Search Results (29)

Lipid transfer proteins (LTPs) play crucial regulatory roles in plant growth, development, and abiotic stress responses. Stipagrostis pennata is a species of grass widely distributed in arid and semi-arid regions, particularly adapted to desert and steppe environments. Under extreme drought conditions, it exhibits a variety of physiological and morphological adaptation mechanisms, making it an important species for studying plant drought tolerance. Recently, LTPs have been found to exhibit upregulated expression under drought stress in plants such as wheat and tobacco, enhancing their drought tolerance. However, the functional role of LTPs in S. pennata remains unexplored. In this study, the SpLTP1 gene was isolated from S. pennata via molecular cloning, encoding a 116-amino acid protein. Phylogenetic analysis revealed that this protein contains a highly conserved nsLTP1 (cd01960) domain and has high sequence similarity with LTPs of Setaria viridis, Setaria italica, Musa acuminata and Phragmites australis. qRT-PCR revealed that SpLTP1 was highly expressed and dynamically regulated under drought, suggesting its potential role in root rhizosheath formation and drought tolerance. To investigate SpLTP1 function, SpLTP1-overexpressing (SpLTP1-OE) and complementation (SpLTP1-atltp) Arabidopsis lines were generated using the floral dip method, in comparison with the existing wild-type (WT) and the LTP-deficient mutant (atltp). Drought stress phenotyping and physiological assays indicated that SpLTP1 likely enhances drought tolerance by elevating antioxidant enzyme activities and osmolyte accumulation. Comparative transcriptome analysis of SpLTP1-OE and WT plants further suggested that SpLTP1 modulates critical pathways, including phenylpropanoid biosynthesis, zeatin biosynthesis, and plant hormone signal transduction, thereby influencing plant growth and stress adaptation. These findings not only provide novel insights into the molecular mechanisms by which SpLTP1 regulates rhizosheath development in S. pennata but also establish a foundation for deciphering its role in extreme drought adaptation. Full article

Sugar beet (Beta vulgaris L.), a biennial sugar crop, provides about 16% of the world’s sucrose production. PEG and Agrobacterium tumefaciens-mediated transformation have been established for sugar beet. However, the traditional transformation of sugar beet is time-consuming, low efficiency, and dependent on tissue regeneration. Recently, the use of Agrobacterium rhizogenes for genetic transformation without tissue culture has become a new possibility. Here, we describe an optimized A. rhizogenes-mediated transformation for the generation of composite sugar beet without tissue culture. By dipping A. rhizogenes K599 colonies onto a wound of hypocotyl and petiole, about 81.7% and 51.1% of shoots and leaves could be induced to produce adventitious roots. Of these, more than 60% of the explants contained transformed adventitious roots. Specifically, we discovered that the transformation efficiency was significantly improved when the MAS promoter was employed instead of the CaMV35S promoter. The transformation in adventitious roots was also validated by qRT-PCR and Western blot at the transcriptional and translational levels. The transformed adventitious roots have great potential for the study of taproot development, sugar accumulation, and resistance to root diseases, which is closely related to sugar beet yield and quality. Full article

Every year, diarrhoea is responsible for >1 million deaths in children with ages from 0 to 5 years, with rotavirus as the leading cause. The regions most affected lack routine rotavirus diagnosis due to high cost, lack of necessary equipment and shortage of trained-personnel for Enzyme-Link-Immunosorbent-Assay (ELISA) and molecular methods. We report the development and evaluation of a cheap, nanoparticle-based immunoassay for routine machine-free rotavirus diagnosis. In this work, optimal conditions for oxidation of cotton swabs and aldehyde production for kit development was confirmed by Fourier-Transform Infrared Spectroscopy (FTIR). Lactoferrin (LF) needed to bind the virus to the cotton swab was immobilised on activated cotton swabs, followed by the capture of commercial rotavirus antigen on LF-immobilised swabs. This was dipped in coloured nanobeads covalently coupled to rotavirus-group-specific monoclonal antibody for visual rotavirus detection. Subsequently, rotavirus detection by nanoassay, commercial ELISA and quantitative reverse transcription PCR were compared using same set of 186 stool samples and subjected to statistical analyses. Optimal oxidisation condition was observed using 48 mg/mL NaIO₄ in 0.1 M sodium acetate buffer at 35 °C for 9 h. Rotavirus detection was confirmed visually by blue colour retention on swabs after several washings. Sensitivity, specificity, positive-predictive-value and negative-predictive-value of ELISA in rotavirus detection were 60%, 84%, 53% and 88%, respectively, while our immunoassay showed performance at 88%, 94%, 82% and 96%. This immunoassay will provide effective rotavirus public health interventions in low-and-middle-income countries with high morbidity/mortality. Full article

