Search Results (425)

The search for substances that increase the immunity of bees is becoming a necessity in the era of various environmental threats and the declining immunocompetence of these insects. Therefore, we tested the biological and physicochemical properties of 7-diethylamino-4-hydroxycoumarin (7DOC). In a cage test, two groups of bees were created: a control group fed with sugar syrup and an experimental group fed with sugar syrup with the addition of 7DOC. In each group, the longevity of the bees was determined and the protein concentrations and antioxidant activities in the bees’ hemolymph were determined. The bees fed with 7DOC lived 2.7 times longer than those in the control group. The protein concentrations and activities of SOD, CAT, GPx and GST, as well as the TAC levels, were significantly higher in the hemolymph of the supplemented workers. To confirm these potent biological properties of 7DOC, the UV-Vis spectra, emission and excitation of fluorescence, synchronous spectra and finally the fluorescence lifetimes of this compound were measured using the time-correlated single photon counting method, in various environments differing in polarity and in the environment applied in bee research. This compound was shown to be sensitive to changes in solvent polarity. The spectroscopic assays were complemented with crystallographic tests of the obtained monocrystals of the aforementioned compounds, which attested to the aggregation effects observed in the spectra measurements for the selected coumarin. The research results confirm that this compound has the potential to be implemented in apiary management, which will be our application goal, but further research into apiary conditions is required. Full article

Background/Objectives: Radiotherapy (RT) is a mainstay of clinical cancer therapy that causes broad immune responses. The complement system is a pivotal effector mechanism in the innate immune response, but the impact of RT is less well understood. This study investigates the interaction between RT and the complement system as a possible approach to improve immune responses in cancer treatment. Methods: Human solid cancer (lung, prostate, liver, breast cancer), lymphoma, and leukemia cells were irradiated using X-rays and treated with polyclonal antibodies or anti-CD20 monoclonal antibodies, respectively. Chromium release assay was applied to measure cell lysis after radiation with or without complement-activating antibody treatment. The expression of membrane-bound complement regulatory proteins (mCRPs; CD46, CD55, CD59), which confer resistance against complement activation, CD20 expression, apoptosis, and radiation-induced DNA double-strand breaks (γH2AX), was measured by flow cytometry. The radiosensitivity of tumor cells was assessed by colony-forming assay. Results: We demonstrate that RT profoundly impacts complement function by upregulating the expression of membrane-bound complement regulatory proteins (mCRPs) on tumor cells in a dose- and time-dependent manner. Impaired complement-mediated tumor cell lysis could thus potentially contribute to radiotherapeutic resistance. We also observed RT-induced upregulation of CD20 expression on lymphoma and leukemic cells. Notably, complement activation prior to RT proved more effective in inducing RT-dependent early apoptosis compared to post-irradiation treatment. While complement modulation does not significantly alter RT-induced DNA-damage repair mechanisms or intrinsic radiosensitivity in cancer cells, our results suggest that combining RT with complement-based anti-cancer therapy may enhance complement-dependent cytotoxicity (CDC) and apoptosis in tumor cells. Conclusions: This study sheds light on the complex interplay between RT and the complement system, offering insights into potential novel combinatorial therapeutic strategies and a potential sequential structure for certain tumor types. Full article

Background/Objectives: Neuropilin-1 (NRP1) is a key co-receptor for SARS-CoV-2, complementing the ACE2 receptor. Several investigations have documented highly conserved sequences in this receptor, supporting the implication of NRP1 as a key mediator in SARS-CoV-2 cellular entry mechanisms. Methods: To investigate this hypothesis, we examined 104,737 SARS-CoV-2 genome fastas from GISAID genomic data, corresponding to isolates collected between 2020 and 2025 in Mexico. Specifically, we focused on the RRAR motif, a known furin-binding site for NRP-1 and the binding site for ACE2 with the spike protein. Our analysis revealed high conservation (>98%) of the RRAR domain compared to a rapidly diminishing ACE2-binding domain. A complementary analysis, using Data from Gene Expression Omnibus (GEO, GSE150316), showed that NRP1 expression in lung tissue remains relatively stable, whereas ACE2 displayed high inter-individual variability and lower abundance compared to NRP1. Based on this evidence, we designed two humans–rats NRP1 siRNAs that were tested in vivo using a melittin-induced lung injury model. Results: The RT-PCR assays confirmed an effective NRP1 knockdown, and the siRNA-treated group showed a significant reduction in the lesions severity. These findings highlight NRP1 as a stable and relevant therapeutic target and suggest the protective potential of siRNA-mediated gene silencing. Conclusions: The evidence presented here supports the rational design of NRP1-directed therapies for multiple circulating SARS-CoV-2 variants in Mexico. Full article

