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11 pages, 761 KiB  
Brief Report
Naked-Eye Molecular Testing for the Detection of Xylella fastidiosa in Mallorca (Balearic Island) Almond Orchards by Colorimetric LAMP
by Amoia Serafina Serena, Ana Falcón-Piñeiro, Milica Pastar, José Manuel Garcìa-Madero, Nicoletta Contaldo, Mikael Muegge, Stéphane Compant, Pasquale Saldarelli and Angelantonio Minafra
Appl. Sci. 2025, 15(2), 739; https://doi.org/10.3390/app15020739 - 13 Jan 2025
Abstract
Xylella fastidiosa (Xf) is a quarantine pathogen heavily affecting economically important crops worldwide. Different sequence types (STs) belonging to Xf subspecies are present in various areas of Spain, including the Balearic Islands, and cause the almond leaf scorch disease (ALSD) in [...] Read more.
Xylella fastidiosa (Xf) is a quarantine pathogen heavily affecting economically important crops worldwide. Different sequence types (STs) belonging to Xf subspecies are present in various areas of Spain, including the Balearic Islands, and cause the almond leaf scorch disease (ALSD) in Prunus spp. The increased demand for rapid tests for early detection of the pathogen should enforce strict containment measures. Molecular detection through isothermal amplification reactions enables simplified instrumentation and the use of raw nucleic acid extracts. Colorimetric loop-mediated isothermal amplification (cLAMP) was applied to rapidly detect Xf in naturally infected almonds on Mallorca Island (Spain), using a quick crude sap extraction without DNA purification. Following tissue homogenization, an alkaline treatment for target DNA extraction was conducted before the cLAMP test. The cLAMP assay was able to detect up to 100 CFU/mL of the Xf bacterial suspension diluted in healthy almond sap. The same crude extracts used in the cLAMP test were also tested by qPCR. An overall positive agreement of about 47% was observed between the results of the two techniques, while a decrease in cLAMP sensitivity was evident as the bacterial titer declined in infected plants over Cq > 26–27. This study shows the potential of the cLAMP application as a rapid and low-cost point-of-care diagnostic method for the timely monitoring of Xf directly in the field. Full article
25 pages, 5204 KiB  
Article
Comparative Evaluation of AI-Based Multi-Spectral Imaging and PCR-Based Assays for Early Detection of Botrytis cinerea Infection on Pepper Plants
by Dimitrios Kapetas, Eleni Kalogeropoulou, Panagiotis Christakakis, Christos Klaridopoulos and Eleftheria Maria Pechlivani
Agriculture 2025, 15(2), 164; https://doi.org/10.3390/agriculture15020164 - 13 Jan 2025
Abstract
Pepper production is a critical component of the global agricultural economy, with exports reaching a remarkable $6.9B in 2023. This underscores the crop’s importance as a major economic driver of export revenue for producing nations. Botrytis cinerea, the causative agent of gray [...] Read more.
Pepper production is a critical component of the global agricultural economy, with exports reaching a remarkable $6.9B in 2023. This underscores the crop’s importance as a major economic driver of export revenue for producing nations. Botrytis cinerea, the causative agent of gray mold, significantly impacts crops like fruits and vegetables, including peppers. Early detection of this pathogen is crucial for a reduction in fungicide reliance and economic loss prevention. Traditionally, visual inspection has been a primary method for detection. However, symptoms often appear after the pathogen has begun to spread. This study employs the Deep Learning algorithm YOLO for single-class segmentation on plant images to extract spatial details of pepper leaves. The dataset included hyperspectral images at discrete wavelengths (460 nm, 540 nm, 640 nm, 775 nm, and 875 nm) from derived vegetation indices (CVI, GNDVI, NDVI, NPCI, and PSRI) and from RGB. At an Intersection over Union with a 0.5 threshold, the Mean Average Precision (mAP50) achieved by the leaf-segmentation solution YOLOv11-Small was 86.4%. The extracted leaf segments were processed by multiple Transformer models, each yielding a descriptor. These descriptors were combined in ensemble and classified into three distinct classes using a K-nearest neighbor, a Long Short-Term Memory (LSTM), and a ResNet solution. The Transformer models that comprised the best ensemble classifier were as follows: the Swin-L (P:4 × 4–W:12 × 12), the ViT-L (P:16 × 16), the VOLO (D:5), and the XCIT-L (L:24–P:16 × 16), with the LSTM-based classification solution on the RGB, CVI, GNDVI, NDVI, and PSRI image sets. The classifier achieved an overall accuracy of 87.42% with an F1-Score of 81.13%. The per-class F1-Scores for the three classes were 85.25%, 66.67%, and 78.26%, respectively. Moreover, for B. cinerea detection during the initial as well as quiescent stages of infection prior to symptom development, qPCR-based methods (RT-qPCR) were used for quantification of in planta fungal biomass and integrated with the findings from the AI approach to offer a comprehensive strategy. The study demonstrates early and accurate detection of B. cinerea on pepper plants by combining segmentation techniques with Transformer model descriptors, ensembled for classification. This approach marks a significant step forward in the detection and management of crop diseases, highlighting the potential to integrate such methods into in situ systems like mobile apps or robots. Full article
(This article belongs to the Section Digital Agriculture)
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Figure 1

Figure 1
<p>A high-level overview of the proposed methodology. The process begins with feeding the RGB image, five hyperspectral images, and five derived indices into the YOLO model to perform single-leaf segmentation across all images. The segmented leaf images are then processed through Transformer models to generate descriptors for each leaf. These descriptors are subsequently classified on a per-leaf, per-wavelength, and per-model basis. A weighted voting ensemble integrates these classifications to determine the final class for each leaf. Finally, the classifications are visualized on the original image, providing a comprehensive overview of the results. The leaves are categorized into three classes: green (healthy), blue (botrytis-invisible), and red (botrytis-visible).</p>
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<p>The eleven images that comprised a single capture: (<b>a</b>) RGB, (<b>b</b>) 460 nm, (<b>c</b>) 540 nm, (<b>d</b>) 640 nm, (<b>e</b>) 775 nm, (<b>f</b>) 875 nm, (<b>g</b>) CVI, (<b>h</b>) GNDVI, (<b>i</b>) NDVI, (<b>j</b>) NPCI, (<b>k</b>) PSRI.</p>
Full article ">Figure 2 Cont.
<p>The eleven images that comprised a single capture: (<b>a</b>) RGB, (<b>b</b>) 460 nm, (<b>c</b>) 540 nm, (<b>d</b>) 640 nm, (<b>e</b>) 775 nm, (<b>f</b>) 875 nm, (<b>g</b>) CVI, (<b>h</b>) GNDVI, (<b>i</b>) NDVI, (<b>j</b>) NPCI, (<b>k</b>) PSRI.</p>
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<p>RGB image overlaid with the ground truth annotations. The leaves are categorized into three classes: green (healthy), blue (botrytis-invisible), and red (botrytis-visible).</p>
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<p>A depiction of an example image of a leaf from datasets (<b>a</b>): as extracted directly from the original image; (<b>b</b>): with a four-times zoom; (<b>c</b>): with a crop around the leaf area. Additionally, (<b>a</b>) portrays how the leaf image was extracted from the original image in a visual manner.</p>
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<p>Disease severity (% of leaf area showing gray mold symptoms) as recorded over a period of 65 dpis on the 2nd artificially inoculated leaf of six plants.</p>
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<p>Disease severity expressed as the percentage of the maximum possible area under disease progress curve (AUDPC) for the whole period of the experiment (65 dpis) on the 2nd artificially inoculated leaf (yellow column) and on the 3rd and up to the 9th naturally and accidentally infected leaves (blue columns) of the six plants used in the bioassay. Bars indicate standard deviation. Columns with the same letter are not different according to Fisher’s LSD multiple range test (<span class="html-italic">p</span> ≤ 0.05).</p>
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<p>Gray mold symptoms: (<b>A</b>) Early and (<b>B</b>,<b>C</b>) late stages of leaf infection. (<b>D</b>,<b>E</b>) Flower and fruit infection.</p>
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<p>The relative <span class="html-italic">Botrytis cinerea</span> fungal abundance determined by RT-qPCR analysis of the fungal reference gene <span class="html-italic">BcRPL5</span> (<span class="html-italic">Bcin01g09620</span>) in mock- and <span class="html-italic">Botrytis cinerea</span>-inoculated second pepper leaves at 1, 2, and 5 dpis. Columns represent the means of three independent leaf samples per treatment (mock- and <span class="html-italic">B. cinerea</span>-inoculated). Columns with different letters are statistically different according to Fisher’s LSD multiple range test (<span class="html-italic">p</span> ≤ 0.05).</p>
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<p>The relative expression of the disease-responsive pepper genes <span class="html-italic">DEF1</span> (<span class="html-italic">Defensin 1</span>) and <span class="html-italic">PR1</span> (<span class="html-italic">Pathogenesis-related protein 1</span>) in mock- and <span class="html-italic">Botrytis cinerea</span>-inoculated second pepper leaves at 1, 2, and 5 dpis via RT-qPCR. Columns represent the means of three independent leaf samples per treatment (mock- and <span class="html-italic">B. cinerea</span>-inoculated). Bars indicate standard deviation and columns with the same letter are not different according to Fisher’s LSD multiple range test (<span class="html-italic">p</span> ≤ 0.05).</p>
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<p>(<b>a</b>) RGB images captured by the camera and (<b>b</b>) corresponding YOLO predictions visualized in red.</p>
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<p>A comparative visualization of three images with their leaf classes drawn on the leaves from (<b>a</b>) the manual annotated process and the (<b>b</b>) result of the whole methodology of this study. The green leaves represent the “healthy” class, the blue leaves represent the “botrytis-invisible” class, and the red leaves represent the “botrytis-visible” class.</p>
Full article ">Figure 12
<p>Classification visualization of individual detached leaves used for estimating fungal biomass via RT-qPCR. Images (<b>a</b>–<b>c</b>) represent mock-inoculated leaves, while images (<b>d</b>–<b>f</b>) depict <span class="html-italic">Botrytis cinerea</span>-inoculated leaves at 1, 2, and 5 dpis, respectively.</p>
Full article ">
16 pages, 3408 KiB  
Article
Molecular Characterization, Oxidative Stress-Mediated Genotoxicity, and Hemato-Biochemical Changes in Domestic Water Buffaloes Naturally Infected with Trypanosoma evansi Under Field Conditions
by Waqas Ahmad, Muhammad Yasin Tipu, Muti ur Rehman Khan, Haroon Akbar, Aftab Ahmad Anjum and Muhammad Ovais Omer
Pathogens 2025, 14(1), 66; https://doi.org/10.3390/pathogens14010066 - 13 Jan 2025
Abstract
(1) Background: Surra is a debilitating disease of wild and domestic animals caused by Trypanosoma evansi (T. evansi), resulting in significant mortality and production losses in the affected animals. This study is the first to assess the genetic relationships of T. [...] Read more.
