Search Results (309)

Coffea canephora cultivation areas in Brazil are frequently exposed to successive cycles of water deficit, triggering plant stress responses. In addition to water deficit, increased air temperature can act as a second stress factor. The recurrence of these stress factors may induce plant tolerance mechanisms, potentially mitigating future stress responses even of a different stress nature. We hypothesized that repeated cycles of water deficit can trigger tolerance mechanisms that make C. canephora leaves more resilient to supra-optimal temperatures. To test this hypothesis, young C. canephora plants were grown under non-limited water conditions for seven months (Ψ_mSoil > −20 kPa), after which they were subjected to two consecutive cycles of water deficit (Ψ_mSoil < −300 kPa), followed by rehydration. Two clones were used, ‘A1’ and ‘3V’, previously classified as drought sensitive and tolerant, respectively, considering the dynamics of physiological and architectural responses. After the second cycle, leaf discs were collected from completely expanded leaves formed during the two stress cycles and exposed to heat treatments (35 °C, 40 °C, 45 °C, 50 °C, and 55 °C) for 15 min in a water bath. Chlorophyll a fluorescence emission was then monitored, and the results were analyzed using OJIP transient kinetics and the JIP_Test. High temperatures induced negative changes in both OJIP kinetics and JIP_Test-derived parameters. A significant increase in F₀ and a reduction in F_M were observed mainly at 50 °C and 55 °C, due to changes in the stages of the OJIP curve. These changes impacted the “energy connectivity” and consequently the electron transport along the electron transfer chain (ETC), increasing energy dissipation, as confirmed by the JIP_Test variables. Despite the high temperature impacts, previous water deficit induced heat tolerance in clone ‘A1’, while it increased sensitivity in clone ‘3V’. This study suggests that selecting drought-resistant varieties should consider their subsequent response to short high-temperature stress to avoid cross-sensitivity caused by selecting for a single environmental factor. Full article

Milk is a primary source of vital nutrients and bioactive components fundamental to the growth and development of both newborn animals and humans. Produced by economically significant livestock species (including cattle, buffaloes, goats, sheep and camels), milk is a complex matrix rich in caseins, vitamins, fats, and proteins. Beyond its classical nutritional profile, milk serves as a pivotal vehicle for milk-derived extracellular vesicles (mEVs). These specialized food-derived EVs (fEVs) exert pleiotropic effects that resonate with the One Health paradigm, linking animal well-being and human nutrition to broader ecosystem stability. mEVs offer unique advantages, such as high biocompatibility and gastrointestinal stability, also rendering them potential therapeutic tools as drug delivery systems. However, challenges remain regarding the standardization of mEVs and the variability of their molecular cargo. This review provides a comprehensive comparative analysis of mEVs across a diverse taxonomic range, including bovines, water buffaloes, yaks, camels, goats, pigs, horses, donkeys, and humans, highlighting their distinct functional signatures. Indeed, a critical issue in mEV research is the isolation process: recommendations to minimize contamination from milk fat globules and casein micelles (which can cover EV signals) are given. Finally, current detection methods and instrumentation, with a specific focus on advancing flow cytometry (FC) approaches are discussed. Key insights include the use of conventional FC (with fluorescence triggering, the necessity of rigorous controls and calibration, and the utility of bead-based assays to overcome resolution limits) and imaging flow cytometry (IFC). In both technical approaches, the application of different EV generic fluorescent markers and the strategic selection of tetraspanins (i.e., CD9, CD63, CD81), is mandatory: we emphasize that selecting the correct antibody clones and accounting for inter-species cross-reactivity are essential steps for ensuring the accuracy and reproducibility of mEV research across mammalian species. Full article