Drought stress poses a major constraint on global crop productivity. Although aspartic proteases (APs) are primarily characterized in plant disease resistance, their roles in abiotic stress adaptation remain largely unexplored. Here, we demonstrate that rice (Oryza sativa) OsAP4 critically regulates drought stress tolerance at the seedling stage. Genetic manipulation through overexpression (OsAP4-OE) or CRISPR knockout (OsAP4-KO) resulted in significantly reduced or enhanced stress tolerance compared to wild-type plants, respectively. Through integrated approaches including yeast two-hybrid, bimolecular fluorescence complementation, pull-down, co-immunoprecipitation, and protein degradation assays, we established that OsAP4 physically interacts with and destabilizes OsCATA/OsCATC, two catalase enzymes responsible for reactive oxygen species (ROS) scavenging. Importantly, OsAP4 modulates ROS production under drought stress treatment conditions. Together, these findings reveal a novel OsAP4-OsCATA/OsCATC regulatory module governing rice drought stress responses. Full article

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea. Preventative vaccines or novel treatments based on a better understanding of the molecular basis of N. gonorrhoeae infection are required as resistance to current antibiotics is widespread. Toxin–antitoxin (TA) systems modulate bacterial physiology by interfering with vital cellular processes; type II TA systems, where both toxin and antitoxin are proteins, are the best-studied. Bioinformatics analysis revealed genes encoding an uncharacterized type II HicAB TA system in the N. gonorrhoeae strain FA1090 chromosome, which were also present in >83% of the other gonococcal genome sequences examined. Gonococcal HicA overproduction inhibited bacterial growth in Escherichia coli, an effect that could be counteracted by the co-expression of HicB. Kill/rescue assays showed that this effect was bacteriostatic rather than bactericidal. The site-directed mutagenesis of key histidine and glycine residues (Gly22, His24, His29) abolished HicA-mediated growth arrest. N. gonorrhoeae FA1090∆hicAB and complemented derivatives that expressed IPTG-inducible hicA, hicB, or hicAB, respectively, grew as wild type, except for IPTG-induced FA1090∆hicAB::hicA. RT-PCR demonstrated that hicAB are transcribed in vitro under the culture conditions used. The deletion of hicAB had no effect on biofilm formation. Our study describes the first characterization of a HicAB TA system in N. gonorrhoeae. Full article