(1) Background: Surra is a debilitating disease of wild and domestic animals caused by Trypanosoma evansi (T. evansi), resulting in significant mortality and production losses in the affected animals. This study is the first to assess the genetic relationships of T. evansi in naturally affected buffaloes from Multan district, Pakistan, using ITS-1 primers and evaluating the effects of parasitemia and oxidative stress on DNA damage and hematobiochemical changes in infected buffaloes. (2) Methods: Blood samples were collected from 167 buffaloes using a multi-stage cluster sampling strategy, and trypomastigote identification was performed through microscopy and PCR targeting RoTat 1.2 and ITS-1 primers. Molecular characterization involved ITS-1 via neighbor-joining analysis. The impact of parasitemia loads was correlated with oxidative stress markers, genotoxicity, and hematobiochemical parameters using Pearson correlation and multivariable regression models. (3) Results: Field-stained thin blood film microscopy and molecular identification revealed 8.98% and 10.18% infection rates, respectively. Phylogenetic analysis based on ITS-1 region sequences of the identified isolates showed close genetic associations with Indian isolates. The mean trypomastigote count observed in the infected buffaloes was 5.15 × 106 (±5.3 × 102)/µL of blood. The parasitemia loads were significantly correlated with the alterations in oxidative stress markers, DNA damage, and changes in hematobiochemical parameters. Infected animals exhibited significant (p < 0.05) alterations in oxidative stress biomarkers, including catalase, nitric oxide, and malondialdehyde concentrations. Noteworthily, a comet assay revealed a significantly (p < 0.0001) higher mean genetic damage index in the infected buffaloes (0.7 ± 0.04) compared with the healthy ones (0.196 ± 0.004). Alongside significant (p < 0.05) reductions in red cell indices, a marked elevation in leukocyte counts and serum hepatic enzyme levels was recorded in the affected buffaloes. (4) Conclusion: T. evansi isolates of buffaloes from Multan, Pakistan, have genetic similarities to Indian isolates. This study also revealed that higher parasitemia loads induce genotoxicity in the infected animals through oxidative stress and cause hematobiochemical alterations under natural field conditions. Full article
(This article belongs to the Collection Pathology and Parasitic Diseases of Animals)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Geographical map highlighting the study area in green with designated sampling sites marked by red circles. The inset map shows location of the Multan district, within Punjab, Pakistan, for regional context. The map was created using ArcMap 10.7.1.</p>
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<p>PCR amplification results for identification of Trypanosoma species. (<b>a</b>) ITS-1 primers produce a 480 bp band across the positive samples (Lanes 1–4) only. (<b>b</b>) Lanes 1–5 show Trypanosoma evansi-type A-specific RoTat 1.2 primers yielding a 205 bp band, confirming T. evansi infection in the positive samples. Lane 6 depicts the results from uninfected buffalo. L: Molecular marker of 100 bp.</p>
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<p>Neighbour-joining phylogenetic tree showing the relationships between <span class="html-italic">T. evansi</span> isolates from different geographical and host origins, including the Pakistani isolates sequenced in this study (highlighted in red). The tree is based on partial sequence analysis of the internal transcribed spacer-1 gene, and the bootstrap values (indicated as percentages) at nodes represent support from 1000 replicates. The scale bar indicates genetic distance. The Pakistani bubaline isolates (1, 2, and 3) clustered closely with Indian and Thai isolates, showing their phylogenetic relationships. The scale bar indicates genetic distance.</p>
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<p>Oxidative stress biomarkers in healthy and infected samples. Panels (<b>a</b>–<b>c</b>) show bar graphs comparing the levels of catalase (U/L), nitric oxide (µmol/L), and malondialdehyde (nmol/L) between healthy and infected groups. Catalase levels are significantly lower in the infected group (**** <span class="html-italic">p</span> &lt; 0.0001), while nitric oxide and MDA levels are significantly higher in the infected group (**** <span class="html-italic">p</span> &lt; 0.0001 for both). Panels (<b>d</b>–<b>f</b>) illustrate the correlation between trypomastigote count (log<sub>10</sub> count/µL) and levels of catalase, nitric oxide, and MDA, respectively. A strong negative correlation was observed between trypomastigote count and catalase levels (r = −0.92), while positive correlations were recorded regarding nitric oxide (r = 0.871) and MDA levels (r = 0.898).</p>
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<p>Comet assay results show DNA integrity in buffaloes’ peripheral blood cells. (<b>a</b>) Cells from healthy animals exhibited intact, undamaged nuclei and appeared as distinct fluorescent spots. (<b>b</b>) DNA damage in cells from <span class="html-italic">T. evansi</span>-infected buffaloes exhibiting comet-like appearance with tails indicating DNA fragmentation.</p>
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<p>Hematological parameter comparison between healthy and <span class="html-italic">T. evansi</span>-infected buffaloes under natural field conditions. (<b>a</b>) RBC count, (<b>b</b>) hemoglobin (Hb), (<b>c</b>) packed cell volume (PCV), (<b>d</b>) mean corpuscular volume (MCV), (<b>e</b>) mean corpuscular hemoglobin concentration (MCHC), (<b>f</b>) total leukocyte count (TLC), (<b>g</b>) lymphocyte count, (<b>h</b>) neutrophil count, (<b>i</b>) monocyte count, (<b>j</b>) eosinophil count, (<b>k</b>) basophil count, and (<b>l</b>) platelet count. Significant differences between groups are marked with asterisks, where * <span class="html-italic">p</span> &lt; 0.05 and **** <span class="html-italic">p</span> &lt; 0.0001.</p>
Full article ">Figure 7
<p>Biochemical parameter comparison between healthy and <span class="html-italic">T. evansi</span>-infected buffaloes under natural field conditions. (<b>a</b>) Total serum protein (TSP), (<b>b</b>) albumin, (<b>c</b>) globulin, (<b>d</b>) albumin/globulin (A/G) ratio, (<b>e</b>) alanine aminotransferase (ALT), (<b>f</b>) aspartate aminotransferase (AST), (<b>g</b>) alkaline phosphatase (ALP), and (<b>h</b>) gamma-glutamyl transferase (GGT). Significant differences between groups are marked with asterisks, where **** <span class="html-italic">p</span> &lt; 0.0001; “ns” indicates a non-significant difference.</p>
Full article ">Figure 8
<p>Correlation matrix of oxidative stress markers, trypomastigote counts, and hematobiochemical parameters. The color scale ranges from blue (negative correlation) to red (positive correlation), with the density plot (top-left) indicating the distribution of correlation values. The dendrograms on the left and top represent hierarchical clustering based on patterns of correlation of the parasitemia load, oxidative stress profile, and hematobiochemical parameters, respectively.</p>
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12 pages, 1974 KiB  
Article
Occurrence and Multi-Locus Genotyping of Giardia duodenalis in Black Goats from Fujian Province, China
by Shou-Xiao Huang, Kai Hu, Peng-Fei Fu, Si-Ang Li, Yang Liu, Zhipeng Niu and Dong-Hui Zhou
Animals 2025, 15(2), 199; https://doi.org/10.3390/ani15020199 - 13 Jan 2025
Abstract
Giardia duodenalis is a zoonotic parasite that causes gastrointestinal diseases in both humans and animals. To evaluate the prevalence and genetic diversity of G. duodenalis in black goats, we collected 539 fecal samples from nine districts in Fujian Province, China. The presence of [...] Read more.
Giardia duodenalis is a zoonotic parasite that causes gastrointestinal diseases in both humans and animals. To evaluate the prevalence and genetic diversity of G. duodenalis in black goats, we collected 539 fecal samples from nine districts in Fujian Province, China. The presence of G. duodenalis was confirmed through nested PCR targeting the SSU rRNA gene, and genotyping was performed at the beta-giardin, glutamate dehydrogenase, and triosephosphate isomerase loci. Among the samples, 115 tested positive, yielding an overall infection rate of 21.34%. Assemblages A and E were identified, with assemblage E being predominant. Statistical analysis revealed significant regional differences in infection rates (p < 0.01), with Zhangzhou exhibiting the highest infection rate (39%) and Fuzhou the lowest (3.13%). No significant differences in infection rates were observed based on age: 24.56% (56/228) for goats <1 year, 14.92% (27/181) for goats 1–2 years, 26.8% (26/97) for goats 2–3 years, and 18.18% (6/33) for goats ≥ 3 years. Similarly, no significant differences were found between sexes: 24.84% (40/161) for males and 19.84% (75/378) for females. Notably, assemblage A, a zoonotic genotype, was detected, indicating a potential risk of cross-species transmission. This study contributes to a deeper understanding of G. duodenalis in black goats and provides critical data for the development of targeted control strategies in Fujian Province. Full article
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Figure 1

Figure 1
<p>Distribution of sampling sites in Fujian.</p>
Full article ">Figure 2
<p>The phylogenetic tree illustrating the evolutionary relationships of <span class="html-italic">G. duodenalis</span> from sheep was constructed based on SSU rRNA gene sequences using maximum likelihood analysis. Sequences representative of each sequence type identified in this study were included in the phylogenetic analysis. The arrow indicates the genotypes identified in this experiment.</p>
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<p>Phylogenetic evolutionary tree diagram of the <span class="html-italic">bg</span> (<b>A</b>), <span class="html-italic">gdh</span> (<b>B</b>), and <span class="html-italic">tpi</span> (<b>C</b>) loci. The arrow indicates the genotypes identified in this experiment.</p>
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15 pages, 786 KiB  
Article
Gene Expression Profiles Reveal Distinct Mechanisms Driving Chronic Obstructive Pulmonary Disease Exacerbations
by Melissa Bello-Perez, Eduardo García-Pachón, Nieves Gonzalo-Jimenez, Montserrat Ruiz-García, Lucía Zamora-Molina, Carlos Baeza-Martinez and Antonio Galiana
Int. J. Mol. Sci. 2025, 26(2), 627; https://doi.org/10.3390/ijms26020627 - 13 Jan 2025
Abstract
Chronic obstructive pulmonary disease (COPD) exacerbations are major contributors to morbidity and mortality, highlighting the need to better understand their molecular mechanisms to improve prevention, diagnosis, and treatment. This study investigated differential gene expression profiles and key biological processes in COPD exacerbations categorized [...] Read more.