Norovirus is a major cause of acute viral gastroenteritis in humans. Molecular biology-based detection methods play a pivotal role in ensuring accurate and specific diagnosis. The inclusion of Qβ phage particles as armored positive controls in these assays can further enhance their reliability and specificity. Herein, we discuss rational design strategies to improve the stability of Qβ bacteriophage capsid proteins armored with RNA using Discovery Studio 2019 protein design software. Amino acid mutation sites were deter-mined based on changes in folding free energy differences (ΔΔGmut). These single-site mutations were subsequently evaluated using molecular dynamics simulations. Wild-type and mutant recombinant expression plasmids were constructed and transformed into Escherichia coli BL21 (DE3) for cloning and expression. The stability of Qβ virus-like particles (VLPs) was assessed using real-time fluorescence RT-qPCR. The results showed that structurally intact and uniformly distributed wild-type and single-site mutant VLPs were successfully obtained. Stability analyses indicated that at 4 °C, 25 °C, 37 °C, 45 °C, and 60 °C, the single-site mutant exhibited a significantly lower rate of degradation than the wild-type. In conclusion, rational design enables the generation of single-site mutant VLPs with enhanced stability, providing a safer and more stable standard reference material for the molecular detection of foodborne viruses. Full article

Rhododendrons naturally inhabit acidic soils where aluminum (Al) toxicity severely restricts plant growth, yet the molecular basis underlying cultivar-dependent differences in Al tolerance remains poorly understood. In this study, we compared the transcriptional and physiological responses of an Al-resistant cultivar (Kangnaixin) and an Al-sensitive cultivar (Baijinpao) under Al stress. Transcriptome analysis was performed to identify Al-responsive differentially expressed genes (DEGs), followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to elucidate functional categories and metabolic pathways involved in stress adaptation. In addition, the Al tolerance-related gene RsALS3 was cloned and functionally characterized through heterologous overexpression in Arabidopsis thaliana. The two cultivars exhibited distinct transcriptional profiles in response to Al stress, with DEGs significantly enriched in abiotic stress responses, membrane-associated functions, and key metabolic pathways, including starch and sucrose metabolism, phenylpropanoid and flavonoid biosynthesis, and photosynthesis-related processes. These results suggest that Al stress disrupts membrane integrity and alters carbon metabolism in Rhododendron. Functional validation demonstrated that RsALS3 overexpression moderately alleviated Al-induced toxicity in A. thaliana, as evidenced by reduced leaf damage and improved photosynthetic efficiency. Although the observed phenotypic differences were modest, and some chlorophyll fluorescence kinetics data did not reach strong statistical significance. The overall physiological trends support a potential role of RsALS3 in Al stress adaptation. Collectively, these findings provide insight into cultivar-specific Al stress responses in Rhododendron and identify RsALS3 as a promising candidate gene for further investigation aimed at improving adaptation to acidic soils. Full article

Background: Dendritic cells (DCs) are heterogeneous antigen-presenting cells that bridge innate and adaptive immunity. Recent classifications of hematolymphoid neoplasms highlight the complex origins of DC-related neoplasms. DCs have also been associated with the progression of multiple myeloma (MM). This report presents the case of a patient with MM in whom bone marrow analysis revealed an unusual additional clonal population of immature cells, in addition to plasmacytoid DCs, that later evolved into plasmacytoid dendritic cell proliferation associated with acute myeloid leukemia (pDC-AML). Methods: The bone marrow of a 69-year-old man with neutropenia and thrombocytopenia was examined by morphology, immunohistochemistry, flow cytometry, cytogenetics, fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS). Serial assessments were performed before and during treatment with bortezomib and dexamethasone for MM, and later with daunorubicin/cytarabine for AML. Results: Initial bone marrow analysis revealed coexisting clonal plasma cells with t(11;14) and a population of CD34+/CD123+/CD45RA+ cells lacking lineage markers, in addition to pDCs, suggestive of precursor DCs rather than acute undifferentiated leukemia. Cytogenetic analysis identified a small clone with isolated del(20q), which corresponded in size to the clone of undifferentiated cells and to the clone with pathogenic variants detected by NGS in the BCOR, RUNX1, and SRSF2 genes. Myeloma therapy decreased both MM and undifferentiated cells; however, within four months, pDC-AML evolved with del(20q) and higher variant allele frequencies of the previously detected gene variants. Remission was achieved with standard AML chemotherapy. Conclusions: This case supports evidence that MM-associated immune dysfunction and bone marrow niche alterations may promote secondary myeloid malignancies independently of cytotoxic therapy. It demonstrates the earliest events in pDC-AML evolution. Furthermore, the immature immunophenotype raises the question of appropriate treatment, since a diagnosis of acute undifferentiated leukemia can be established. Full article