Background/Objectives: Type 2 diabetes mellitus (T2DM) is a prevalent metabolic disease characterized by chronic hyperglycemia and progressive microvascular complications, including retinopathy, nephropathy, and neuropathy. While traditional markers like HbA1c capture average glycemic control, they often fail to predict microvascular damage risk. Glycated CD59 (GCD59), a complement regulatory protein modified under hyperglycemic conditions, has emerged as a promising biomarker reflecting complement dysregulation and endothelial injury. This study aimed to examine the relationship between plasma GCD59 levels and the presence of microvascular complications in patients with type 2 diabetes mellitus and to evaluate whether GCD59 shows potential for future use as a predictive biomarker, pending prospective validation. Methods: In this single-center, prospective case–control study, 246 participants were enrolled: 82 healthy controls, 82 T2DM patients without microvascular complications (DM − MC), and 82 T2DM patients with microvascular complications (DM + MC). Microvascular complications were defined based on standardized criteria for retinopathy, nephropathy, and neuropathy. Plasma GCD59 levels were measured using validated ELISA methods. Receiver operating characteristic (ROC) analyses, forest plots, and odds ratio calculations were employed to assess the discriminatory performance of GCD59. Statistical significance was set at p < 0.05. Results: Plasma GCD59 levels were significantly elevated across all diabetic groups compared to healthy controls (p < 0.001), with the highest levels in the DM + MC group (median 4.5 ng/mL) versus DM − MC (median 1.9 ng/mL) and controls (median 1.2 ng/mL). ROC analysis demonstrated excellent diagnostic performance for distinguishing DM + MC from healthy controls (AUC = 0.946, sensitivity 89%, specificity 97.6%) and good performance for distinguishing DM + MC from DM − MC (AUC = 0.849, sensitivity 72%, specificity 87.8%). Forest plot analyses confirmed significantly elevated odds ratios for GCD59 across all microvascular subgroups. Importantly, GCD59 levels correlated positively with inflammatory markers (CRP, ESR, leukocyte count), suggesting a combined role of complement dysregulation and chronic inflammation in diabetic microangiopathy. Conclusions: Plasma GCD59 may be a promising biomarker for identifying T2DM patients who may be at increased risk for microvascular complications, independent of conventional glycemic markers. Given the cross-sectional design of this study, causal inference is not possible; prospective validation is required. The observed strong discriminatory performance highlights potential future clinical utility, pending further validation of diagnostic thresholds, assay standardization, and feasibility in routine care settings. Full article

BEL1-like homeodomain protein 3 (BLH3) plays a crucial role in plant development. However, its involvement in the salt stress response has not been studied. In this study, we investigated the molecular mechanism underlying the response of LpBLH3 to salt stress in Lilium pumilum (L. pumilum) using various techniques, including quantitative PCR (RT-qPCR), determination of physiological indices of plant after Saline-Alkali stress, yeast two-hybrid screening, luciferase complementation imaging (LCI), and chromosome walking to obtain the promoter sequence, analyzed by PlantCARE, electrophoretic mobility shift assay (EMSA), and then dual-luciferase reporter assay(LUC). RT-qPCR analysis revealed that LpBLH3 is most highly expressed in the leaves of L. pumilum. The expression of LpBLH3 peaks at 24 or 36 h in the leaves under different saline stress. Under various treatments, compared to the wild type (WT), the LpBLH3 overexpression lines exhibited less chlorosis and leaf curling and stronger photosynthesis. The overexpression of LpBLH3 can enhance lignin accumulation in root and stem by positively modulating the expression of crucial genes within the lignin biosynthesis pathway. Y2H and LCI analyses demonstrated that LpBLH3 interacts with LpKNAT3. Additionally, EMSA and LUC analyses confirmed that LpBLH3 can bind to the promoter of LpABI5 and upregulate the expression of ABI5 downstream genes (LpCAT1/LpATEM/LpRD29B). In summary, LpBLH3 enhances the plant’s salt tolerance through the ABA pathway and lignin synthesis. This study can enrich the functional network of the BLH transcription factor family, obtain Lilium pumilum lines with good saline-alkali resistance, expand the planting area of Lilium pumilum, and improve its medicinal and ornamental values. Additionally, the functional analysis of the BLH transcription factor family provides new insights into how crops adapt to the extreme growth environment of saline-alkali soils. Full article

The aim of this study was to investigate the biological relevance of predicted sites involved in protein–protein interaction formation by bHLH transcription factors associated with gynoecium development in Arabidopsis (Arabidopsis thaliana). We used AlphaFold2 to generate three-dimensional protein structures of the bHLH proteins SPATULA (SPT), HECATE1 (HEC1), and INDEHISCENT (IND). These structures were subjected to molecular docking using the HawkDock server, enabling the identification of potential interaction sites. PCR-based site-directed mutagenesis was used to modify the predicted interaction sites, followed by testing for protein–protein interaction formation using Bimolecular Fluorescence Complementation (BiFC) assays. Furthermore, these modified versions were overexpressed in Arabidopsis to observe whether gynoecium and fruit development would be affected. BiFC assays with the modified versions revealed a complete loss of the SPT-HEC1 interaction and a strong reduction in the SPT-IND interaction. The overexpression experiments in Arabidopsis showed that the 35S::SPT-4A line exhibited strong phenotypes in the development of the medial tissues of the gynoecium, resulting in reduced seed number and shorter fruits. In the 35S::HEC1-2A line, a reduced seed number and shorter fruits were also observed, but no other obvious defects were observed. Finally, the 35S::IND-3A line was less affected than the 35S::IND line. In the latter, medial tissue development was strongly affected, while in the 35S::IND-3A line, it was only slightly affected; however, a reduced seed number and shorter fruits were observed. In summary, the predicted interaction sites are relevant and, when modified, affect gynoecium development in Arabidopsis. The findings demonstrate that predictive computational tools represent a viable strategy for a deeper understanding of protein–protein interactions. Full article