Chronic obstructive pulmonary disease (COPD) exacerbations are major contributors to morbidity and mortality, highlighting the need to better understand their molecular mechanisms to improve prevention, diagnosis, and treatment. This study investigated differential gene expression profiles and key biological processes in COPD exacerbations categorized based on sputum microbiome profiling. An observational study was performed on a cohort of 16 COPD patients, who provided blood and sputum samples during exacerbations, along with five stable-state samples as controls. Exacerbations were classified using 16S rRNA sequencing to analyze the sputum microbiota and multiplex PCR to detect respiratory viruses. Blood transcriptomic profiling was conducted using Oxford Nanopore technology, followed by differential gene expression and pathway enrichment analyses. A total of 768 regulated genes were identified across the exacerbation groups, with 35 shared genes associated with neutrophil activation. Bacterial exacerbations activated pathways related to phagocytosis and toll-like receptor signaling, while viral exacerbations were linked to pro-inflammatory responses and mitochondrial damage. Exacerbations of unknown origin showed activation of pathways involved in protozoan defense and neutrophilic asthma. Biomarkers such as IFITM3 and ISG15 for bacterial exacerbations, DEFA3 for viral, and CD47 for unknown-origin exacerbations were identified. These findings highlight distinct transcriptomic profiles and biological pathways in COPD exacerbations, emphasizing the central role of neutrophil-driven inflammation and identifying potential biomarkers for improved differential diagnosis and personalized management. Full article
23 pages, 814 KiB  
Article
Neuroprotective Effects of Myrtle Berry By-Product Extracts on 6-OHDA-Induced Cytotoxicity in PC12 Cells
by Debora Dessì, Giacomo Fais, Paolo Follesa and Giorgia Sarais
Antioxidants 2025, 14(1), 88; https://doi.org/10.3390/antiox14010088 - 13 Jan 2025
Abstract
The rising global focus on healthy lifestyles and environmental sustainability has prompted interest in repurposing plant-based by-products for health benefits. With increasing life expectancy, the incidence of neurodegenerative diseases—characterized by complex, multifactorial mechanisms such as abnormal protein aggregation, mitochondrial dysfunction, oxidative stress, and [...] Read more.
The rising global focus on healthy lifestyles and environmental sustainability has prompted interest in repurposing plant-based by-products for health benefits. With increasing life expectancy, the incidence of neurodegenerative diseases—characterized by complex, multifactorial mechanisms such as abnormal protein aggregation, mitochondrial dysfunction, oxidative stress, and inflammation—continues to grow. Medicinal plants, with their diverse bioactive compounds, offer promising therapeutic avenues for such conditions. Myrtus communis L., a Mediterranean plant primarily used in liquor production, generates significant waste rich in antioxidant and anti-inflammatory properties. This study explores the neuroprotective potential of Myrtus berry by-products in a cellular model of neurodegeneration. Using PC12 cells exposed to 6-hydroxydopamine (6-OHDA), we assessed cell viability via MTT assay and measured reactive oxygen species (ROS) production using DCFDA fluorescence. Additionally, we analyzed the expression of genes linked to oxidative stress and neuronal function, including AChE, PON2, Grin1, Gabrd, and c-fos, by RT-PCR. Our findings reveal that Myrtus extract significantly protects against 6-OHDA-induced cytotoxicity, reduces ROS levels, and modulates the expression of key stress-related genes, underscoring its potential as a neuroprotective agent. These results highlight the therapeutic promise of Myrtus extracts in mitigating neurodegenerative processes, paving the way for future interventions. Full article
13 pages, 2459 KiB  
Article
Establishment of Pathogen-Free Rhipicephalus bursa Colonies Under Laboratory Conditions for the Vector Competence Studies
by Mehmet Can Ulucesme, Sezayi Ozubek and Munir Aktas
Vet. Sci. 2025, 12(1), 54; https://doi.org/10.3390/vetsci12010054 - 13 Jan 2025
Abstract
Rhipicephalus bursa, the primary vector of Babesia ovis, is also considered to transmit Theileria, Babesia, and Anaplasma spp. These claims are based on pathogen detections rather than experimental validation. To confirm vector competence, sterile ticks must acquire pathogens from [...] Read more.
Rhipicephalus bursa, the primary vector of Babesia ovis, is also considered to transmit Theileria, Babesia, and Anaplasma spp. These claims are based on pathogen detections rather than experimental validation. To confirm vector competence, sterile ticks must acquire pathogens from infected hosts and transmit them to other hosts. The basic step is establishing a pathogen-free tick colony. In this study, engorged R. bursa females were collected from 12 infested livestock and allowed to lay eggs. The carcasses and larvae were screened for tick-borne pathogens using nPCR. The 0.150 g pathogen-free F1 larvae were fed on New Zealand rabbits, resulting in 592 engorged nymphs that molted into F1 adults. Eighty F1 adults were fed on pathogen-free splenectomized sheep, producing the next larval generation (F2). This protocol was repeated to produce F3 larvae. At the end of all developmental stages, ticks were screened via nPCR and found to be negative for tick-borne pathogens. The sheep were monitored for 63 days with no clinical signs or positive nPCR results, confirming F3 larvae as pathogen-free and suitable for vector competence studies. The R. bursa life cycle was completed in 72–153 days, providing a reliable model for vector competence research and offering valuable insights into its biological parameters. Full article
(This article belongs to the Topic Ticks and Tick-Borne Pathogens)
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Figure 1

Figure 1
<p>The representative scheme of the study design. (<b>A</b>) Collecting engorged ticks from cattle, sheep, and goats naturally infested with ticks, oviposition of <span class="html-italic">R. bursa</span> engorged female adults, hatching larvae, and the determination of pathogen-free (<span class="html-italic">Babesia</span>, <span class="html-italic">Theileria</span>, <span class="html-italic">Anaplasma</span>, and <span class="html-italic">Ehrlichia</span> spp.) larval bathes. (<b>B</b>,<b>C</b>) Infestation of the rabbits and splenectomized sheep by immature and adult stages of <span class="html-italic">R. bursa,</span> respectively, and screening by nPCR for the detection of <span class="html-italic">Babesia</span> spp., <span class="html-italic">Theileria</span> spp., <span class="html-italic">Anaplasma</span> spp., and <span class="html-italic">Ehrlichia</span> spp. <a href="#vetsci-12-00054-f001" class="html-fig">Figure 1</a> was created using BioRender.com (<a href="https://www.biorender.com/" target="_blank">https://www.biorender.com/</a> accessed on 14 November 2024).</p>
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<p>Gel imaging of nPCR results of carcasses and larvae from engorged <span class="html-italic">R. bursa</span> females collected from animals. (<b>A</b>) Gel image showing positive and negative nPCR amplification products representing <span class="html-italic">Babesia</span> and <span class="html-italic">Theileria</span> species obtained using Nbab1F/Nbab1R [<a href="#B47-vetsci-12-00054" class="html-bibr">47</a>] and RLB-F2/RLB-R2 [<a href="#B48-vetsci-12-00054" class="html-bibr">48</a>] primers. M: 100 bp marker, N: negative control (distilled water), P: <span class="html-italic">Babesia ovis</span> (EF092454), Lanes 1, 3, 5, 7, 9, 11, and 13 show DNA from engorged <span class="html-italic">R. bursa</span> females (#202-1, #204-1, #207-1, #209-1, #211-1, #212-1, and #216-1, respectively). Lanes 2, 4, 6, 8, 10, 12, and 14 represent their corresponding larval pools. (<b>B</b>) Gel image showing positive and negative nPCR amplification products representing <span class="html-italic">Anaplasma</span> and <span class="html-italic">Ehrlichia</span> species obtained using Ec9/Ec12a [<a href="#B45-vetsci-12-00054" class="html-bibr">45</a>] and 16S8FE/BGA1B [<a href="#B46-vetsci-12-00054" class="html-bibr">46</a>] primers. P: <span class="html-italic">Anaplasma ovis</span> (MG693754). Sample order is identical to Panel A.</p>
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<p>Monitoring body temperature of splenectomized sheep (#026 and #934) infested with <span class="html-italic">R. bursa</span> unfed adults (F1 and F2 generations). The red line (40 °C) represents the maximum body temperature observed in healthy sheep. DPI: Day post infestation.</p>
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<p>Nested PCR analysis of <span class="html-italic">Babesia</span> and <span class="html-italic">Theileria</span> (Panel <b>A</b>) and <span class="html-italic">Anaplasma</span> and <span class="html-italic">Ehrlichia</span> (Panel <b>B</b>) in splenectomized sheep #026 and #934 up to 63 days post-infestation. All results were negative for tick-borne pathogens after infestation with F1 and F2 unfed R. bursa adult ticks.</p>
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14 pages, 1707 KiB  
Article
Ehrlichia Species in Dromedary Camels (Camelus dromedarius) and Ruminants from Somalia
by Aamir M. Osman, Ahmed A. Hassan-Kadle, Marcos R. André, Flávia C. M. Collere, Amir Salvador Alabí Córdova, Fabiano Montiani-Ferreira, Thállitha S. W. J. Vieira, Abdalla M. Ibrahim, Abdulkarim A. Yusuf, Rosangela Z. Machado and Rafael F. C. Vieira
Pathogens 2025, 14(1), 65; https://doi.org/10.3390/pathogens14010065 - 13 Jan 2025
Abstract
Ehrlichioses, caused by Ehrlichia species, are tick-borne diseases (TBDs) that affect animals and humans worldwide. This study aimed to investigate the molecular occurrence of Ehrlichia spp. in 530 animals (155 Dromedary camels, 199 goats, 131 cattle, and 45 sheep) in the Benadir and [...] Read more.
Ehrlichioses, caused by Ehrlichia species, are tick-borne diseases (TBDs) that affect animals and humans worldwide. This study aimed to investigate the molecular occurrence of Ehrlichia spp. in 530 animals (155 Dromedary camels, 199 goats, 131 cattle, and 45 sheep) in the Benadir and Lower Shabelle regions of Somalia. Blood DNA samples were tested for PCR targeting dsb and sodB genes of Ehrlichia spp. and PCS20 and map1 genes of E. ruminantium. The obtained sequences were submitted for phylogenetic analyses. Ehrlichia spp. were detected in 26.4% (140/530) of animals by dsb-PCR, with the highest prevalence in dromedary camels (54.8%), followed by cattle (29.8%), goats (7.0%), and sheep (4.4%). Dromedary camels, cattle, and goats had significantly higher infection odds compared to sheep (p < 0.05). Among dsb-PCR-positive samples, 76.9% (30/39) of cattle tested sodB-positive, while other species were negative. E. ruminantium was detected in 13.7% (18/131) of cattle by pCS20-PCR, but none were positive for the map1 gene. Phylogenetic analysis confirmed E. minasensis in camels, sheep, and goats and E. ruminantium in cattle, marking the first molecular evidence of E. minasensis in dromedary camels, sheep, and goats globally, and E. ruminantium in cattle from Somalia. These findings emphasize the need for further research on its economic and public health impact. Full article
(This article belongs to the Section Bacterial Pathogens)
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<p>Phylogenetic tree inferred by using maximum likelihood inference and GTR+G evolutionary model based on an alignment of 390 bp of the <span class="html-italic">dsb</span> gene. The sequences detected are highlighted in red in the present study. <span class="html-italic">Ehrilichia muris</span> was used as the outgroup.</p>
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<p>Phylogenetic tree inferred by using maximum likelihood inference and GTR+G evolutionary model based on an alignment of 300 bp of the <span class="html-italic">sodB</span> gene. The sequences detected are highlighted in red in the present study. The numbers at the nodes correspond to posterior probability values higher than 50% accessed with 1000 replicates. <span class="html-italic">A. marginale</span> was used as outgroups.</p>
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<p>Phylogenetic tree inferred by using maximum likelihood inference and GTR+G evolutionary model based on an alignment of 280 bp of the <span class="html-italic">E. ruminantium PCS20</span> gene. The sequences detected are highlighted in red in the present study. The numbers at the nodes correspond to posterior probability values higher than 50% accessed with 1000 replicates. <span class="html-italic">E. canis</span> was used as the outgroup.</p>
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19 pages, 4733 KiB  
Article
Genome-Wide Analysis and Expression Profiling of Watermelon VQ Motif-Containing Genes Under Abiotic and Biotic Stresses
by Yanjun He, Jia Shen, Xinyang Xu and Weisong Shou
Horticulturae 2025, 11(1), 81; https://doi.org/10.3390/horticulturae11010081 - 13 Jan 2025
Abstract
Valine-glutamine (VQ) motif-containing proteins play important roles in diverse plant developmental processes and signal transduction in response to biotic and abiotic stresses. However, no systematic investigation has been conducted on VQ genes in watermelon. In this study, we identified 31 watermelon VQ genes, [...] Read more.
