Search Results (677)

Leaves represent an important organ in plant photosynthesis, and moderately rolled leaves would be beneficial in establishing an ideal plant architecture and thereby increasing rice yields. In this study, a stable inherited rolled leaf mutant was obtained via ethyl methanesulfonate (EMS) mutagenesis from japonica variety WYJ27, which was named rll2 (rolling leaf 2). rll2 showed a leaf-rolling phenotype at the seedling stage, which increased with growth. Compared with the wild type, the leaves at all levels of rll2 were significantly shorter and narrower, and the leaf-rolling index gradually decreased from the highest leaf to the third-highest leaf. Semi-thin sections showed that the bulliform cells of rll2 were significantly larger than those of the wild type, and the number of cells was significantly higher than that of the wild type. Genetic analysis showed that rll2 is controlled by a pair of recessive nuclear genes. Map-based cloning revealed that RLL2 encodes a conserved and plant-specific calpain-like cysteine proteinase. RLL2 was mainly expressed in young roots, shoots, spikelets, and panicles. Transcriptome sequencing showed that a total of 104 genes were differentially expressed in the wild type and rll2. Moreover, several transcription factor genes were significantly altered in the rll2 mutant. Taken together, our findings indicate that RLL2 plays an important role in leaf rolling by regulating bulliform cells, which may be useful in breeding rice with an ideal plant architecture. Full article

Due to the high toxicity and widespread distribution of aflatoxin B1 (AFB1), there is significant interest in efficient, safe, and environmentally friendly microbial degradation methods. Rhodococcus opacus PD630 cell-free supernatant (RCFS) shows excellent activity in degrading AFB1, but its active components and mechanisms remain unclear. We assessed the feasibility of ethanol precipitation to enrich active components in RCFS and characterized the ethanol precipitate (RCFSC-EP). Metabolomics and proteomics were used to elucidate the active components, mechanisms, and products of AFB1 degradation by RCFS. The results indicate that ethanol precipitation enriches over 80% of the active components for AFB1 degradation in RCFS. RCFSC-EP exhibits excellent heat resistance, and inhibitors like EDTA-2Na and proteinase K significantly inhibit its activity. Multi-omics analysis suggests that active components in RCFS metabolize AFB1 into six products through four potential pathways, three of which withstand 135 °C for 20 min. The AFB1-degrading activity of RCFS is an intrinsic, constitutive trait of R. opacus PD630 during normal growth. The active components are diverse proteins or enzymes, including glutathione S-transferases, aldo/keto reductase, peroxidases, and carbonyl reductases. This study enriches and reveals the active components, pathways, and products of AFB1 degradation by RCFS, providing a basis for developing RCFS as a biological agent for AFB1 degradation. Full article

Metarhizium anisopliae, an entomopathogenic fungus, can produce four extracellular proteases, subtilisin (Pr1), trypsin (Pr2), metalloproteases (Pr3), and cysteine proteases (Pr4), which are important for pathogenicity of M. anisopliae in target hosts. In order to understand their function in M. anisopliae pathogenicity, third-instar nymphs of Locusta migratoria were fed with a diet containing either conidia of M. anisopliae strain IPPM202 or in combination with one of the four inhibitors (TPCK: tosyl-phenylalanine chloromethyl-ketone, inhibitor of Pr1; EDTA: ethylenediaminetetraacetic acid, inhibitor of Pr3; APMSF: 4-amidinophenyl methanesulfonyl fluoride, inhibitor of Pr2; CI1: cathepsin inhibitor 1, inhibitor of Pr4). The effects on mortality, midgut integrity, and the gut enzymes peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and phenol oxidase (PO) were examined. The results indicated that exposure to IPPM202/TPCK and IPPM202/CI1 caused decreased mortality to L. migratoria with no loss of midgut epithelial cellular integrity. On the other hand, exposure to IPPM202/APMSF or IPPM202/EDTA mixtures resulted in higher mortality similar to PPM202, with severely damaged epithelial gut cells with fragmented microvilli, broken endoplasmic reticulum, and disrupted nucleus membrane. The activity of the protective enzymes POD, SOD, CAT, and PO all increased significantly when L. migratoria was treated with IPPM202 only, but decreased when any one of the inhibitors was added. We further concluded that TPCK, a subtilisin (Pr1) inhibitor, and CI1, a cysteine protease (Pr4) inhibitor, played important roles in the pathogenicity of the M. anisopliae strain IPPM202. Conversely, trypsin (Pr2) and metalloproteases (Pr3) did not have a role in the given process. We further concluded that trypsin (Pr2) and metalloproteases (Pr3) do not contribute to the fungal infection process, while the subtilisin (Pr1) inhibitor TPCK and cysteine protease (Pr4) inhibitor CI1 play critical roles in the pathogenicity of Metarhizium anisopliae strain IPPM202, thus providing a foundation for targeted biocontrol strategies. Full article