Background/Objectives: The complement system (particularly C5b-9) is an instrumental part of the induction and progression of atherosclerosis. The fluid phase C5b-9, also known as soluble C5b-9 (sC5b-9), is a reliable indicator of terminal complement pathway activation. Response Gene to Complement (RGC)-32 is a C5b-9 effector involved in cell cycle regulation and differentiation, immunity, tumorigenesis, obesity, and vascular lesion formation. RGC-32 regulates the expression of Sirtuin1 (SIRT1), known to delay vascular aging. The aim of this study was to assess the levels of sC5b-9, RGC-32, and SIRT1 in patients with atherosclerotic chronic and acute ischemic coronary syndromes. Methods: We determined the levels of sC5b-9, serum RGC-32, and SIRT1 by enzyme-linked immunosorbent assays (ELISAs) in 41 patients with chronic atherosclerotic coronary syndromes, 36 patients with acute ischemic coronary syndromes, and 21 asymptomatic controls with no history of ischemic heart disease. Results: sC5b-9 was significantly higher in patients with acute coronary syndrome as compared to the control group (p = 0.020, AUC = 0.702). In chronic coronary ischemia patients, serum RGC-32 was correlated with the extension of coronagraphically visualized atherosclerotic lesions (r = 0.352, p = 0.035) as well as with sC5b-9 levels (r = 0.350, p = 0.025). RGC-32 concentration was significantly lower in patients with acute coronary syndrome than in the control group (p = 0.020). We also observed significantly lower serum SIRT1 concentrations in patients with chronic ischemic heart disease than in the control group (p = 0.025). Conclusions: sC5b-9 may function as a possible biomarker for myocardial tissue damage in acute coronary syndrome. In acute coronary syndrome settings, low levels of RGC-32 may indicate a protective, antifibrotic function of RGC-32 in the ischemia-damaged myocardium; however, in stable chronic disease, RGC-32 serum values appear to correlate with the extent of atherosclerotic lesions, suggesting a pro-atherogenic role for RGC-32. Chronic myocardial ischemia decreases SIRT1 protein levels in serum, which underscores the use of SIRT1-modulating drugs in these patients. Full article

The cap-binding protein complex (CBC), comprising Cbp20 and Cbp80, is crucial for gene expression, yet its role in the notorious crop pathogen Botrytis cinerea remains unclear. Immunoprecipitation coupled with LC-MS/MS demonstrated that BcCbp20 interacts with BcCbp80. Yeast two-hybrid, GST pull-down, and Split-luciferase complementation assays confirmed that the conserved RNA recognition motif (RRM, 54–127 aa) of BcCbp20 and the N-terminal MIF4G domain (1–370 aa, 1–577 aa) of BcCbp80 constitute the core interaction regions. Genetic transformation experiments revealed that BcCBP80 exerts a more dominant role than BcCBP20 in regulating hyphal morphology, growth rate, conidiophore development, and conidial yield. Furthermore, BcCBP20 and BcCBP80 differentially regulate sclerotium formation to maintain sclerotial quantity. Based on pathogenicity assays, BcCBP80 associated with infection cushion development, with this phenotypic alteration possibly being among the factors correlated with altered pathogenicity. However, the increased sensitivity of ΔBccbp20 to various stress factors may be the primary reason for the diminished pathogenicity. Taken together, these results indicate that BcCBP20 and BcCBP80 play important roles in multiple aspects of B. cinerea growth, development, stress response, and pathogenicity. Full article

