporidia with promising potential for controlling agricultural pests, including Locusta migratoria manilensis. However, it has the disadvantage of having a slow insecticidal rate, and how P. locustae infection impacts the host immune response is currently unknown. The present study investigated the effect of P. locustae on the natural immune response of L. migratoria and the activities of enzymes that protect against oxidative stress. Infection with P. locustae increased the hemocytes and nodulation number of L. migratoria at the initial stage of infection. The hemocyte-mediated modulation of immune response was also affected by a decrease in the number of hemocytes 12 days postinfection. Superoxide dismutase activity in locusts increased in the early stages of infection but decreased in the later stages, whereas the activities of peroxidase (POD) and catalase (CAT) showed opposite trends may be due to their different mechanisms of action. Furthermore, the transcription levels of mRNA of antimicrobial peptide-related genes and phenoloxidase activity in hemolymph in L. migratoria were suppressed within 15 days of P. locustae infection. Overall, our data suggest that P. locustae create a conducive environment for its own proliferation in the host by disrupting the immune defense against it. These findings provide useful information for the potential application of P. locustae as a biocontrol agent.
Subject(s)
Locusta migratoria , Microsporidia , Animals , Locusta migratoria/genetics , Microsporidia/physiology , PeroxidaseABSTRACT
Microsporidia are a group of eukaryotic intracellular parasitic organisms that infect almost all vertebrates and invertebrates. Paranosema locustae are specialized parasites of Orthoptera that are often used as biological controls of locusts, with slow effects of action. In this study, we found that after infection with P. locustae, changes in energy metabolism in male and female Locusta migratoria as were consistent, with no gender differences. During the first 8 days of infection, L. migratoria used sugar as a source of energy. After 8 days, lipids and proteins were consumed to provide energy when the spore load was considerably heavy, and energy supply was insufficient. With increasing infection concentration and time, energy conversion from sugar, fats, and proteins was improved, which may explain why high mortality did not occur until about 15 days after P. locustae infection. The tandem mass tag-based quantitative proteomics analysis revealed that most altered metabolism-related proteins were upregulated (27 of 29 in the metabolic pathway). This result suggests that P. locustae infection accelerated metabolism in L. migratoria, which facilitated the pathogen's life cycle, inhibiting the growth and development of the locusts and eventually killing them. Our findings will be useful to better understand of the chronic pathogenic mechanisms of P. locustae and inform on applications of P. locustae to control locusts.
Subject(s)
Locusta migratoria , Microsporidia , Orthoptera , Female , Male , Animals , SugarsABSTRACT
Objective: We aimed to examine the prevalence of multidrug-resistant tuberculosis (MDR-TB) in Luoyang, China, identify related risk factors, inform clinical practices, and establish standardized anti-tubercular treatment regimens. Methods: We conducted a retrospective analysis of high-resolution melting curve (HRM) data from 17,773 cases (2,748 of which were positive) between June 2019 and May 2022 to assess the prevalence of MDR-TB and to identify its associated risk factors. Results: Between June 2019 and May 2022, out of the 17,773 HRM results, 2,748 were HRM-positive, and 312 were MDR-TB cases. The detection rates for HRM-positive and MDR-TB were 17.0 and 12.1% for males, and 12.4 and 8.2% for females, respectively. The MDR-TB detection rate was higher in the urban areas (14.6%) than in the rural areas (10.6%) and more common among individuals under 51 years of age (14.1%) than those over 50 years of age (9.3%). Notably, the rate of detecting MDR-TB was 18.3% higher in new male patients than in new female patients, which was at 10.6%, and this difference was statistically significant (p < 0.001). Moreover, the rate of MDR detection in females who had received anti-tuberculosis treatment (21.3%) was higher than that in males (16.9%). In the multivariate model that considered the results of the sputum smear and detection time, MDR-TB was positively correlated with a history of tuberculosis (TB) treatment, being male, being younger than 51 years, and living in urban areas. Conclusion: Local TB infections are complex and diverse; therefore, more comprehensive monitoring methods are needed to curb the spread of MDR-TB.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Multidrug-Resistant , Humans , Male , Female , Middle Aged , Antitubercular Agents/therapeutic use , Retrospective Studies , Microbial Sensitivity Tests , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/epidemiology , Tuberculosis, Multidrug-Resistant/diagnosis , Risk Factors , China/epidemiologyABSTRACT