The global rise of antimicrobial resistance represents a critical challenge to public health, with Escherichia coli emerging as one of the most significant contributors due to its high adaptability and prevalence of extended-spectrum β-lactamase (ESBL) production. Outer membrane vesicles (OMVs), nanoscale structures released by Gram-negative bacteria, have recently been implicated in the dissemination of resistance determinants and direct antibiotic inactivation. This study investigated the role of OMVs derived from ESBL-producing E. coli in mediating resistance to ampicillin. Clinical strains harboring CTX-M-15 resistance genes were cultured under selective pressure, and OMVs were purified via size-exclusion chromatography. Characterization using tunable resistive pulse sensing (TRPS) and cryo-transmission electron microscopy confirmed vesicle integrity, with sizes ranging from 80 to 150 nm. DNA quantification and PCR analysis revealed the presence of CTX-M-15 genes within vesicles, which remained protected from DNase digestion, confirming encapsulation. Functional assays demonstrated β-lactamase activity within OMVs, with proteinase K treatment indicating localization primarily within vesicles rather than on their surface. Importantly, OMVs inactivated ampicillin in a dose-dependent manner, significantly reducing its efficacy against susceptible E. coli. Disc diffusion and microtiter plate assays confirmed that β-lactamase-positive OMVs protected susceptible strains from antibiotic killing, promoting bacterial survival and growth. This study uniquely demonstrates that OMVs from CTX-M-15–producing Escherichia coli carry both resistance genes and active β-lactamase enzymes, thereby facilitating both genetic dissemination and direct antibiotic inactivation. Targeting OMV biogenesis may represent a novel strategy to combat antimicrobial resistance. Full article

Helper component-proteinase (HC-Pro), encoded by tobacco vein banding mosaic virus (TVBMV), can cause various viral symptoms and even abortion. HC-Pro counteracts host-mediated inhibition by interfering with the accumulation of microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, it is unclear whether the abortion phenotype of transgenic plants expressing HC-Pro is related to the abnormal expression of TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING cell factors (TCPs), which are involved in regulating fertility. In this study, the molecular mechanisms through which HC-Pro causes various sterile phenotypes in plants were investigated. Reverse transcription–quantitative polymerase chain reaction (RT–qPCR) and Northern blotting revealed that in HC-Pro transgenic plants, the expression levels of TCP4 and TCP24 significantly increased. The increased expression of TCP4 further upregulated LIPOXYGENASE2 (LOX2), a gene encoding a key enzyme in the synthesis of jasmonic acid (JA) precursors. Further studies confirmed that the aberrant expression of TCP3, TCP4 and TCP24 blocks the elongation of petals and anthers and that the aberrant expression of TCP4 and TCP24 blocks the release of pollen. This study demonstrated that HC-Pro affects the expression levels of the miR319-targeted genes TCP2, TCP3, TCP4, TCP10 and TCP24, thereby affecting the normal development of floral organs and resulting in plant abortion. Both tobacco and Arabidopsis thaliana were used as model systems in this study on virus-mediated fertility, which provides important information for understanding how viral pathogenicity affects the regulation of fertility in crops. Full article

Background/Objectives: Mesalazine agents are essential drugs for treating ulcerative colitis (UC). Biomarkers that can differentiate mesalazine intolerance from exacerbated UC are needed because of the similarity of their symptoms and increasing prevalence of mesalazine intolerance. The study aim was to assess the usefulness of proteinase 3 antineutrophil cytoplasmic antibody (PR3-ANCA) to identify mesalazine intolerance in patients with UC. Methods: In this single-center retrospective study, patients with UC in whom serum PR3-ANCA was measured were included, and the serum levels were compared between the mesalazine-intolerant and -tolerant patient groups. The predictability of the marker to discriminate between these patients was analyzed. Results: Among 406 patients with UC with measured serum PR3-ANCA levels, 68 (17%) had mesalazine intolerance. The PR3-ANCA levels were significantly higher in the intolerance group than in the tolerance group [4.5 U/mL (0.8–26.2 U/mL) vs. 1.5 U/mL (0.0–8.5 U/mL), p = 0.001]. The area under the curve of the receiver operating characteristic curve analysis of the predictability of PR3-ANCA in differentiating mesalazine-intolerant patients from clinically active patients with UC was 0.755 (95% confidence interval: 0.634–0.876, cutoff value: 15.05 U/mL; sensitivity: 0.625, specificity: 0.813). Multivariate logistic regression analysis using various clinical factors revealed that serum PR3-ANCA > 15.0 U/mL was an independent risk factor of mesalazine intolerance (odds ratio: 8.25, 95% confidence interval: 2.52–27.02, p < 0.001). Conclusions: Serum PR3-ANCA could be a useful marker to identify mesalazine-intolerant patients with UC. Full article