E2F transcription factors regulate cell cycle progression by influencing the expression of proteins required for the G₁-S phase transition and DNA synthesis with its heterodimeric partners (DP1 or DP2). The dimerization domain is the E2Fs and DP1 protein interaction interface and is believed to function in protein dimerization. In this study, eight E2F transcription factors (PcE2F1–8) of large yellow croaker Pseudosciaena crocea and one dimerization partner (PcDP1) are identified in the genome of large yellow croakers. The prediction of E2Fs conserved domains revealed that PcE2F1–6 has one DNA-binding domain (DBD) and one dimerization-binding domain (DD), while PcE2F7–8 only possess two duplicate DBDs but not DD, indicating that E2F7–8 cannot form the E2F/DP1 heterodimer. To explore whether PcDP1 is a partner of PcE2F1–6, the ORF of PcE2F1–6 was cloned. Subsequently, its sequence characteristics, the expression pattern in healthy fish, and subcellular co-localization were analyzed, and an interaction between PcDP1 and PcE2F1–6 were detected directly by yeast two-hybrid and BiFC. The PcE2F1, PcE2F2, PcE2F3, PcE2F4, PcE2F5, and PcE2F6 genes encode a protein of 454, 448, 444, 392, 362, and 396 amino acids, respectively, with accession numbers QFZ93593.1, QFZ93594.1, QFZ93595.1, QFZ93596.1, QFZ93597.1, and QFZ93598.1, respectively. Sequence characteristics analysis found that PcE2F1–5 but not PcE2F6 proteins share the pocket protein-binding domain sequestering in dimerization domains and transactivation domains. The PcE2F1,2,4 proteins possess one nuclear localization signal (NLS), and PcE2F3 protein possess two NLSs, but there is no NLS in PcE2F5 and 6 protein. Moreover, PcE2F4 also contains one NES. However, PcE2F1–6 proteins were all located in nucleus by using Euk-mPloc 2.0 programs and were confirmed by performing the Cherry and EGFP reporter assay. Regarding co-expression of DP1, only E2F4 can transfer DP1’s subcellular location from cytoplasm to the nucleus. RT-qPCR analysis indicated that PcE2F1–6 are constitutively and tissue specifically expressed in all of the tissues tested of a healthy large yellow croaker. The PcE2F1–6, except for PcE2F3, mRNA levels were all detected higher in the liver. PcE2F1–4 were also highly specifically expressed in the kidney, PcE2F4,6 in the brain, and PcE2F5 in the spleen of a healthy large yellow croaker, respectively. Using a yeast two-hybrid system, PcE2F4 interacting with PcDP1 was identified. The interaction between PcE2F4 and PcDP1 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Collectively, these results indicate that an interaction between PcE2F4 and PcDP1 was detected, which may form heterodimer E2F4/DP1 to regulate cell cycles and immune-related pathways in large yellow croakers. Full article

CRISPR-Cas proteins from bacteria are powerful tools for gene editing and molecular diagnostics. Expanding capacity of CRISPR to low cost, multiplexed assays of biomarkers is a key to future disease diagnostics, since multiple biomarker detection is essential for reliable diagnostics. Herein we describe a multiplexed assay in a 3D-printed 96-well plate with CRISPR-Cas13a immobilized in each well to target three circulating blood biomarker microRNAs (miRNAs 34c-5p, 200c-3p, and 30e-5p) for Alzheimer’s disease (ALZ). Immobilized Cas13a is equipped with different crRNAs complementary to each miRNA target. MiRNA binding to crRNA complements activates the collateral RNase activity of Cas13a, cleaving a quenched fluorescent reporter (RNaseAlert) with fluorophore and quencher connected by an RNA oligonucleotide to enable fluorescence measurements. We achieved ultralow limits of detection (LOD) of 0.74 fg/mL for miRNA 34c-5p, 0.70 fg/mL for miRNA 30e-5p, and 7.4 fg/mL for miRNA 200c-3p, with dynamic ranges from LODs up to about 1800 pg/mL. The accuracy of the assay was validated by spike-recovery studies and good correlation of levels of patient plasma samples vs. a referee method. This new approach provides selective, sensitive multiplex miRNA biosensing, and simultaneously accommodates analysis of standards and controls. Full article