(1) Background: The challenges in Implantology involve the development of alternative methods to enhance bone repair in patients with systemic conditions, such as osteoporosis. This study aimed to evaluate the effect of a local anti-sclerostin monoclonal antibody (Scl-Ab) on the functionalization of titanium implant surfaces through a dip-coating technique in peri-implant bone repair. (2) Methods: A total of 32 female rats were separated into four groups (n = 8): SHAM NT (Sham surgery), OVX NT (ovariectomy), SHAM Scl-Ab (SHAM; implants functionalized with Scl-Ab), and OVX Scl-Ab (OVX; implants functionalized with Scl-Ab). Implant surgery was executed 30 days after ovariectomy, and the rats were euthanized 28 days postoperatively. The right tibia was used for removal torque and RT-PCR, while the left tibia was collected for micro-CT and laser confocal microscopy. (3) Results: Functionalization with Scl-Ab significantly increased the gene expression of bone markers, especially ALP, in the SHAM Scl-Ab group compared to the other groups (p < 0.05). (4) Conclusions: Some parameters of this study indicate that implants functionalized with anti-sclerostin bone anabolic drug enhance peri-implant bone repair, especially in healthy rats. However, more studies must be carried out to confirm the therapeutic benefits of this approach. Full article

Our previous work identified several differentially expressed miRNAs (DEmiRNAs) in plasma exosomes from Takayasu’s arteritis (TAK) patients. This study aimed to validate these findings and explore the correlation between DEmiRNAs and clinical parameters in untreated TAK. Plasma exosomes were isolated from 30 untreated TAK patients and 20 healthy controls. qPCR was used to quantify miR-34a-5p, miR-143-3p, miR-22-3p, miR-200c-3p, and miR-21-5p expression. Correlations between miRNA levels, clinical data, inflammation markers, and T helper cell frequencies were analyzed. The target genes of validated DEmiRNAs were identified using mirDIP, and pathway enrichment analysis was performed using GO/KEGG. The effect of validated DEmiRNAs on the MAPK pathway and proliferation in human aortic endothelial cells (HAECs) was investigated in vitro. Only miR-200c-3p expression was validated as significantly downregulated in plasma exosomes from untreated TAK patients. Lower miR-200c-3p levels correlated negatively with ITAS-2010 scores and were associated with relapsed disease. MiR-200c-3p levels also negatively correlated with circulating Th17.1 cell frequencies. In vitro, the TAK exosome treatment activated ERK1/2 and JNK pathways and promoted HAEC proliferation, which was inhibited by the miR-200c-3p mimic. The pathway enrichment analysis showed that the MAPK pathway may be involved. This study confirms the reduced miR-200c-3p expression in plasma exosomes from TAK patients, suggesting its potential as a biomarker for vascular inflammation. MiR-200c-3p may exert protective effects in TAK by suppressing MAPK pathway activation and EC proliferation. Full article