The understanding and control of protein crystallization are crucial in structural biology, drug development, and biomaterial design. This study introduces a unified framework for modeling and comparing crystallization kinetics using selected growth functions. Experimental datasets from the literature for four proteins, Lysozyme, Thaumatin, Ribonuclease A, and Proteinase K, under Hanging Drop and Langmuir–Blodgett conditions were analyzed. Five kinetic models, Avrami, Kashchiev, Hill, Logistic, and Generalized Sigmoid (GSM), were fitted to size–time data of the four benchmark proteins. From each fit, four descriptors were extracted: crystallization half-time, time of maximum growth, width at half-maximum, and peak growth rate. These metrics summarize crystallization dynamics and enable cross-comparison of proteins and methods. Langmuir–Blodgett templating accelerated onset and improved synchrony, though the effect varied by protein and model. Logistic, Hill, and GSM models provided consistent fits across most conditions, while Avrami and Kashchiev were more sensitive to early or late deviations. Notably, descriptor extraction remained reliable even with limited or uneven sampling, revealing kinetic regimes such as synchrony, asymmetry, or prolonged nucleation, not evident in raw data. This transferable analytical framework supports quantitative evaluation of crystallization behavior, aiding screening, process optimization, and time-resolved structural studies. Full article

Expansion microscopy (ExM) enables conventional light microscopes to achieve nanoscale resolution by physically enlarging biological specimens. While ExM has been widely applied in neurobiology, it has not been adapted for the enteric nervous system (ENS). Here, we provide a detailed and reproducible protocol for applying ExM to mouse colonic ENS tissue. The procedure includes preparation of the external muscle layers with the myenteric plexus, histochemical staining for NADPH-diaphorase, immunostaining for glial fibrillary acidic protein (GFAP), anchoring of biomolecules, gelation, proteinase K digestion, and isotropic expansion in a swellable polymer matrix. Step-by-step instructions, required reagents, and critical parameters are described to ensure robustness and reproducibility. Using this protocol, tissues expand 3–5-fold, allowing neuronal somata, fibers, and glial cell processes to be clearly visualized by standard brightfield or fluorescence microscopy. The tissue architecture is preserved, with distortion in the X–Y plane of about 7%. This protocol provides a reliable framework for high-resolution structural analysis of the ENS and can be readily adapted to other peripheral tissues. Full article

Dearth periods associated with less floral resources negatively impact Apis mellifera colony performance. Artificial diets offer nutritional supplements to sustain bee colonies under stressful conditions. An eight-week feeding trial was conducted using various artificial diets (eight diets, including a control diet), formulated with varying quantities of pulses, yeast, fenugreek powder, vegetable oil, dry apricot powder, and powdered sugar. Colony performance of bees subjected to different artificial diets was evaluated based on diet consumption, brood area, adult bee population, worker bee longevity, honey production, and enzymatic activity. Diet-7, which uniquely combined lupin, mung bean, and chickpea flours, proved the most efficacious and was the most consumed diet (84.29 ± 1.61 g), while diet-1 showed the lowest consumption (35.30 ± 1.08 g). Maximum brood area was recorded in colonies which were offered diet-7 (1385.95 ± 14.91 cm²), followed by diet-6, whereas the lowest was observed in the control (831.03 ± 18.95 cm²). The adult bee population was highest in diet-7 (21,594.50 ± 94.55 bees/hive), while lowest in the control (diet-0) (12,625.43 ± 385.06 bees/hive). Worker bee longevity was greatest in diet-7 (49.40 ± 0.41 days) and lowest in the control group (37.01 ± 0.39 days). Honey production was also highest in diet-7 (8.86 ± 0.21 kg), while lowest in the control (2.79 ± 0.35 kg). The results further showed that the enzymatic activities of bees were significantly improved due to diet-7, with the highest values for amylase (48.62 ± 0.23 U/mg), lipase (16.85 ± 0.20 U/mg), proteinase (25.21 ± 0.18 U/mg), and α-glucosidase (39.21 ± 0.21 U/mg). In conclusion, statistical analyses confirmed that diet-7 emerged as the most effective artificial diet, enhancing colony performance across all evaluated parameters. Future research should aim to optimize diet formulations and evaluate their effectiveness on colony health, including gut microbiome and immune function, across different seasons and ecological regions. Full article