Valine-glutamine (VQ) motif-containing proteins play important roles in diverse plant developmental processes and signal transduction in response to biotic and abiotic stresses. However, no systematic investigation has been conducted on VQ genes in watermelon. In this study, we identified 31 watermelon VQ genes, which were classified into six subfamilies (I–VI). All of the deduced proteins contained a conserved FxxxVQxL/F/VTG motif. Eleven ClVQs were involved in segment duplication, which was the main factor in the expansion of the VQ family in watermelon. Numerous stress- and hormone-responsive cis-elements were detected in the putative promoter region of the ClVQ genes. Green fluorescent protein fusion proteins for ten selected ClVQs were localized in the nucleus, but three ClVQs also showed signals in cell membranes and the cell wall, thus confirming their predicted divergent functionality. Quantitative real-time PCR (qRT-PCR) analysis indicated that the majority of ClVQ genes were specifically or preferentially expressed in certain tissues or organs, especially in the male flower. Analyses of RNA-sequencing data under osmotic, cold, and drought stresses and Cucumber green mottle mosaic virus (CGMMV) infection revealed that the majority of ClVQ genes, especially those from subfamily IV, were responsive to these stresses. The results provide useful information for the functional characterization of watermelon ClVQ genes to unravel their biological roles. Full article
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<p>Phylogenetic relationships, gene structure, and conserved motifs of all VQs identified in watermelon. (<b>A</b>) Unrooted phylogenetic tree generated based on the deduced amino acid sequences using the neighbor-joining method implemented in MEGA 5. Bootstrap support values from 1000 replicates are provided for each branch. (<b>B</b>) Gene structure analyzed using the Gene Structure Display Server. Yellow boxes indicate exons, and lines indicate introns. (<b>C</b>) Motif analysis performed using MEME 4.0 software. Different-colored boxes represent different motifs in the corresponding position of each ClVQ protein.</p>
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<p>Amino acid sequence alignment of the VQ domain from watermelon VQs. Sequences were aligned using Clustal X. Conserved motifs are marked.</p>
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<p>Phylogenetic relationships among VQ proteins of <span class="html-italic">Arabidopsis</span>, rice, maize, soybean, Chinese cabbage, watermelon, and cucumber. Phylogenetic trees were constructed using the neighbor-joining method implemented in MEGA 5.0. Bootstrap support values from 1000 replicates are provided for each branch. The bar represents relative divergence of the sequences examined. Different subgroups of VQ are highlighted in different colors.</p>
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<p>Chromosomal distribution of <span class="html-italic">VQ</span> genes in watermelon. Chromosome number is indicated at the top of each chromosome. Black box highlights pairs of tandemly duplicated genes. Duplicated gene pairs are linked by a black line.</p>
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<p>Relationship of syntenic <span class="html-italic">VQ</span> genes in chromosomal synteny regions distributed in watermelon and cucumber. Chromosomes of watermelon and cucumber are represented by blue and red arcs according to their own sizes. Different-colored lines link representative syntenic <span class="html-italic">VQ</span> genes in watermelon and cucumber.</p>
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<p>Expression analysis of <span class="html-italic">ClVQ</span> genes in different tissues of watermelon and at different stages of watermelon fruit development. (<b>A</b>) Expression analysis of the <span class="html-italic">ClVQ</span> genes in different tissues of watermelon detected by quantitative real-time PCR. (<b>B</b>) Transcriptome profiles of <span class="html-italic">ClVQ</span> genes in fruit at four stages: 10, 18, 26, and 34 days after pollination (DAP) during watermelon fruit development.</p>
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<p>Transcriptome profiles of watermelon <span class="html-italic">VQs</span> in response to abiotic stresses. (<b>A</b>) The digital expression profiles of watermelon <span class="html-italic">VQs</span> in response to osmotic stress. Roots from watermelon ‘M08’ with four true leaves were collected under osmotic stress. (<b>B</b>) The expression patterns of <span class="html-italic">ClVQ</span> genes in response to cold stress in watermelon. Cold stress was imposed by placing watermelon in cold chamber at 4 °C and leaves were collected. (<b>C</b>) The digital expression profiles of <span class="html-italic">ClVQ</span> genes in leaves of two watermelon cultivars (‘Y34’ and ‘M20’) subjected to drought stress (for 4 d and 8 d) and without drought stress (control, 4 d, and 8d).</p>
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<p>Transcriptome profiles of <span class="html-italic">ClVQ</span> genes in watermelon fruit in response to <span class="html-italic">Cucumber green mottle mosaic virus</span> (CGMMV) infection.</p>
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<p>Subcellular localization of ClVQ proteins. Green fluorescent protein (GFP) fusion proteins were transiently expressed in tobacco leaf epidermal cells. After 48 h of incubation, the GFP signal was detected with a fluorescence microscope. Bright-field, endoplasmic nucleus marker, fluorescence, and merged images of p35S::<span class="html-italic">ClVQ02</span>-GFP (<b>A</b>); p35S::<span class="html-italic">ClVQ05</span>-GFP (<b>B</b>); p35S::<span class="html-italic">ClVQ07</span>-GFP (<b>C</b>); p35S::<span class="html-italic">ClVQ11</span>-GFP (<b>D</b>); p35S::<span class="html-italic">ClVQ16</span>-GFP (<b>E</b>); p35S::<span class="html-italic">ClVQ18</span>-GFP (<b>F</b>); p35S::<span class="html-italic">ClVQ22</span>-GFP (<b>G</b>); p35S::<span class="html-italic">ClVQ24</span>-GFP (<b>H</b>); p35S::<span class="html-italic">ClVQ25</span>-GFP (<b>I</b>); and p35S::<span class="html-italic">ClVQ26</span>-GFP (<b>J</b>).</p>
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<p>Gene Ontology (GO) analysis of <span class="html-italic">VQ</span> genes in watermelon. The <span class="html-italic">ClVQ</span> genes were categorized into three groups: molecular function (A); biological process (B); and cell component (C). Note: 1, negative regulation of molecular function; 2, regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process; 3, regulation of macromolecule biosynthetic process; 4, regulation of cellular biosynthetic process; 5, regulation of nitrogen compound metabolic process; 6, regulation of primary metabolic process; 7, regulation of macromolecule metabolic process.</p>
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18 pages, 4616 KiB  
Article
The AP2/ERF Transcription Factor ERF56 Negatively Regulating Nitrate-Dependent Plant Growth in Arabidopsis
by Guoqi Yao, Chunhua Mu, Zhenwei Yan, Shijun Ma, Xia Liu, Yue Sun, Jing Hou, Qiantong Liu, Bing Cao, Juan Shan and Bingying Leng
Int. J. Mol. Sci. 2025, 26(2), 613; https://doi.org/10.3390/ijms26020613 - 13 Jan 2025
Abstract
ERF56, a member of the APETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/ERF) transcription factor (TF) family, was reported to be an early nitrate-responsive TF in Arabidopsis. But the function of ERF56 in nitrate signaling remains not entirely clear. This study aimed to investigate the role of [...] Read more.