Background/Objectives: DNA methylation is a key epigenetic mark involved in regulating gene expression. Aberrant DNA methylation contributes to various human diseases, including cancer, autoimmune disorders, atherosclerosis, and cardiovascular diseases. While whole-genome bisulfite sequencing and methylated DNA immunoprecipitation (MeDIP) are standard techniques for studying DNA methylation, they are typically limited to a few samples per run, making them expensive and low-throughput. Therefore, an automation-friendly method is needed to increase throughput and reduce costs without compromising data quality. Methods and Results: We developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), which can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed a 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis. Conclusions: Mx-MeDIP-Seq is a cost-effective and efficient method for high-throughput DNA methylation analysis, offering faster processing and reduced sample requirements. This technique makes DNA methylome analysis more accessible for large-scale studies. Full article

Crown gall is a soil-borne bacterial disease caused by Agrobacterium tumefaciens, leading to significant economic losses in many plant species. For the assessment of the biological and chemical products on crown gall, each plant’s crown region and roots were wounded, and then were dipped into their respective treatments. After the treatments, the plants were inoculated with a suspension of pathogenic A. tumefaciens isolate FBG1034 and maintained in a greenhouse for six months to assess them for gall formation. A quantitative real-time PCR assay was performed to quantify the A. tumefaciens using the chvE gene. Biological products such as the Agrobacterium radiobacter strain K1026, and strains 1 and 2, resulted in the lowest average root gall diameter and significantly reduced the crown gall diameter to stem diameter ratio, and the chemical product copper octanoate reduced the number of crown and root galls as well as the crown and root gall diameter compared to the inoculated, non-treated control. Moreover, both the A. radiobacter strain K1026 and strain 1 treatments resulted in an approximately 85% and 65% reduction in crown and root gall incidence, respectively, in both of the trials compared to the inoculated, non-treated plants. The findings of this study indicate that the use of biological and chemical products could help to suppress crown and root gall disease in rose plants. Full article

The combination of multiplex polymerase chain reaction (mPCR) and microfluidic technologies demonstrates great significance in biomedical applications. However, current microfluidics-based molecular diagnostics face challenges in multi-target detection due to their limited fluorescence channels, complicated fabrication process, and high cost. In this research, we proposed a cost-effective sandblasting method for manufacturing silicon microchips and a chip-based microdevice for field mPCR detection. The atomic force microscopy (AFM) images showed a rough surface of the sandblasted microchips, leading to poor biocompatibility. To relieve the inhibitory effect, we dip-coated a layer of bovine serum albumin (BSA) on the irregular substrate. The optimized coating condition was determined by scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS) (65 °C for 60 min). After sufficient coating, we performed on-chip PCR tests with 500 copies/mL Coronavirus Disease 2019 (COVID-19) standard sample within 20 min, and the sandblasted microchip displayed a higher amplification rate compared to dry etching chips. Finally, we achieved a 50 min mPCR for screening five resistance genes of the endophthalmitis pathogens on our microdevices, with strong specificity and reliability. Thus, this sandblasted microchip-based platform not only provides a rapid, accessible, and effective solution for multiplex molecular detection but also enables large-scale microfabrication in a low-cost and convenient way. Full article

Type 2 diabetes mellitus (T2DM) patients suffer premature development of cardiovascular disease and commonly require cardiac revascularization using the autologous saphenous vein (SV). Smooth muscle cells (SMCs) are the principal cell type within the vascular wall and are dysfunctional in T2DM SV-SMCs, yet the mechanisms underpinning this are incompletely understood. The purpose of this study was to interrogate differential microRNA (miRNA) expression in SV-SMCs to enhance our understanding of T2DM SV-SMC phenotypic change. miRNA expression in primary human SV-SMCs from T2DM and non-diabetic (ND) donors was determined using an array (n = 6 each of ND and T2DM SV-SMCs). Differentially expressed miRNAs were ranked, and functional annotation of the 30 most differentially expressed miRNAs using DAVID and KEGG analysis revealed pathways related to SMC phenotype, including proliferation, migration, cytokine production and cell signaling. After selecting miRNAs known to be involved in SMC phenotypic regulation, miR-17, miR-29b-2, miR-31, miR-130b and miR-491 were further validated using qRT-PCR (n = 5 each of ND and T2DM SV-SMC), with miR-29b-2 subsequently being removed from further investigation. Potential mRNA targets were identified using mirDIP. Predicted target analysis highlighted likely dysregulation in transcription, epigenetic regulation, cell survival, intracellular signaling and cytoskeletal regulation, all of which are known to be dysfunctional in T2DM SV-SMCs. In conclusion, this paper identified four miRNAs that are dysregulated in T2DM SV-SMCs and are implicated in functional changes in the behavior of these cells. This provides a step forward in our understanding of the molecular and epigenetic regulation of vascular dysfunction in T2DM. Full article