Background. Routine lymphocyte counting requires quality assurance validation using quality controls (QCs) that closely resemble fresh human blood leukocytes. This study aimed to evaluate a novel artificial product designed to mimic leukocyte light scatters and marker expression. Methods. FlowCytes and TruCytes, “artificial cell mimics” (Slingshot Biosciences, Emeryville, CA, USA), were tested on CE-IVD-certified systems, namely FACSCanto, FACSLyric (BD Biosciences), Navios, and DxFlex (Beckman Coulter), using routine staining, lysing, fixation, and no-wash procedures for T, B, and NK counting. Results. FlowCytes and TruCytes provided forward and side scatter profiles comparable to human leukocytes on the FACSCanto, FACSLyric, and DxFlex systems but not on Navios despite adapting the process to be slightly different from the manufacturer’s recommendations. TruCytes demonstrated robust immunolabeling of CD3, CD4, CD8, and CD19 on the FACSCanto, FACSLyric, and DxFlex systems, with fluorescence intensities and subset distributions being similar to those usually observed in fresh human blood. However, CD16 and CD56 labeling was inconsistent and depended on the antibody clones used. Regrettably, monocyte and granulocyte mimics lacked expression of CD4, CD16, and CD14. TruCytes also displayed significantly lower concentrations of TBNK lymphocyte subsets compared to healthy human blood. Conclusions. FlowCytes and TruCytes show promises as internal quality controls for T cell and B cell immunophenotyping, but not NK cells. They are compatible with most CE-IVD cytometers, even when using lysis/fixation/no-wash routine diagnosis procedures. Further multicentric studies are warranted to assess their performance relative to existing products, such as stabilized human blood. Full article

RNA interference (RNAi) is a crucial antiviral defense mechanism in plants, where Argonaute (AGO) proteins play a central role. However, the function of AGO proteins in the interaction between Soybean mosaic virus (SMV) and Nicotiana benthamiana remains unclear. In this study, SMV pathogenicity was confirmed using an SMV-GFP infectious clone, with typical symptoms and systemic GFP fluorescence observed 14 days post-inoculation. Real-time quantitative reverse transcription polymerase chain reaction analysis revealed dynamic regulation of multiple NbAGO genes upon infection. Notably, NbAGO1a, NbAGO1b, and NbAGO2 were significantly upregulated and positively correlated with viral accumulation, suggesting their critical roles in antiviral defense. Based on these findings, these three genes were selected as targets for artificial microRNA (amiRNA) silencing. Three amiRNAs were designed for each gene using the Arabidopsis miR1596 backbone, with the most effective sequences exhibiting silencing efficiencies ranging from 75.2% to 98.1%. A polycistronic tRNA-amiRNA (PTA) cassette was constructed using Golden Gate cloning technology to simultaneously target all three genes. Co-infection assays indicated that the PTA cassette enhanced SMV accumulation more effectively than single amiRNAs, as evidenced by increased GFP fluorescence (49.1–60.5%) and pronounced leaf necrosis. The PTA system downregulated the expression of NbAGO1a, NbAGO1b, and NbAGO2 by 18.4–26.7%. Furthermore, silencing NbAGO2 alone resulted in severe necrosis, underscoring its essential role in this antiviral defense mechanism. This study elucidates the importance of NbAGO1a, NbAGO1b, and NbAGO2 in antiviral immunity and demonstrates the utility of the PTA system for efficient multi-gene silencing, offering valuable insights for developing RNAi-based antiviral strategies. Full article