Fluorene-9-bisphenol (BHPF), a bisphenol A (BPA) substitute, has been increasingly used as a material in syntheses of polymers that are widely used in road markings, artificial tracks, coating floors, building paints, etc., increasing the likelihood of BHPF contamination in the aquatic environment due to its release from the products. However, to date, it is unknown whether it may have actual impacts on fish in real environments. In this study, a 105-day exposure experiment of BHPF at various concentrations (0.01, 0.1, 1, and 10 µg/L) on Chinese medaka (Oryzias sinensis) was performed under laboratory conditions and found decreased fecundity, such as lower egg qualities and quantities, retarded oogenesis, and atretic follicles in the fish and deformed eyes and bodies in its F1 generation. Toxico-transcriptome analyses showed that estrogen-responsive genes were significantly suppressed by BHPF, indicating that antagonist properties of BHPF on estrogen receptors might be causes for the decreased fecundity. Field investigations (Beijing) demonstrated that BHPF was detectable in 60% surface waters, with a mean concentration of 10.49 ± 6.33 ng/L, by gas chromatography-mass spectrometry, and similar effects in wild Chinese medaka were also observed, some of which the parameters were found to be obviously correlated with the BHPF levels in corresponding waters.
Subject(s)
Oryzias , Water Pollutants, Chemical , Animals , Fluorenes/toxicity , Fluorenes/chemistry , Reproduction , Water Pollutants, Chemical/toxicityABSTRACT
Ecological toxicity assessments of contaminants in aquatic environments are of great concern. However, a dilemma in ecological toxicity assessments often arises when linking the effects found in model animals in the laboratory and the phenomena observed in wild fishes in the field due to species differences. Chinese medaka (Oryzias sinensis), widely distributed in East Asia, is a satisfactory model animal to assess aquatic environment in China. Here, we domesticated this species and assembled its genome (814 Mb) using next-generation sequencing (NGS). A total of 21,922 high-confidence genes with 41,306 transcripts were obtained and annotated, and their expression patterns in tissues were determined by RNA-sequencing. Six mostly sensitive biomarker genes, including vtg1, vtg3, vtg6, zp3a.2, zp2l1, and zp2.3 to estrogen exposure were screened and validated in the fish exposed to concentrations of estrone (E1), 17ß-estradiol (E2), and estriol (E3) under laboratory condition. Field investigations were then performed to evaluating the gene expression of biomarkers in wild Chinese medaka and levels of E1, E2, and E3 in the fish habitats. It was found that in 40 sampling sites, the biomarker genes were obviously highly expressed in the wild fish from about half sites, and the detection frequencies of E1, E2, and E3, were 97.5%, 42.5%, and 45% with mean concentrations of 82.48, 43.17, 52.69 ng/L, respectively. Correlation analyses of the biomarker gene expressions in the fish with the estrogens levels which were converted to EEQs showed good correlation, indicating that the environmental estrogens and estrogenicity of the surface water might adversely affect wild fishes. Finally, histologic examination of gonads in male wild Chinese medaka was performed and found the presence of intersex in the fish. This study facilitated the uses of Chinese medaka as a model animal for ecotoxicological studies.
Subject(s)
Oryzias , Water Pollutants, Chemical , Animals , Male , Biomarkers/metabolism , Estrogens/analysis , Estrone/analysis , Oryzias/genetics , Oryzias/metabolism , Transcriptome , Water Pollutants, Chemical/analysisABSTRACT
Whether under anaerobic or aerobic conditions, glycolysis results in production of lactate. Increasing evidence suggests that lactate serves as a multifunctional signaling molecule that develops non-metabolic activities in addition to serving as a key metabolite to link glycolysis and oxidative phosphorylation. Histone posttranslational modification patterns (HPTMs) are essential epigenetic processes controlling a variety of biological activities. Proteomics based on mass spectrometry (MS) has been used to progressively reveal new HPTMs. Recent discoveries of histone lactylation modification mediated by lactate and subsequent research demonstrating its involvement in cancer, inflammation, lung fibrosis, and other conditions suggest that it plays a significant role in immune regulation and homeostasis maintenance. This review provides a brief overview of the complicated control of histone lactylation modification in both pathological and physiological conditions.