ERF56, a member of the APETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/ERF) transcription factor (TF) family, was reported to be an early nitrate-responsive TF in Arabidopsis. But the function of ERF56 in nitrate signaling remains not entirely clear. This study aimed to investigate the role of ERF56 in nitrate-dependent plant growth and nitrate signaling. We confirmed with reverse transcription quantitative PCR (RT-qPCR) that the transcription of ERF56 is quickly induced by nitrate. ERF56 overexpressors displayed decreased nitrate-dependent plant growth, while erf56 mutants exhibited increased plant growth. Confocal imaging demonstrated that ERF56 is localized into nuclei. Assays with the glucuronidase (GUS) reporter showed that ERF56 is mainly expressed at the region of maturation of roots and in anthers. The dual-luciferase assay manifested that the transcription of ERF56 is not directly regulated by NIN-LIKE PROTEIN 7 (NLP7). The transcriptome analysis identified 1038 candidate genes regulated by ERF56 directly. A gene ontology (GO) over-representation analysis showed that ERF56 is involved in the processes of water transport, inorganic molecule transmembrane transport, secondary metabolite biosynthesis, and cell wall organization. We revealed that ERF56 represses nitrate-dependent growth through regulating the processes of inorganic molecule transmembrane transport, the secondary metabolite biosynthesis, and cell wall organization. Full article
(This article belongs to the Section Molecular Plant Sciences)
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<p>Transcription of <span class="html-italic">ERF56</span> is highly induced by NO<sub>3</sub><sup>−</sup>. (<b>a</b>) The relative mRNA levels of <span class="html-italic">ERF56</span>, <span class="html-italic">ERF57</span>, <span class="html-italic">NIR1</span>, and <span class="html-italic">NRT2.1</span> in the seedlings of 10-day-old wild-type plants deficient in nitrate treated with 10 mM KNO<sub>3</sub> or KCl (mock treatment) for 2 h. Values are means ± SE (<span class="html-italic">n</span> = 3). ** and * indicate <span class="html-italic">p</span> &lt; 0.01 and <span class="html-italic">p</span> &lt; 0.05 with Student’s <span class="html-italic">t</span>-test, respectively. ACT8 was used as a reference gene. (<b>b</b>) A phylogenetic tree of the <span class="html-italic">ERF56</span> homologs. (<b>c</b>) Dynamic relative mRNA levels of <span class="html-italic">ERF56</span> in the seedlings of 10-day-old wild-type plants deficient in nitrate treated with 10 mM KNO<sub>3</sub> or KCl. Values are means ± SE (<span class="html-italic">n</span> = 3).</p>
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<p>Confirmation of <span class="html-italic">ERF56</span> OEs and <span class="html-italic">erf56</span> mutants. (<b>a</b>) Transcription of <span class="html-italic">GFP</span> in leaves of 20-day-old WT (control) and OE T2 plants; in the OEs, the C-terminal of <span class="html-italic">ERF56</span> was fused in frame with <span class="html-italic">GFP</span>; and values are means ± SE (<span class="html-italic">n</span> = 3). <span class="html-italic">ACT8</span> was used as the reference gene. (<b>b</b>) Mutation sites in <span class="html-italic">ERF56</span> of two <span class="html-italic">erf56</span> mutants. The red frames indicated the inserted bases.</p>
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<p>Influence of <span class="html-italic">ERF56</span> on plant growth under conditions with different concentrations of KNO<sub>3</sub>. (<b>a</b>–<b>h</b>) Quantitative analysis of seedlings growth of <span class="html-italic">ERF56</span> OEs, <span class="html-italic">erf56</span> mutants, and the WT type on the modified 1/2× MS medium containing 1 mM KNO<sub>3</sub> or 20 mM KNO<sub>3</sub>. Data are means ± SE (<span class="html-italic">n</span> = 4 replicates for 1 mM KNO<sub>3</sub>, <span class="html-italic">n</span> = 10 replicates for 20 mM KNO<sub>3</sub>, and each replicate has 7 seedlings). Data were analyzed with two-way ANOVA. Multiple comparisons of genotype effects were performed using LSD-test with significant differences being indicated by letters (<span class="html-italic">p</span> ≤ 0.05). (<b>i</b>) Representative seedlings grown on the modified 1/2× MS medium containing 1 mM KNO<sub>3</sub> or 20 mM KNO<sub>3</sub>. Bar = 1 cm.</p>
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<p>ERF56 protein is localized into nuclei in <span class="html-italic">Arabidopsis</span>. Confocal imaging was performed with roots of <span class="html-italic">35S:ERF56:GFP</span> transgenic plants. DF, the dark field image with laser-aided confocal laser scanning. BF, the bright field image with transmitted light. The top and bottom panels show the results for two different transformants, respectively.</p>
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<p>GUS staining images of <span class="html-italic">proERF:GUS</span> transgenic plants. (<b>a</b>) The image of an 8-day-old seedling. (<b>b</b>) The image of an inflorescence of an adult plant.</p>
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<p>Interaction of NLP7 protein with the promoter of <span class="html-italic">ERF56</span>. (<b>a</b>) Schematic illustration of constructs used in the Dual-LUC assay. The <span class="html-italic">35S:REN-proERF56:LUC</span> reporter plasmid was transiently expressed in the leaves of <span class="html-italic">Nicotiana benthamiana</span> plants together with the <span class="html-italic">35S:NLP7</span> effector plasmid or empty control plasmid. The reporter plasmid contains an <span class="html-italic">LUC</span> gene derived by the promoter of <span class="html-italic">ERF56 proERF56</span> to monitor the effect of NLP7 on the <span class="html-italic">proERF56</span> and a 35S-derived <span class="html-italic">REN</span> used as an internal control. Grey, red and blue boxes indicate sequences of promoters, CDS, CAMV terms, respectively. (<b>b</b>) The expression of reporter genes. Data are means ± SE (<span class="html-italic">n</span> = 5), and <span class="html-italic">p</span> = 0.608 using the Student’s <span class="html-italic">t</span>-test.</p>
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<p>Identification of genes targeted by ERF56. (<b>a</b>) Volcano plots of log2 fold changes and negative log10 P-values of gene expression in the inducible <span class="html-italic">ERF56</span> OE line <span class="html-italic">ER8-ERF56-1</span> and WT after 3 h ß-estradiol treatments, respectively. The 10-day-old plants deficient in nitrate were transferred to a fresh nitrate-free medium either containing 2 uM 17-ß-estradiol or not, as a mock treatment (MOCK), for 3 h. Three biological replicates, each of which had 20 plants, were used for the transcriptome analysis. DEGs were determined using <span class="html-italic">p</span>-value ≤ 0.01 and |log<sub>2</sub>fold change| ≥ 1, with up-regulated genes in samples treated with ß-estradiol being plotted as red points, down-regulated genes being plotted as green points, and the others being plotted as grey points. (<b>b</b>) Top over-presented GO terms in biological process (BP), cellular component (CC), and the molecular function (MF) for the 1038 candidate genes targeted by ERF56. The 1038 candidate genes were selected from 1221 DEGs in <span class="html-italic">ER8-ERF56-1</span>, which were not differentially expressed in the WT in response to ß-estradiol (<span class="html-italic">p</span>-value ≤ 0.01) and did not include <span class="html-italic">ERF56.</span> A list of each of the top 20 significantly enriched terms in BP and MF, and the only 2 enriched in CC selected with the lowest P-value and the number of genes mapped within each term are shown, with enriched terms being determined by <span class="html-italic">p</span>-value ≤ 0.05.</p>
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<p>Identification of cis-regulatory elements putatively targeted by ERF56. Weight matrix representation of the motifs was retrieved using the soft MEME from 1000-bp promoter sequences upstream the start codons of the 483 induced and 517 repressed nuclear genes regulated by ERF56, respectively.</p>
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19 pages, 2847 KiB  
Article
Effective Mixed-Type Tissue Crusher and Simultaneous Isolation of RNA, DNA, and Protein from Solid Tissues Using a TRIzol-Based Method
by Kelly Karoline dos Santos, Isabelle Watanabe Daniel, Letícia Carani Delabio, Manoella Abrão da Costa, Júlia de Paula Dutra, Bruna Estelita Ruginsk, Jeanine Marie Nardin, Louryana Padilha Campos, Fabiane Gomes de Moraes Rego, Geraldo Picheth, Glaucio Valdameri and Vivian Rotuno Moure
J 2025, 8(1), 3; https://doi.org/10.3390/j8010003 - 13 Jan 2025
Abstract
One of the major challenges of studying biomarkers in tumor samples is the low quantity and quality of isolated RNA, DNA, and proteins. Additionally, the extraction methods ideally should obtain macromolecules from the same tumor biopsy, allowing better-integrated data interpretation. In this work, [...] Read more.
One of the major challenges of studying biomarkers in tumor samples is the low quantity and quality of isolated RNA, DNA, and proteins. Additionally, the extraction methods ideally should obtain macromolecules from the same tumor biopsy, allowing better-integrated data interpretation. In this work, an in-house, low-cost, mixed-type tissue crusher combining blade and beating principles was made and the simultaneous isolation of macromolecules from human cells and tissues was achieved using TRIzol. RT-qPCR, genotyping, SDS-PAGE, and Western blot analysis were used to validate the approach. For tissue samples, RNA, DNA, and proteins resulted in an average yield of 677 ng/mg, 225 ng/mg, and 1.4 µg/mg, respectively. The same approach was validated using cell lines. The isolated macromolecule validation included the detection of mRNA levels of ATP-binding cassette (ABC) transporters through RT-qPCR, genotyping of TNFR1 (rs767455), and protein visualization through SDS-PAGE following Coomassie blue staining and Western blot. This work contributed to filling a gap in knowledge about TRIzol efficiency for the simultaneous extraction of RNA, DNA, and proteins from a single human tissue sample. A low-cost, high yield, and quality method was validated using target biomarkers of multidrug resistance mechanisms. This approach might be advantageous for future biomarker studies using different tissue specimens. Full article
(This article belongs to the Section Biology & Life Sciences)
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<p>The flow chart illustrates the steps in analyzing RNA, DNA, and proteins obtained from a single sample using a low-cost tissue crusher (in-house prototype) and TRIzol reagent.</p>
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<p>Prototype and overview of TRIzol sample processing. (<b>A</b>) Photos of the tissue crusher, denominated as the prototype. Some components and details appear in zoom images. (<b>B</b>) Overview of the tissue disruption using the mixed-type tissue crusher combining blade and beating principles and the TRIzol strategy. The illustration shows the coupling of nylon in the grooves of the stainless steel rod, the steel beads, and the coupling of the stainless steel rod in the quick-change chuck from a drill motor.</p>
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<p>RNA bands, amplification plots, and melting curves. Agarose gel electrophoresis of 28S and 18S ribosomal RNA (rRNA) bands from RNA samples extracted using the TRIzol method for HEK293-ABCG2 and H460 cells (<b>A</b>) and fragment of breast tissues (<b>D</b>). Amplification plots (relative fluorescence of SYBR green versus cycle number) represent the accumulation of product over the duration of the real-time PCR experiment. The horizontal line indicates the threshold placement. The targets are indicated by color in the figures, in which the same color represents a single gene. Different samples were analyzed, including cells (<b>B</b>), breast tissues (<b>E</b>), and breast or stomach tissues (<b>G</b>). A melting curve (60–95 °C; in increments of 0.5 °C) was generated to verify the specificity of primer amplification. Melting curves from different samples were analyzed, including cells (<b>C</b>), breast tissue (<b>F</b>), and breast or stomach tissues (<b>H</b>).</p>
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<p>Allelic discrimination plot. Genotyping of rs767455 was performed using the TaqMan™ SNP genotyping method on a 7500 Fast Real-time PCR. The assay mix include unlabeled PCR primers and FAM™- and VIC<sup>®</sup> dye-labeled probes. Each dot corresponds to a different sample. Non-circled samples correspond to the DNA from human blood cells representing the genotypic controls to alleles CC (blue), TC (green), and TT (red). Circled samples correspond to five samples of human breast tissues (turquoise, AC297, AC305, AC188, AC270, and AC276). DNA was obtained using the TRIzol-based method.</p>
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<p>SDS-PAGE and Western blot. (<b>A</b>) Comparison of protein recovery from HEK293-ABCG2 cells and tissue cancer samples (AC148, AC42, and AC297). Proteins were extracted with TRIzol, resuspended in 1% SDS and 8 M urea in Tris–HCl, pH 8.0, and then solubilized using sonication. Protein concentration was determined by BCA and gels were Coomassie Blue-stained. (<b>B</b>) Representative Western blot analysis of total ABCG2 and GAPDH from HEK293-ABCG2 cells. The experiments were repeated at least three times, and the most representative image is shown. Total protein was loaded on 8% SDS-PAGE. PVDF membranes were incubated with an antibody against ABCG2 or GAPDH.</p>
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<p>Integrative review of experimental studies on human tumor tissue extracted by a TRIzol-based method and reported yield of each macromolecule. (<b>A</b>) The studies were searched in the PubMed and Web of Science databases with a search strategy with the words TRIzol reagent (and analogs), cancer, RNA, DNA, protein, human, and tissue (<span class="html-italic">n</span> = 45). Based on the search findings (<b>A</b>), the arrow indicates (<b>B</b>) the number of studies reporting the number of macromolecules yield was assessed (<span class="html-italic">n</span> = 7).</p>
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16 pages, 12401 KiB  
Article
Glucose-Sensing Carbohydrate Response Element-Binding Protein in the Pathogenesis of Diabetic Retinopathy
by Christopher R. Starr, Assylbek Zhylkibayev, Oleg Gorbatyuk, Alli M. Nuotio-Antar, James Mobley, Maria B. Grant and Marina Gorbatyuk
Cells 2025, 14(2), 107; https://doi.org/10.3390/cells14020107 - 13 Jan 2025
Abstract
Glucose-sensing ChREBP and MondoA are transcriptional factors involved in the lipogenic, inflammatory, and insulin signaling pathways implicated in metabolic disorders; however, limited ocular studies have been conducted on these proteins. We aimed to investigate the potential role of ChREBP in the pathogenesis of [...] Read more.