Updated information on the distribution and abundance of Aedes aegypti and Aedes albopictus is crucial to prepare African countries, such as Benin, for possible arboviral disease outbreaks. This study aims to evaluate the geographical distribution, abundance and biting behaviour of these two vectors in Benin. Three sampling techniques were used in this study. The collection of Aedes spp. adults were made through human landing catch (HLC), immatures were captured with the use of ovitraps, and a dipping technique was used for the collection of Aedes spp. in 23 communes located along the North–South and East–West transect of Benin. Adult Aedes mosquitoes were collected indoors and outdoors using HLC. Mosquito eggs, larvae and pupae were collected from containers and ovitraps. The adult mosquitoes were morphologically identified, then confirmed using a polymerase chain reaction (PCR). Overall, 12,424 adult specimens of Aedes spp. were collected, out of which 76.53% (n = 9508) and 19.32% (n = 2400) were morphologically identified as Ae. aegypti and Ae. albopictus, respectively. Geographically, Ae. aegypti was found across the North–South transect unlike Ae. albopictus, which was only encountered in the southern part of the country, with a great preponderance in Avrankou. Furthermore, an exophagic behaviour was observed in both vectors. This updated distribution of Aedes mosquito species in Benin will help to accurately identify areas that are at risk of arboviral diseases and better plan for future vector control interventions. Full article

Polycystic ovary syndrome (PCOS) is an endocrine disease associated with infertility and metabolic disorders in reproductive-aged women. In this study, we evaluated the expression of eight genes related to endometrial function and their DNA methylation levels in the endometrium of PCOS patients and women without the disease (control group). In addition, eight of the PCOS patients underwent intervention with metformin (1500 mg/day) and a carbohydrate-controlled diet (type and quantity) for three months. Clinical and metabolic parameters were determined, and RT-qPCR and MeDIP-qPCR were used to evaluate gene expression and DNA methylation levels, respectively. Decreased expression levels of HOXA10, GAB1, and SLC2A4 genes and increased DNA methylation levels of the HOXA10 promoter were found in the endometrium of PCOS patients compared to controls. After metformin and nutritional intervention, some metabolic and clinical variables improved in PCOS patients. This intervention was associated with increased expression of HOXA10, ESR1, GAB1, and SLC2A4 genes and reduced DNA methylation levels of the HOXA10 promoter in the endometrium of PCOS women. Our preliminary findings suggest that metformin and a carbohydrate-controlled diet improve endometrial function in PCOS patients, partly by modulating DNA methylation of the HOXA10 gene promoter and the expression of genes implicated in endometrial receptivity and insulin signaling. Full article

Betalains can be conveniently observed and quantified and, accordingly, have the potential as naked-eye visual screening reporters during plant transformation. RUBY is a new reporter system that uses “2A” peptides to fuse three key genes, CYP76AD1, DODA, and glucosyl transferase, for betalain biosynthesis, and has been successfully used for transformation of rice, Arabidopsis, and cotton, but its potential applications in the genetic transformation of various other plant species remain to be verified. In this study, RUBY was transferred into the hairy roots of Plukenetia volubilis and Nicotiana benthamiana, and was transferred into Arabidopsis by the floral-dip method. The expression levels of CYP76AD1, DODA, and glucosyl transferase were detected by RT−PCR and RT−qPCR, the relationship between the expression level of RUBY and red coloration was analyzed, and the genetic stability of RUBY in transgenic Arabidopsis was studied. The results showed that the expression of RUBY could reconstruct the betalain biosynthesis pathway in the hairy roots of P. volubilis, N. benthamiana, and Arabidopsis plants, indicating that it has the potential for versatile use across species. As a reporter, betalain did not affect callus induction, plant regeneration, development, or fertility. However, when used in plant transformation for observation and visual screening, it needed to accumulate to a certain extent to show red coloration, and it was positively correlated with gene expression. In general, RUBY is a convenient reporter for plant transformation, and has no obvious side effects during plant growth and development. However, the potential application of RUBY for visual screening is highly determined by the expression level, and further improvement is needed. Full article