Prostate cancer tissue usually involves either well formed glands, poorly formed glands or a combination of the two morphologies, which can be correlated with metabolic differences and tumor heterogeneity. This is particularly important for metastatic castration-resistant prostate cancer, where the heterogeneity and metabolic changes drive cancer progression and treatment refractory properties. Sortilin and syndecan-1 expression accurately define the two different morphologies in prostate cancer tissue, are critical to the process of metabolic regulation, and exhibit mechanistic/functional interactions during prostate cancer progression. As trans-membrane proteins that recycle from endocytic compartments to the cell surface, sortilin and syndecan-1 are attractive targets for therapeutic intervention that address the two major forms of prostate cancer. In this study, we describe an antibody-drug conjugate (ADC) strategy that utilizes monoclonal antibodies which bind to specific extracellular domains of these integral membrane proteins to elicit anticancer activity in prostate cancer cell lines. Anti-sortilin (clone 11H8) and anti-syndecan-1 (clone 6D11) monoclonal antibodies demonstrated high specificity for epitopes on the extracellular, N-terminal domains of these respective proteins and were effectively internalized into prostate cancer cell endocytic compartments. Monomethyl aurastatin E (MMAE)-conjugated ADCs exhibited low nanomolar cytotoxicity in LNCaP and PC-3 prostate cancer cells. Mechanistically, 11H8-MMAE and 6D11-MMAE triggered cytotoxicity and morphological alterations in androgen-sensitive and androgen-insensitive cells. However, the uptake of fluorescent labelled 11H8 and 6D11 antibodies appeared to be high, whereas the killing capacity of the MMAE-conjugated antibodies was less impressive, suggesting the need for further ADC development. These promising proof-of-concept ADCs are designed to exploit molecular and metabolic vulnerabilities in prostate cancer and may have utility for overcoming treatment resistance by simultaneously targeting different forms of the cancer. Full article

Cacti of the genera Opuntia and Nopalea exhibit morphophysiological and biochemical characteristics that favor their adaptation to semiarid environments, such as crassulacean acid metabolism (CAM) and cladode succulence. These strategies reduce water loss and allow the maintenance of photosynthesis under stress conditions. In this study, we evaluated the seasonal variation in the physiological and photochemical responses of forage cactus clones grown in semiarid environments, considering the rainy, dry, and transition seasons. The net photosynthetic rate (P_n) and chlorophyll fluorescence parameters varied significantly as a function of water availability and microclimatic conditions. We found higher CO₂ assimilation rates during the rainy season, while the dry season resulted in a strong impairment of photosynthetic activity, with reductions of 65% in stomatal conductance, 37% in transpiration, 20% in maximum quantum efficiency of photosystem II, and 19% in the electron transport rate. Furthermore, during these periods, we observed an increase in initial fluorescence and non-photochemical dissipation, demonstrating the activation of photoprotective mechanisms against excess light energy. During the transition seasons, the cacti exhibited rapid adjustments in gas exchange and energy dissipation, indicating the adaptive plasticity of CAM pathway. The MIU (Nopalea cochenillifera (L.) Salm-Dyck), OEM (Opuntia stricta (Haw.) Haw.), and IPA (Nopalea cochenillifera (L.) Salm-Dyck) clones demonstrated greater resilience, maintaining greater stability in P_n, instantaneous water use efficiency, and photochemical parameters during the drought. In contrast, the OEA (Opuntia undulata Griffiths) clone showed high sensitivity to water and heat stress, with marked reductions in physiological and photochemical performance. In summary, the photosynthetic efficiency and chlorophyll fluorescence of CAM plants result from the interaction between water availability, air temperature, radiation, and genotypic traits. This study provides a new scientific basis for exploring the effects of environmental conditions on the carbon and biochemical metabolism of cacti grown in a semiarid environment. Full article

In this study, we successfully cloned the fluorescent proteins eGFP and DsRed in-frame with the antimicrobial peptide epinecidin-1 (FIFHIIKGLFHAGKMIHGLV) at the N-terminal. The cloning strategy involved inserting the fluorescent reporters into the expression vector, followed by screening for positive clones through visual fluorescence detection and molecular validation. The visually identified fluorescent colonies were confirmed as positive by PCR and plasmid migration assays, indicating successful cloning. This fusion of fluorescent reporters with a short antimicrobial peptide enables real-time visualization and monitoring of the peptide’s mechanism of action on membranes and within cells, both in vivo and in vitro. The fusion of eGFP and DsRed to epinecidin-1 did not impair the expression or fluorescence of the reporter protein. Full article