In plants, ferric-chelate reductase (FRO) plays a critical role in mediating extracellular iron (Fe) reduction, a process essential for cellular Fe homeostasis and abiotic stress tolerance. However, the biological functions and regulatory mechanisms of FRO proteins in fruit crops remain poorly characterized. Here, six VvFRO genes were identified in the table grape cultivar ‘Yanhong’. Transcriptional analysis revealed that root expression of these genes was mainly induced under Fe deficiency, Fe depletion, NaCl stress, and PEG-induced drought stress, respectively, but remained unchanged by low temperature (4 °C) or heat treatment (45 °C). Among them, VvFRO3 exhibited the highest constitutive expression, predominantly in leaves, and was significantly up-regulated under Fe deficiency, Fe depletion, or NaCl treatment. Functional complementation assays demonstrated that heterologous overexpression of VvFRO3 in the Arabidopsis thaliana fro2 knockout mutant rescued its growth retardation phenotype, particularly under Fe-deficient conditions. This study advances our understanding of Fe uptake, transport, and homeostasis mechanisms in perennial fruit crops. Full article

Cases of new COVID-19 infection, which manifested in 2019 and caused a global socioeconomic crisis, still continue to be registered worldwide. The high mutational activity of SARS-CoV-2 leads to the emergence of new antigenic variants of the virus, which significantly reduces the effectiveness of COVID-19 vaccines, as well as the sensitivity of diagnostic test systems based on variable viral antigens. These problems may be solved by focusing on highly conserved coronavirus antigens, for example nucleocapsid (N) protein, which is actively expressed by coronavirus-infected cells and serves as a target for the production of virus-specific antibodies and T cell responses. It is known that anti-N antibodies are non-neutralizing, but their protective potential and functional activity are not sufficiently studied. Here, the protective effect of anti-N antibodies was studied in Syrian hamsters passively immunized with polyclonal sera raised to N(B.1) recombinant protein. The animals were infected with 10⁵ or 10⁴ TCID₅₀ of SARS-CoV-2 (B.1, Wuhan or BA.2.86.1.1.18, Omicron) 6 h after serum passive transfer, and protection was assessed by weight loss, clinical manifestation of disease, viral titers in the respiratory tract, as well as by the histopathological evaluation of lung tissues. The functional activity of anti-N(B.1) antibodies was evaluated by complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC) assays. The protection of anti-N antibodies was evident only against a lower dose of SARS-CoV-2 (B.1) challenge, whereas almost no protection was revealed against BA.2.86.1.1.18 variant. Anti-N(B.1) monoclonal antibodies were able to stimulate both CDC and ADCC. Thus, anti-N(B.1) antibodies possess protective activity against homologous challenge infection, which is possibly mediated by innate Fc-mediated immune reactions. These data may be informative for the development of N-based broadly protective COVID-19 vaccines. Full article

We used the data from a successful therapeutic assay that used icatibant in patients with hypoxemic COVID-19 pneumonia (the ICAT·COVID trial) to explore pathophysiological mechanisms. We performed concurrent-type, criterion-related validity analyses to assess the discriminative ability of a panel of nine potential serum markers (interleukin 6, ferritin, lactate dehydrogenase, C reactive protein, fibrin fragment D (D-dimer), complement 1 esterase inhibitor (antigenic and functional), complement 4 factor, and lymphocyte count) to predict the clinical milestones. Consistent with previous research, we evidenced a significant relationship between interleukin 6, lactate dehydrogenase and the lymphocyte count, and the clinical events. Furthermore, exposure to icatibant, a bradykinin B2 receptor antagonist (which improved pneumonia and mortality in the aforementioned randomised trial), attenuated this relationship, although this effect faded over time. The results reinforce the key role that the angiotensin-converting enzyme 2 has on COVID-19 pathophysiology as a point of convergence between the renin–angiotensin and kallikrein–kinin systems. This was shown clinically by the successful blocking of inflammatory pathways by icatibant at the bradykinin effector loop level early during the acute hyperinflammatory stage of the disease. Full article