Glucose-sensing ChREBP and MondoA are transcriptional factors involved in the lipogenic, inflammatory, and insulin signaling pathways implicated in metabolic disorders; however, limited ocular studies have been conducted on these proteins. We aimed to investigate the potential role of ChREBP in the pathogenesis of diabetic retinopathy (DR). We used diabetic human and mouse retinal cryosections analyzed by immunohistochemistry. qRT-PCR was performed to quantify gene expression. To explore the role of ChREBP in rods, we generated caChREBPRP mice with constitutively active (ca) ChREBP. These mice underwent retinal functional testing, which was followed by proteomic analysis using LC-MS. Furthermore, ARPE-19 cells were infected with lentiviral particles expressing human ChREBP (ARPE-19ChREBP) and subjected to global proteomics. Our results demonstrate that both proteins were expressed across the retina, although with distinct distribution patterns: MondoA was more prominently expressed in cones, while ChREBP was broadly expressed throughout the retina. Elevated expression of both proteins was observed in DR. This may have contributed to rod photoreceptor degeneration, as we observed diminished scotopic ERG amplitudes in caChREBPRP mice at P35. The retinal proteomic landscape revealed a decline in the KEGG pathways associated with phototransduction, amino acid metabolism, and cell adhesion. Furthermore, rod-specific caChREBP induced TXNIP expression. Consistent with altered retinal proteomics, ARPE-19ChREBP cells exhibit a metabolic shift toward increased glyoxylate signaling, sugar metabolism, and lysosomal activation. Our study demonstrates that ChREBP overexpression causes significant metabolic reprogramming triggering retinal functional loss in mice. Full article
(This article belongs to the Special Issue Mechanism of Cell Signaling during Eye Development and Diseases)
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Figure 1
<p>The immunohistochemical analysis of human control and diabetic retinas. (<b>A</b>) The retinas were subjected to treatment with anti-ChREBP antibody. The ChREBP immunoreactivity in human control (A,C) and diabetic (B,D) retinas. In the diabetic retina, robust staining of ChREBP (green) was detected in the photoreceptors, the cells of the INL (E), endothelial cells (F,H), retinal pigment epithelial cells (G), and retinal ganglion cells (I). The co-localization of ChREBP in the nuclei (red) of retinal cells is indicated in yellow. (<b>B</b>) The MondoA immunoreactivity in human control (A) and diabetic (B) retinas is shown in green. In the diabetic retina, robust staining was detected in the cones (C) and the cells of the INL (C). The localization of MondoA in the nuclei is shown in yellow (D).</p>
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<p>Expression of Mondo family proteins in hyperglycemic mouse retinas. (<b>A</b>) The RNAscope technique revealed enhanced ChREBP mRNA expression (green) in 12-week-old db/db retinas. The retinal ganglion cells (RGCs), Müller cells (highlighted in right inserts), and photoreceptors are responsive to hyperglycemia, showing increased ChREBP expression. (<b>B</b>) Mouse retinal explants were cultured in a medium supplemented with either high glucose or an equimolar concentration of mannitol (control) for 24 h. High-glucose, but not high-mannitol, culture conditions resulted in significant increases in both Chrebpα and Chrebpβ mRNAs, indicating that high glucose stimulates ChREBP expression ex vivo. (<b>C</b>) To confirm the ex vivo findings, diabetic retinas were isolated for qRT-PCR analysis to evaluate ChREBP and MondoA gene expression. The qRT-PCR results show that retinas from 12-week-old db/db and Akita mice exhibit increased expression of both Chrebp and MondoA mRNAs. Statistical significance is indicated as * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01 (<span class="html-italic">n</span> = 4 per group).</p>
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<p>Transgenic expression of constitutively active ChREBP in mouse rods leads to vision loss. (<b>A</b>) i-Cre-mediated recombination in rod photoreceptors of caChREBPRP transgenic mice resulted in deletion of eGFP and expression of FLAG-tagged caChREBP, detected by anti-FLAG antibody, shown in red. Nuclei are shown in blue. (<b>B</b>) Expression of caChREBP in rod photoreceptors leads to reduction in scotopic a- and b-wave amplitudes in caChREBP<sup>RP</sup> versus control (eGFP<sup>flox/wt</sup>-caChREBP or CghREBP<sup>f/wt</sup>) mice at postnatal day 35. Representative waveforms are shown on right. * <span class="html-italic">p</span> &lt; 0.05, <span class="html-italic">n</span> = 3–6 per group.</p>
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<p>Increased ChREBP activity in rods alters retinal proteomics and KEGG signaling in caChREBP<sup>RP</sup> mice. (<b>A</b>) Heatmap showing major altered proteins in caChREBP<sup>RP</sup> compared with eGFP<sup>flox/wt</sup>-caChREBP control (Control) retinas (<span class="html-italic">n</span> = 3–4 per group), highlighting significant changes in retinal protein expression. (<b>B</b>) Expression of constitutively active ChREBP (caChREBP) in rods leads to increase in TXNIP levels at postnatal day 35 (P35). Statistical significance is indicated by * <span class="html-italic">p</span> &lt; 0.05, with <span class="html-italic">n</span> = 3–4 per group. (<b>C</b>) Major downregulated KEGG signaling pathways in caChREBP-expressing retinas are identified, showing significant pathway alterations due to sustained ChREBP activity in rods.</p>
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<p>Overexpression of ChREBP in human ARPE-19 cells. (<b>A</b>) The images of ARPE-19 cells overexpressing human ChREBP and control cells treated with the empty virus. The direct fluorescence emitted by GFP indicates successful infection and ChREBP expression. (<b>B</b>) The heatmap displays the major altered proteins in ARPE-19 cells with sustained ChREBP expression (<span class="html-italic">n</span> = 3–4 per group), highlighting significant protein expression changes due to ChREBP overexpression. (<b>C</b>) The results from the proteomic analysis were analyzed using the Shiny GO program to generate diagrams of altered KEGG pathways. Both decreased and increased pathways are shown, reflecting the impact of sustained ChREBP expression on cellular signaling networks in ARPE-19 cells.</p>
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<p>ChREBP overexpression alters molecular function and affects cellular components of human ARPE-19 cells. (<b>A</b>) The molecular functions that are decreased or increased due to ChREBP overexpression in ARPE-19 cells are shown, highlighting the functional alterations induced by sustained ChREBP expression. (<b>B</b>) The cellular components affected by ChREBP expression in ARPE-19 cells are presented, demonstrating how ChREBP overexpression leads to changes in the cellular architecture and composition (<span class="html-italic">n</span> = 3–4 per group).</p>
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22 pages, 3986 KiB  
Article
Lipopolymers as the Basis of Non-Viral Delivery of Therapeutic siRNA Nanoparticles in a Leukemia (MOLM-13) Model
by Panadda Yotsomnuk, Amarnath Praphakar Rajendran, Daniel Nisakar Meenakshi Sundaram, Luis Carlos Morales, Cezary Kucharski, Mohammad Nasrullah, Wanwisa Skolpap, Xiaoyan Jiang, Spencer B. Gibson, Joseph Brandwein and Hasan Uludağ
Biomolecules 2025, 15(1), 115; https://doi.org/10.3390/biom15010115 - 13 Jan 2025
Abstract
Small interfering RNA (siRNA) therapy in acute myeloid leukemia (AML) is a promising strategy as the siRNA molecule can specifically target proteins involved in abnormal cell proliferation. The development of a clinically applicable method for delivering siRNA molecules is imperative due to the [...] Read more.
Small interfering RNA (siRNA) therapy in acute myeloid leukemia (AML) is a promising strategy as the siRNA molecule can specifically target proteins involved in abnormal cell proliferation. The development of a clinically applicable method for delivering siRNA molecules is imperative due to the challenges involved in effectively delivering the siRNA into cells. We investigated the delivery of siRNA to AML MOLM-13 cells with the use of two lipid-substituted polyethyleneimines (PEIs), a commercially available reagent (Prime-Fect) and a recently reported reagent with improved lipid substitution (PEI1.2k-PHPA-Lin9). The siRNAs utilized in this study were targeting the oncogenes FLT3 and KMT2A::MLLT3. Both lipopolymers gave similar-size siRNA complexes (210–220 nm) with positive ζ-potentials (+17 to +25 mV). While the binding efficiency of both lipopolymers to siRNA were similar, PEI1.2k-PHPA-Lin9 complexes were more resistant to heparin-induced dissociation. The quantitative analysis of gene silencing performed by qPCR as well as immunostaining/flow cytometry indicated significant reduction in both FLT3 expression and FLT3 protein after specific siRNA delivery. The desired inhibition of cell growth was attained with both FLT3 and KMT2A::MLLT3 siRNAs, and the combination provided more potent effects in both cell growth and colony formation assays. Induction of apoptosis was confirmed after specific siRNA treatments using the Annexin V assay. Using Luc(+) MOLM-13 cells, the growth of the xenografted cells was shown to be retarded with Prime-Fect-delivered FLT3 siRNA, unlike the siRNA delivered with PEI1.2k-PHPA-Lin9. These results demonstrate the potential of designed lipopolymers in implementing RNAi (via delivery of siRNA) for inhibition of leukemia growth and provide evidence for the feasibility of targeting different oncogenes using siRNA-mediated therapy. Full article
(This article belongs to the Special Issue The Role of Nanoparticles in Tumor Treatment)
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Figure 1
<p>(<b>a</b>) siRNA binding profiles of complexes at different ratios of polymer/siRNA were evaluated using the SYBR Green II dye exclusion assay (<span class="html-italic">n</span> = 3). (<b>b</b>) Dissociation of polymer/siRNA complexes incubated with different concentrations of heparin. The insert displays the variation in DC<sub>50</sub> values relative to the polymer/siRNA ratio. (<b>c</b>) Hydrodynamic size in nm (mean + SD) and <span class="html-italic">ζ</span>-potential (mean ± SD) of Prime-Fect and PEI1.2k-PHPA-Lin9 complexes (<span class="html-italic">n</span> = 3). (<b>d</b>) Morphology of polymer/siRNA complexes at a 6:1 ratio (<span class="html-italic">w</span>/<span class="html-italic">w</span>) by TEM: (i) Prime-Fect/siRNA and (ii) PEI1.2k-PHPA-Lin9/siRNA.</p>
Full article ">Figure 1 Cont.