Gynaephora qinghaiensis is a major grassland pest common in the alpine meadows of the western plateau of China, and its biological behavior is affected by the synergy of a variety of chemicals in the environment. OBPs can dissolve and transport odor molecules such as volatile plant compounds through lymphatic fluid, which plays an important olfactory-to-olfactory role. However, the specific function of OBPs in the interaction mechanism between moths and volatile plant compounds is still unknown. The purpose of this study was to analyze the binding characteristics of GqinOBP10 and its volatile plant compounds in moths and to explore its role in the olfactory perception mechanism of moths so as to study the corresponding target ligands and achieve green control. The purified GqinOBP10 was subjected to fluorescence competitive binding to eight ligands. The 3D modeling of GqinOBP10 was carried out by the SWISS-MODEL website, and the molecular docking was carried out by Autodock 4.2.6 software, and the binding of GqinOBP10 to eight ligands was simulated and verified. The results showed that the cloned strain with the full length of GqinOBP10 was cloned. The fluorescence competition binding results showed that GqinOBP10 had strong binding ability to eight volatile plant compounds, among which the binding ability to 2-Amino-1-phenylethanol and 2-Oleoylglycerol was the strongest, and had high binding ability with the other six ligands. The molecular docking results showed that the binding energy of GqinOBP10 and eight odorant molecules was negative, and all of them could form 1~4 hydrogen bond for binding, among which the binding performance with 2-Oleoylglycerol was the best. The findings suggest that dsOBP10 injection leads to a notable decrease in both the expression levels of GqinOBP10 and the antennal potential response in male and female tissues. This indicates that GqinOBP10 is likely crucial for the localization and recognition of host plants in G. qinghaiensis. By silencing GqinOBP10, the olfactory perception of host volatiles is significantly impaired, highlighting the protein’s importance in the caterpillars’ ability to detect and respond to their environment. These insights provide a valuable basis for developing targeted attractants, potentially enhancing pest management strategies by manipulating olfactory cues in these caterpillars. Further research could explore the specific mechanisms by which GqinOBP10 influences olfactory perception and host plant selection. Full article

Aroma is a crucial quality trait in ornamental flowers; however, the molecular mechanisms by which hormones regulate fragrance in Rhododendron remain poorly understood. In this study, Gibberellin (GA₃)-treated petals of Rhododendron fortunei Lindl were used as experimental materials to integrate volatile metabolomics with RNA-seq analysis, aiming to investigate aroma changes and their underlying molecular regulatory mechanisms. We cloned and characterized RfHMGR1, which encodes a key enzyme in the Mevalonate (MVA) pathway, and verified its function. Subcellular localization analysis showed that the Green Fluorescent Protein (GFP) signal of the RfHMGR1-GFP fusion protein was mainly distributed in the cytoplasm. Transient overexpression of RfHMGR1 in petals of two Rhododendron species (R. fortunei and Rhododendron hybrida) significantly increased the accumulation of the terpenoid linalool, whereas gene silencing reduced linalool accumulation. Furthermore, the purified recombinant RfHMGR1 protein exhibited HMGR-specific reductase activity in vitro. Our results confirmed that GA₃ regulates the terpenoid fragrance of R. fortunei by targeting the MVA pathway gene RfHMGR1. Collectively, these findings provide new insights into the fragrance regulation mechanisms in R. fortunei and identify molecular targets for breeding strategies aimed at improving floral scent. Full article

Glucagonoma, a rare neuroendocrine tumor, lacks targeted treatment drugs. Excessive secretion of glucagon is the main cause of its clinical syndrome. To explore targeted therapeutic drugs that can inhibit glucagon secretion and tumor proliferation, we investigated the effect of Trametenolic Acid (TA) on mouse pancreatic alpha TC1 clone 6 (αTC1-6) cells and its regulatory role in the PI3K/AKT signaling pathway. Cell viability of αTC1-6 cells was assessed via the MTT assay. Glucagon content in cell culture supernatants was measured using an Enzyme-Linked Immunosorbent Assay (ELISA). Autophagic vacuoles were visualized through Monodansylcadaverine (MDC) staining. The expression of autophagy-related proteins including Atg7, LC3 Ⅱ and PI3K/AKT signaling pathway-related proteins mTOR and FoxO1 were determined by Western blot. The results showed that the proliferation of αTC1-6 cells was significantly inhibited by TA in a dose- and time-dependent manner, and the IC₅₀ was 140.71, 26.77 and 1.99 μM after treatment of 12, 24, and 48 h, respectively. The secretion of glucagon was significantly inhibited by TA. The MDC staining results showed that the fluorescent labeled autophagic vesicles in the TA group were increased. The Western blot results showed that the expression of Atg7 and LC3 Ⅱ was promoted by TA in a dose-dependent manner, the phosphorylation of PI3K, AKT, mTOR and FoxO1 was significantly inhibited, and the expression of FoxO1 protein was increased. These results demonstrated that TA can inhibit glucagon secretion, induce autophagy, and suppress cell proliferation in αTC1-6 cells. The mechanism may be associated with the PI3K/AKT signaling pathway. Full article