Background: The gonads of Chrysemys picta, a turtle with temperature-dependent sex determination (TSD), exhibit differential DNA methylation between males and females, but whether the same is true in somatic tissues remains unknown. Such differential DNA methylation in the soma would provide a non-lethal sex diagnostic for TSD turtle hatchings who lack visually detectable sexual dimorphism when young. Methods: Here, we tested multiple approaches to study DNA methylation in tail clips of Chrysemys picta hatchlings, to identify differentially methylated candidate regions/sites that could serve as molecular sex markers To detect global differential methylation in the tails we used methylation-sensitive ELISA, and to test for differential local methylation we developed a novel hybrid method by sequencing immunoprecipitated and bisulfite converted DNA (MeDIP-BS-seq) followed by PCR validation of candidate regions/sites after digestion with a methylation-sensitive restriction enzyme. Results: We detected no global differences in methylation between males and females via ELISA. While we detected inter-individual variation in DNA methylation in the tails, this variation was not sexually dimorphic, in contrast with hatchling gonads. Conclusions: Results highlight that differential DNA methylation is tissue-specific and plays a key role in gonadal formation (primary sexual development) and maintenance post-hatching, but not in the somatic tail tissue. Full article

FGF5 (Fibroblast Growth Factor) is a member of the fibroblast growth factor family, which not only regulates growth and development but also inhibits hair regeneration. The oil-body expression vector pOTB-hFGF5 was constructed by the genetic engineering method and it was transformed into Arabidopsis by flora dip. T3 homozygous transgenic Arabidopsis was obtained after screening and propagation by the PCR and Western blot methods. The recombinant oil-body-expressed oleosin-hFGF5 can inhibit the proliferation of hair follicle epithelial cells and it exhibits the pharmacological activity of inhibiting hair regeneration in vivo by protein hybridization and imunohistochemistry. At the same time, the potential mechanism of recombinant oil-body-expressed oleosin-hFGF5 inhibiting hair growth was also revealed by RNA-Seq. This implies that the recombinant oil-body-expressed oleosin-hFGF5 has a good effect on inhibiting hair growth. Full article

Background: Although DNA methylation in the gene promoters usually represses gene expression, the TERT hypermethylated oncological region (THOR) located 5′ of the hTERT gene is hypermethylated when hTERT is expressed in diverse cancer types, including urothelial cancer (UC). Methods: Comprehensive MeDIP and DNA methylation array analyses complemented by the technically independent method of bisulfite genomic sequencing were applied on pathologically reviewed and classified urothelial carcinoma specimens and healthy urothelial tissue samples to reveal the methylation status of THOR in detail. Results: The detailed DNA methylation profiles reveal the exact positions of differentially methylated CpG dinucleotides within THOR in urothelial cancer and provide evidence ofa diverging role of methylation of these CpGs in the regulation of hTERT. In particular, our data suggest a regulating mechanism in which THOR methylation acts on hTERT expression through epigenetic silencing of the lncRNA hTERT antisense promoter-associated (hTAPAS), which represses hTERT. Conclusions: These findings precisely define the most differentially methylated CpGs of THOR in early urothelial cancer, enabling optimal design of Methylation-Specific PCR (MSPCR) primers to reliably probe these methylation differences for diagnostic and prognostic purposes. In addition, this strategy presents a prime example that is also applicable to many other malignancies. Finally, the first evidence for the underlying epigenetic mechanism regulating hTERT expression through the methylation status of THOR is provided. Full article