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Genomic selection (GS) has great potential to increase genetic gain in aquaculture breeding; however, its implementation is hindered owing to high genotyping cost and the large number of individuals to genotype. This study investigated the efficiency of genomic prediction in four aquaculture species. In total, 749 to 1481 individuals with records for disease resistance and growth traits were genotyped using SNP arrays ranging from 12K to 40K. We compared the prediction accuracies and bias of breeding values obtained from BLUP, genomic BLUP (GBLUP), Bayesian mixture (BayesR), weighted GBLUP (WGBLUP), and genomic feature BLUP (GFBLUP). For GFBLUP, the genomic feature matrix was constructed based on prior information from genome-wide association studies. Fivefold cross-validation was performed with 20 replicates. Moreover, to reduce the cost of GS, we reduced the SNP density based on linkage disequilibrium as well as the reference population size. The results showed that the methods with marker information produced more accurate predictions than the pedigree-based BLUP method. For the genomic model, BayesR performed prediction with a similar or higher accuracy compared to GBLUP. For the four traits, WGBLUP yielded an average of 1.5% higher accuracy than GBLUP. However, the accuracy of genomic prediction decreased by an average of 6.2% for GFBLUP compared to GBLUP. When the density of SNP panels was reduced to 3K, which was sufficient to obtain accuracies similar to those using the whole dataset in the four species, the cost of GS was estimated to be 50% lower than that of genotyping all animals with high-density panels. In addition, when the reference population size was reduced by 10%, evenly from full-sib family, the accuracy of genomic prediction was almost unchanged, and the cost reduction was 8% in the four populations. Our results have important implications for translating the benefits of GS to most aquaculture species.
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Three forms of gonadotropin-releasing hormones (GnRHs), ArGnRH1, ArGnRH2, and ArGnRH3, were identified in sterlet. Compared with their orthologue, ArGnRH1 and ArGnRH2 have conserved core decapeptide but show low identity in the signal peptide and the rest of the sequences. The existence of the GnRH3 paralogue of sturgeon was predicted for the first time with TBLASTN by using the amino acid sequences of catshark and whale shark GnRH3 precursor as queries against the whole genome and transcript data of sterlet. The predicted ArGnRH3 cDNA sequence was composed of three exons containing all the elements of the GnRH family. The successful molecular cloning of GnRH3 from sterlets verified its expression in the brain of sturgeons. The analysis of the ArGnRH3 amino acid sequence revealed a completely conserved decapeptide sequence that shows 100% identity with the sequence of teleosts and differs in one amino acid with that of the cartilaginous fish (catshark and whale shark) at the 5th position. The structure of the phylogenetic tree showed that a total of 52 vertebrate GnRH sequences were clustered into three main clades corresponding to GnRH1, GnRH2, and GnRH3. The ArGnRH3 sequence is the oldest GnRH3 identified in teleosts. The tissue distribution analysis showed that ArGnRH1 was expressed in all the 13 examined tissues of females and in most of the tested tissues of male fish, with the highest expression in the pituitary and hypothalamus. ArGnRH2 is only expressed in the pituitary, hypothalamus, and gonads of both female and male sterlets. ArGnRH3 mRNA could be detected in the pituitary, hypothalamus, and gonad in both female and male fish. It is also present in the spleen, head kidney, and gill in female fish and in kidney and heart in male fish. However, the ArGnRH3 only showed weak expression in all the positive tissues. ArGnRH1 and ArGnRH2 active decapeptides were synthesized to investigate their roles on the regulation of LH/FSH using a mixed brain cell line from a sexually mature female sterlet. The results showed that ArGnRH1 and ArGnRH2 exerted different effects on the gene expression and release of gonadotropins. ArGnRH1 promoted the expression of fshß significantly around 48 h, and the expression was suppressed when the treatment time was extended to 72 h. ArGnRH1 had no significant effects on the level of either mRNA or secreted lh in any of the tested treatment length or concentrations. Moreover, ArGnRH1 did not stimulate the activity of gonadotropins in the maturation stage of female sturgeons. ArGnRH2 promoted the expression of fshß at 24 h and 48 h and increased mRNA level of lhß at 6 h and 48 h, accompanied by the significant secretion of LH at 72 h, although the high mRNA level of fsh did not correlate with the secretion of FSH in ArGnRH2-treated groups. In conclusion, ArGnRH2 plays an important role in the maturation stage of female sterlets. Therefore, ArGnRH2 has the potential to induce ovulation and spermiation in sturgeons.