This study reports the construction and validation of a CAFLUX (Chemically Activated Fluorescent Expression) HepG2 reporter cell line engineered to express a histone H2B–green fluorescent protein (H2B–GFP) fusion protein under the control of a dioxin-responsive cytochrome P450 1A1 (CYP1A1) promoter. A lentiviral construct containing a synthetic promoter with multiple dioxin-responsive elements (DREs) upstream of the H2B–EGFP coding sequence was cloned into the pFUGW vector, packaged in human embryonic kidney (HEK) 293FT cells, and used to transduce HepG2 hepatocellular carcinoma cells. Stable clones obtained by limiting dilution were screened for GFP expression in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The resulting CAFLUX HepG2 cells exhibited dose-dependent nuclear GFP fluorescence when exposed to aryl hydrocarbon receptor (AhR) agonists, with limits of detection of approximately 0.01 pM for TCDD and 0.1 pM for benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH). This reporter activity correlated with endogenous CYP1A1 mRNA expression as determined by quantitative polymerase chain reaction (qPCR), confirming that GFP signals reflected native transcriptional responses. In functional assays, curcumin suppressed GFP expression in a concentration-dependent manner and induced apoptotic morphology at higher doses, while extracellular vesicles (EVs) derived from adipose-derived stem cells (ADSCs) significantly reduced both GFP fluorescence and CYP1A1 mRNA levels, suggesting an inhibitory effect on AhR-driven transcription. The CAFLUX HepG2 reporter system therefore provides a sensitive and reproducible platform for real-time, nuclear-localized monitoring of AhR-mediated gene expression. Its responsiveness to both agonists and antagonists underscores its potential utility in toxicological evaluation, drug discovery, and the investigation of EV-mediated signaling in liver cancer models. Full article

Extracellular vesicles (EVs) from mesenchymal stem cells hold therapeutic promise for inflammatory and degenerative diseases; however, limited delivery and targeting capabilities hinder their clinical use. In this study, we sought to enhance the anti-inflammatory and chondroprotective effects of EVs through CAP-LAMP2b (chondrocyte affinity peptide fused to an EV membrane protein) engineering and ADAMTS4 gene editing hybrid vesicle formation. Human umbilical cord MSCs (hUCMSCs) were characterized via morphology, immunophenotyping, and trilineage differentiation. EVs from control and CAP-LAMP2b-transfected hUCMSCs were fused with liposomes carrying CRISPR-Cas9 ADAMTS4 gRNA. DiI-labeled EV uptake was assessed via fluorescence imaging. CAP-LAMP2b was expressed in hUCMSCs and their EVs. EVs exhibited the expected size (~120 nm), morphology, and exosomal markers (CD9, CD63, CD81, HSP70). CAP-modified hybrid EVs significantly enhanced chondrocyte uptake compared to control EVs and liposomes. IL-1β increased ADAMTS4 expression, whereas CAP-LAMP2b-ADAMTS4 EVs, particularly clone SG3, reversed these effects by reducing ADAMTS4 and restoring aggrecan. Western blotting confirmed suppressed ADAMTS4 and elevated aggrecan protein. CAP-LAMP2b-ADAMTS4 EVs, therefore, showed superior uptake and therapeutic efficacy in inflamed chondrocytes, attenuating inflammatory gene expression and preserving matrix integrity. These results support engineered EVs as a promising cell-free approach for cartilage repair and osteoarthritis treatment. Full article