<p>(<b>a</b>) siRNA binding profiles of complexes at different ratios of polymer/siRNA were evaluated using the SYBR Green II dye exclusion assay (<span class="html-italic">n</span> = 3). (<b>b</b>) Dissociation of polymer/siRNA complexes incubated with different concentrations of heparin. The insert displays the variation in DC<sub>50</sub> values relative to the polymer/siRNA ratio. (<b>c</b>) Hydrodynamic size in nm (mean + SD) and <span class="html-italic">ζ</span>-potential (mean ± SD) of Prime-Fect and PEI1.2k-PHPA-Lin9 complexes (<span class="html-italic">n</span> = 3). (<b>d</b>) Morphology of polymer/siRNA complexes at a 6:1 ratio (<span class="html-italic">w</span>/<span class="html-italic">w</span>) by TEM: (i) Prime-Fect/siRNA and (ii) PEI1.2k-PHPA-Lin9/siRNA.</p>
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<p>Cellular uptake of Prime-Fect/FAM-siRNA and PEI1.2k-PHPA-Lin9/FAM-siRNA complexes in MOLM-13 cells after transfection with complexes at 60 nM siRNA concentration and using 6:1 polymer/siRNA ratios for 24 h. (<b>a</b>) Visualization of FAM-siRNA uptake by epifluorescent fluorescence microscopy. The scale bar represents 100 μm. (<b>b</b>) Quantification of intracellular uptake after treatment with FAM-labeled siRNA complexes by flow cytometry. (i) Mean fluorescence intensity of FAM-siRNA per cell. (ii) Percentage of FAM-siRNA-positive cell population. Mean + 1 SD is shown (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> ≤ 0.05 versus C-siRNA.</p>
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<p>(<b>a</b>) Effect of the polymer/siRNA ratio on cell viability. The percentage of cell viability is summarized for MOLM-13 cells treated with single siRNA (C-siRNA and FLT3 siRNA) complexes with Prime-Fect and PEI1.2k-PHPA-Lin9 prepared at different ratios of polymer/siRNA (2, 4, and 6). An MTT assay was conducted to assess cell viability after 72 h of transfection, and it was normalized with the untreated group (i.e., taken as 100% cell viability as a control). * <span class="html-italic">p</span> ≤ 0.05 versus C-siRNA. (<b>b</b>) FLT3 gene silencing in MOLM-13 cells as evaluated by qPCR. Cells were transfected with Prime-Fect/siRNA and PEI1.2k-PHPA-Lin9/siRNA complexes for 1 day (i) and 3 days (ii) using 60 nM siRNA concentration and a ratio of polymer/siRNA at 6:1. C-siRNA was employed as a control and significance was determined with * <span class="html-italic">p</span> ≤ 0.05 in comparison with C-siRNA. (<b>c</b>) FLT3 protein levels in MOLM-13 cells were determined by immunochemistry/flow cytometry analysis. (i) FLT-3-positive cell population and (ii) mean FLT3 levels per cell (given by arbitrary fluorescence units of FLT3 antibody) after 24 h of treatment. (iii) FLT-3-positive cell population and (iv) mean FLT3 levels per cell after 72 h of treatment. * <span class="html-italic">p</span> ≤ 0.05 versus control siRNA.</p>
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<p>The percentage of cell viability is summarized for MV4-11 cells treated with single siRNA (C-siRNA and FLT3 siRNA) complexes with Prime-Fect (<b>left</b>) and PEI1.2k-PHPA-Lin9 (<b>right</b>) prepared at polymer/siRNA ratios of 5 and 7.5. The MTT assay was conducted to assess cell viability after 72 h of transfection, and it was normalized with the untreated group (i.e., with 100% cell viability taken as a control). * <span class="html-italic">p</span> ≤ 0.05 versus control siRNA.</p>
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<p>Inhibition of MOLM-13 cell growth with siRNA complexes targeting FLT3 and KMT2A::MLLT3. Percentages of cell viabilities are summarized with treatment of single and combinational siRNA (C-siRNA, siFLT3, and KMT2A::MLLT3) complexes, prepared with (<b>a</b>) Prime-Fect, and (<b>b</b>) PEI1.2k-PHPA-Lin9 at different siRNA concentrations (40, 60, and 80 nM).</p>
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<p>Colony formation in MOLM-13 cells after siRNA treatment. The treatment consisted of polymer complexes with C-siRNA, FLT3 siRNA, KMT2A::MLLT3 siRNA, and an FLT3+ KMT2A::MLLT3 siRNA combination at 60 nM (total siRNA concentration) and a ratio of polymer/siRNA of 6:1. The colonies were counted after two weeks of treatment. (<b>a</b>) The representative images of the colony formation after being stained with the MTT solution. (<b>b</b>) Quantification of the colony numbers, showing the differences in colony formation reduction compared to the untreated group. Mean + SD (<span class="html-italic">n</span> = 3) values are reported, with statistically significant groups noted at * <span class="html-italic">p</span> &lt; 0.05 vs. C-siRNA.</p>
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<p>Assessment of early and late apoptosis in MOLM-13 cells using an Annexin-V/PI apoptosis assay after treatment with the complexes. MOLM-13 cells were treated with the complexes targeting FLT3, KMT2A::MLLT3, or their combination at 60 nM. The apoptosis assay was conducted after 72 h of treatment. (<b>a</b>) Representative (%) number of cells undergoing early-stage apoptosis. (<b>b</b>) Representative (%) number of cells in the late stage of apoptosis. Mean + SD is provided from triplicate experiments with significance indicated by *: <span class="html-italic">p</span> ≤ 0.05, **: <span class="html-italic">p</span> ≤ 0.01, and ***: <span class="html-italic">p</span> ≤ 0.001.</p>
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<p>Effects of FLT3 siRNA complexed with Prime-Fect and PEI1.2k-PHPA-Lin9 on Luc+ MOLM-1l xenografts in NCG mouse model. (<b>a</b>) Timing scheme for the siRNA treatments and the whole-body imaging. (<b>b</b>,<b>d</b>) Whole-body bioluminescence images of mice on days 5, 9, and 13, showing leukemia engraftment extent in vivo. (<b>c</b>,<b>e</b>) Quantification of in vivo bioluminescence to evaluate the reduction in leukemia burden in mice (an error occurred in the whole-body imaging of the fourth mouse in the PEI1.2k-PHPA-Lin9/siFLT3 group on day 13; consequently, this was excluded from the analysis). Data are presented as mean ± SD (<span class="html-italic">n</span> = 5). Significant differences from the C-siRNA group were analyzed by multiple <span class="html-italic">t</span>-tests (Holm–Sidak test).</p>
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23 pages, 9743 KiB  
Article
Development of a Duplex PCR-NALFIA Assay for the Simultaneous Detection of Macrophomina phaseolina and Verticillium dahliae Causal Agents of Crown and Root Rot of Strawberry
by Viola Papini, Angelo Meloni and Susanna Pecchia
Agriculture 2025, 15(2), 160; https://doi.org/10.3390/agriculture15020160 - 13 Jan 2025
Abstract
Strawberry crown and root rot diseases are caused by soil-borne pathogens including Macrophomina phaseolina (Mp) and Verticillium dahliae (Vd). The symptoms caused by these pathogens are very similar and difficult to distinguish, and traditional culture-based detection methods are laborious, [...] Read more.