Subject(s)
Gonadotropin-Releasing Hormone , Luteinizing Hormone, beta Subunit , Animals , Female , Fishes/genetics , Fishes/metabolism , Follicle Stimulating Hormone/metabolism , Follicle Stimulating Hormone, beta Subunit/metabolism , Gonadotropin-Releasing Hormone/genetics , Gonadotropin-Releasing Hormone/metabolism , Luteinizing Hormone, beta Subunit/metabolism , Male , Phylogeny , Pituitary Gland/metabolism , Pyrrolidonecarboxylic Acid/analogs & derivatives , RNA, Messenger/geneticsABSTRACT
In this study, we identified a new citrus vein enation virus (CVEV) isolate (named CVEV-DT1) through sRNA high-throughput sequencing and traditional sequencing. Phylogenetic analysis based on whole genome sequences of all known CVEV isolates revealed that CVEV-DT1 was in an evolutionary branch with other isolates from China. Molecular variation analysis showed that the single nucleotide variability along CVEV full-length sequences was less than 8%, with more transitions (60.55%) than transversions (39.43%), indicating a genetically homogeneous CVEV population. In addition, non-synonymous nucleotide mutations mainly occurred in ORF1 and ORF2. Based on disorder analysis of all encoded ORF by CVEV-DT1, we identified that the CVEV-DT1 coat protein (CP) formed spherical granules, mainly in the cell nucleus and partly throughout the cytoplasm, with liquid properties through subcellular localization and photobleaching assay. Furthermore, we also confirmed that the CVEV P0 protein has weak post-transcriptional RNA-silencing suppressor activity and could elicit a strong hypersensitive response (HR) in tobacco plants. Collectively, to the best of our knowledge, our study was the first to profile the genomic variation in all the reported CVEV isolates and reveal the functions of CVEV-DT1-encoded proteins.
Subject(s)
Citrus , Luteoviridae , Citrus/virology , Genome, Viral , Genomics , Luteoviridae/genetics , Nucleotides , PhylogenyABSTRACT
FluorAcryl 3298 (FA) is a UV-curable fluoroacrylate polymer commonly employed as a chemically resistant, hydrophobic, and oleophobic coating. Here, FA was used in a cleanroom-based microstructuring process to fabricate hydrophilic-in-hydrophobic (HiH) micropatterned surfaces containing femtoliter-sized well arrays. A short protocol involving direct UV photopatterning, an etching step, and final recovery of the hydrophobic properties of the polymer produced patterned substrates with micrometer resolution. Specifically, HiH microwell arrays were obtained with a well diameter of 10 µm and various well depths ranging from 300 nm to 1 µm with high reproducibility. The 300 nm deep microdroplet array (MDA) substrates were used for digital immunoassays, which presented a limit of detection in the attomolar range. This demonstrated the chemical functionality of the hydrophilic and hydrophobic surfaces. Furthermore, the 1 µm deep wells could efficiently capture particles such as bacteria, whereas the 300 nm deep substrates or other types of flat HiH molecular monolayers could not. Capturing a mixture of bacteria expressing red- and green-fluorescent proteins, respectively, served as a model for screening and selection of specific phenotypes using FA-MDAs. Here, green-fluorescent bacteria were specifically selected by overlaying a solution of gelatin methacryloyl (GelMA) mixed with a photoinitiator and using a high-magnification objective, together with custom pinholes, in a common fluorescence microscope to cross-link the hydrogel around the bacteria of interest. In conclusion, due to the straightforward processing, versatility, and low-price, FA is an advantageous alternative to more commonly used fluorinated materials, such as CYTOP or Teflon-AF, for the fabrication of HiH microwell arrays and other biphilic microstructures.