Strawberry crown and root rot diseases are caused by soil-borne pathogens including Macrophomina phaseolina (Mp) and Verticillium dahliae (Vd). The symptoms caused by these pathogens are very similar and difficult to distinguish, and traditional culture-based detection methods are laborious, time-consuming, and slow in providing results. In this work, we developed a duplex PCR-NALFIA assay using two pairs of species-specific primers labeled at the 5′ end with different molecules for the simultaneous identification of Mp and Vd. For the NALFIA assay, a lateral flow device (LFD) for the detection of two analytes was used. The method was developed by single and duplex PCR (Mp, Vd, Mp + Vd) using increasingly complex biological systems: (i) DNA from pure cultures of the pathogens; (ii) DNA from artificially inoculated cut melon stems; and (iii) DNA from artificially inoculated strawberry plants cv. Aromas. The duplex PCR protocol was effective in detecting the two pathogens within melon tissues and provided good results with strawberry crown tissues only when the DNA samples were purified by removing the PCR inhibitors. The amplicons were used for both agarose gel electrophoresis (AGE) and NALFIA assays and demonstrated the greater sensitivity of the NALFIA assay (10 pg) for simultaneous detection of the two pathogens. Full article
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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Figure 1
<p>Artificial inoculation of a cut melon stem with <span class="html-italic">Verticillium dahliae</span> (<b>left side</b>) and <span class="html-italic">Macrophomina phaseolina</span> (<b>right side</b>).</p>
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<p>Artificial inoculation method of strawberry crowns with infected wooden toothpick tips. Colonies of <span class="html-italic">Verticillium dahliae</span> (<b>a</b>) and <span class="html-italic">Macrophomina phaseolina</span> (<b>c</b>) used for toothpick tip inoculation. Stereomicroscope images (bars = 750 µm) of a toothpick tip colonized by <span class="html-italic">Verticillium dahliae</span> (<b>b</b>) and <span class="html-italic">Macrophomina phaseolina</span> (<b>d</b>) on which fungal hyphae and the production of microsclerotia are evident. (<b>e</b>) Strawberry crown subjected to double inoculation treatment with toothpick tips colonized by <span class="html-italic">Verticillium dahliae</span> and <span class="html-italic">Macrophomina phaseolina.</span> (<b>f</b>) Strawberry plant inoculated with <span class="html-italic">Verticillium dahliae</span> and <span class="html-italic">Macrophomina phaseolina</span> showing evident symptoms of collapse. (<b>g</b>) Strawberry control plant inoculated with uncolonized toothpick tips.</p>
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<p>Lateral flow device for the detection of two analytes used in this study. B = biotin; D = Digoxygenin; FITC = fluorescein isothiocyanate; G = conjugated gold nanoparticles.</p>
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<p>Specificity of PCR using the primer pair MP102F/MP102R labeled with biotin and FITC for the detection of <span class="html-italic">Macrophomina phaseolina</span> and the primer pair Vd7b/Vd10 labeled with digoxigenin and FITC for the detection of <span class="html-italic">Verticillium dahliae</span>. (<b>a</b>,<b>b</b>) = agarose gel electrophoresis; (<b>c</b>,<b>d</b>) = NALFIA assay. (<b>a</b>,<b>c</b>). 1–3 = <span class="html-italic">Macrophomina phaseolina</span> (10726, PVS-Mp1, DAFE SP19-24); 4 = <span class="html-italic">Diplodia seriata</span> DAFE SP19-25; 5 = <span class="html-italic">Neofusicoccum parvum</span> DAFE SP19-26; 6 = negative control (no DNA); M = 100 bp DNA ladder. (<b>b</b>,<b>d</b>) 1–3 = <span class="html-italic">Verticillium dahliae</span> (10361, 10357, 10355); <span class="html-italic">Verticillium nubilum</span> 10464; <span class="html-italic">Verticillium tricorpus</span> PD593; 6 = negative control (no DNA); M = 100 bp DNA ladder. The NALFIA assay was carried out with the same amplicons used in the agarose gel electrophoresis. The white arrows indicate the 500 bp band of the marker.</p>
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<p>Gel electrophoresis (<b>left panel</b>) and NALFIA assay (<b>right panel</b>) of single and duplex PCR using DNA extracted from mycelium of <span class="html-italic">Verticillium dahliae</span> and <span class="html-italic">Macrophomina phaseolina</span>. Primer pair Vd7b/Vd10 labeled with digoxigenin and FITC was used for the detection of <span class="html-italic">Verticillium dahliae</span> (lane 1) and primer pair MP102F/MP102R labeled with biotin and FITC was used for the detection of <span class="html-italic">Macrophomina phaseolina</span> (lane 5). Both primer pairs were used for duplex PCR (lane 3). A negative control (no DNA) was performed for each PCR reaction (lanes 2, 4 and 6). M = 100 bp DNA ladder. The white arrow indicates the 500 bp band of the marker. The NALFIA assay was carried out with the same amplicons used in agarose gel electrophoresis. <span class="html-italic">Vd</span> = <span class="html-italic">Verticillium dahliae</span>; <span class="html-italic">Mp</span> = <span class="html-italic">Macrophomina phaseolina</span>; C = control of duplex PCR.</p>
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<p>Sensitivity of duplex PCR using the primer pair MP102F/MP102R labeled with biotin and FITC for the detection of <span class="html-italic">Macrophomina phaseolina</span> and the primer pair Vd7b/Vd10 labeled with digoxigenin and FITC for the detection of <span class="html-italic">Verticillium dahliae</span>. The assay was performed using 10-fold serial dilutions of template DNAs ranging from 1.0 ng and 1.0 pg. Gel electrophoresis (<b>left panel</b>) and NALFIA assay (<b>right panel</b>). 1 = 1 ng of template DNAs; 2 = 100 pg of template DNAs; 3 = 10 pg of template DNAs; 4 = 1 pg of templated DNAs; 5 = negative control (no DNA); M = 100 bp DNA ladder. The white arrow indicates the 500 bp band of the marker.</p>
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<p>Gel electrophoresis (<b>left panel</b>) and NALFIA assay (<b>right panel</b>) of single and duplex PCR using DNA extracted from melon stem tissues inoculated with <span class="html-italic">Verticillium dahliae</span> and <span class="html-italic">Macrophomina phaseolina</span>. Primer pair Vd7b/Vd10 labeled with digoxigenin and FITC was used for the detection of <span class="html-italic">Verticillium dahliae</span> (1) and primer pair MP102F/MP102R labeled with biotin and FITC was used for the detection of <span class="html-italic">Macrophomina phaseolina</span> (5). Both primer pairs were used for duplex PCR (3). A negative control (no DNA) was performed for each PCR reaction (2, 4 and 6). M = 100 bp DNA ladder. The white arrow indicates the 500 bp band of the marker. The NALFIA assay was carried out with the same amplicons used in agarose gel electrophoresis.</p>
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<p>Isolation frequency (%) of culturable fungal genera recovered from crowns and petioles of strawberry plants.</p>
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<p>Gel electrophoresis (<b>left panel</b>) and NALFIA assay (<b>right panel</b>) of PCR using DNA extracted from strawberry crown tissues inoculated with <span class="html-italic">Verticillium dahliae</span> (<b>a</b>) and <span class="html-italic">Macrophomina phaseolina</span> (<b>b</b>). Primer pair Vd7b/Vd10 labeled with digoxigenin and FITC was used for the detection of <span class="html-italic">Verticillium dahliae</span> and primer pair MP102F/MP102R labeled with biotin and FITC was used for the detection of <span class="html-italic">Macrophomina phaseolina</span>. 1 = samples of strawberry crowns inoculated with <span class="html-italic">Verticillium dahliae</span> (<b>a</b>) and <span class="html-italic">Macrophomina phaseolina</span> (<b>b</b>). 2 = DNA extracted from mycelium of <span class="html-italic">Verticillium dahliae</span> (<b>a</b>) and <span class="html-italic">Macrophomina phaseolina</span> (<b>b</b>). 3 = samples of non-inoculated strawberry crown. M = 100 bp DNA ladder. The white arrow indicates the 500 bp band of the marker. The NALFIA assay was carried out with the same amplicons used in agarose gel electrophoresis.</p>
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<p>Gel electrophoresis (<b>left panel</b>) and NALFIA assay (<b>right panel</b>) of duplex PCR using DNA extracted from strawberry crown tissues inoculated with <span class="html-italic">Verticillium dahliae</span> and <span class="html-italic">Macrophomina phaseolina</span>. Primer pair Vd7b/Vd10 labeled with digoxigenin and FITC was used for the detection of <span class="html-italic">Verticillium dahliae</span> and primer pair MP102F/MP102R labeled with biotin and FITC was used for the detection of <span class="html-italic">Macrophomina phaseolina</span>. Both primer pairs were used for duplex PCR. 1 and 2 = samples of strawberry crowns inoculated with both pathogens. 3 = DNA extracted from mycelium of <span class="html-italic">Verticillium dahliae</span> and <span class="html-italic">Macrophomina phaseolina.</span> 4 = sample of non-inoculated strawberry crown. M = 100 bp DNA ladder. The white arrow indicates the 500 bp band of the marker. The NALFIA assay was carried out with the same amplicons used in agarose gel electrophoresis. <span class="html-italic">Vd</span> = <span class="html-italic">Verticillium dahliae</span>; <span class="html-italic">Mp</span> = <span class="html-italic">Macrophomina phaseolina</span>; C = control non-inoculated sample.</p>
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14 pages, 10406 KiB  
Article
Integration of Metabolomics and Transcriptomics to Reveal the Antitumor Mechanism of Dendrobium officinale Polysaccharide-Based Nanocarriers in Enhancing Photodynamic Immunotherapy in Colorectal Cancer
by Shengchang Tao, Huan Wang, Qiufeng Ji, Yushan Yang, Gang Wei, Ruiming Li and Benjie Zhou
Pharmaceutics 2025, 17(1), 97; https://doi.org/10.3390/pharmaceutics17010097 - 13 Jan 2025
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Abstract
Background: The mechanism of Dendrobium officinale polysaccharide-based nanocarriers in enhancing photodynamic immunotherapy in colorectal cancer (CRC) remains poorly understood. Methods: The effects of TPA-3BCP-loaded cholesteryl hemisuccinate–Dendrobium officinale polysaccharide nanoparticles (DOP@3BCP NPs) and their potential molecular mechanism of action in a [...] Read more.
Background: The mechanism of Dendrobium officinale polysaccharide-based nanocarriers in enhancing photodynamic immunotherapy in colorectal cancer (CRC) remains poorly understood. Methods: The effects of TPA-3BCP-loaded cholesteryl hemisuccinate–Dendrobium officinale polysaccharide nanoparticles (DOP@3BCP NPs) and their potential molecular mechanism of action in a tumor-bearing mouse model of CRC were investigated using non-targeted metabolomics and transcriptomics. Meanwhile, a histopathological analysis (H&E staining, Ki67 staining, and TUNEL assay) and a qRT-PCR analysis revealed the antitumor effects of DOP@3BCP NPs with and without light activation. Results: Through metabolomics and transcriptomics analysis, we found an alteration in the metabolome and functional pathways in the examined tumor tissues. The metabolic analysis showed 69 and 60 differentially expressed metabolites (DEMs) in positive- and negative-ion modes, respectively, in the treated samples compared to the Control samples. The transcriptomics analysis showed that 1352 genes were differentially expressed among the three groups. The differentially regulated functional pathways were primally related to the antitumor immune response. The results of the pathological histology assay and qRT-PCR analysis verified the findings of the integrated metabolomics and transcriptomics analysis. Conclusions: Overall, our findings elucidate the potential antitumor mechanisms of the D. officinale polysaccharide-based nanocarrier in enhancing photodynamic immunotherapy in CRC. Full article
(This article belongs to the Special Issue Functional Nanomaterials for Drug Delivery in Photodynamic Therapy)
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Graphical abstract

Graphical abstract
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<p>Effects of DOP@3BCP NP-mediated PDT on metabolite profiling of tumor among three groups. Score plot of OPLS-DA in positive ion mode (<b>A</b>) and in negative ion mode (<b>B</b>) from tumor tissue metabolomics data; Heatmap of DEMs in positive- (<b>C</b>) and negative-ion mode (<b>D</b>) from tumor tissue metabolomics data; Venn diagram of in positive- (<b>E</b>) and negative-ion mode (<b>F</b>) from tumor tissue metabolomics data.</p>
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<p>Bubble diagram of top 20 KEGG pathway enrichments in the positive- and negative-ion mode (<span class="html-italic">n</span> = 5): (<b>A</b>–<b>C</b>), in positive-ion mode; (<b>D</b>–<b>F</b>), in negative-ion mode.</p>
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<p>Profiling of DEGs in tumor tissues from different groups (<span class="html-italic">n</span> = 3). (<b>A</b>) Clustered heatmap for DEGs of different groups. (<b>B</b>) Venn diagram of DEGs from different groups. (<b>C</b>) Stacked bar plots for DEGs from different groups. (<b>D</b>–<b>F</b>) Volcano plot for DEGs from different groups.</p>
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<p>Functional enrichment of DEGs in tumor tissues from different groups (<span class="html-italic">n</span> = 3): (<b>A</b>–<b>C</b>)<b>,</b> GO functional enrichment analysis; (<b>D</b>–<b>F</b>)<b>,</b> KEGG functional enrichment analysis.</p>
Full article ">Figure 5
<p>(<b>A</b>) Representative images of H&amp;E, Ki67, and TUNEL staining of tumor slices in three groups. (<b>B</b>) Relative mRNA expression in the tumor was detected by real-time PCR, GAPDH served as a loading control. Data are expressed as means ± SD. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, and ns means no significance.</p>
Full article ">Figure 6
<p>Schematic diagram illustrating the potential mechanism of DOP@3BCP NP-mediated PDT in CRC.</p>
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