Few studies have investigated gastrointestinal (GI) and external parasites, as well as environmental fungi, in rabbits using a One Health approach. Between September 2023—May 2024, fecal, hair and skin scraping samples were collected from 72 rabbits that attended clinical consultations and from private owners and breeders in Portugal. Diagnostic techniques included Mini-FLOTAC, direct immunofluorescence antibody, and the analysis of the virulence profile of fur fungi. A total of 58% of the rabbits were positive for GI parasites, namely Eimeria spp. (45%), Cryptosporidium spp. (32%), Trichostrongylus retortaeformis (17%), Passalurus ambiguus (13%), Graphidium strigosum (13%), and Giardia spp. (9%), with only 12% of the infected animals showing clinical signs (diarrhea). In addition, 10% of the animals were positive for Cheyletiella sp. infestations. Environmental fungi of the genera Penicillium, Rhizopus, and Scopulariopsis were isolated from 7% of these animals, with the Scopulariopsis sp. isolate S1 testing positive for proteinase, lecithinase, and gelatinase activities. Frequent sanitization and regular deworming emerged as essential factors to minimize parasitic frequency. This integrated diagnosis procedure proved to be effective in the search for parasitic and fungal agents in rabbit medicine. Further research is needed to improve the knowledge on the transmission and pathogenicity of these agents in rabbits. Full article

Background/Objectives: Binding of bactericidal/permeability-increasing (BPI) protein to Gram-negative (GN) bacteria plays a major role in bacterial elimination. The relationship between BPI-antineutrophil cytoplasmic autoantibodies (ANCA), persistent infections and immunoinflammatory diseases has not been elucidated. Methods: In total, 193 ANCA-positive patients detected by IIF with ANCA-associated vasculitides (AAV, n-40), connective tissue diseases (CTD, n-28), drug-induced vasculitides (DIV, n-17), ulcerative colitis (UC, n-24), UC with primary sclerosing cholangitis (UC/PSC, n-14), Crohn’s disease (CD, n-10), autoimmune hepatitis (AIH, n-19) and chronic infections (n-41) were tested using the BPI-ANCA quantitative and semiquantitative ELISA (ANCA-profile: BPI, proteinase 3, myeloperoxidase, elastase, cathepsin G, lactoferrin). BPI-ANCA were analyzed in 52 healthy persons. Results: A total of 46/193 (23.8%) patients had BPI-ANCA positivity. BPI-ANCA were more frequently present in patients with prolonged GN bacterial infections and inflammatory bowel diseases than in AAV, DIV, AIH, CTD and healthy controls (p < 0.001). UC/PSC patients more frequently had BPI-ANCA than UC and CD patients (p < 0.001). GN bacterial infections more frequently had BPI-ANCA than Gram-positive bacterial infections (p < 0.001). Infections caused by Pseudomonas aeruginosa and Mycobacterium tuberculosis had monospecific BPI-ANCA (sensitivity 79% and 71%, respectively). UC/PSC and chronic GN bacterial infections caused by Klebsiella pneumoniae, Proteus mirabilis, or Escherichia coli had multispecific BPI-ANCA (sensitivity 64% and 100%, respectively). Odds ratio analysis showed that patients with IBD who were positive for multispecific BPI-ANCA had a 13.5-fold increased risk of UC/PSC (95% CI 2.98–61.18). Conclusions: Monospecific BPI-ANCA may be a valuable biomarker for persistent Pseudomonas aeruginosa and Mycobacterium tuberculosis infections. In contrast, multispecific BPI-ANCA are associated with UC/PSC and persistent infections caused by intestinal Gram-negative bacteria. Suppression of antimicrobial function by multispecific BPI-ANCA could impair the elimination of Gram-negative bacteria, sustaining the immunoinflammation. Dysregulated antimicrobial response might be the target of immunomodulatory therapy in the initial phase of BPI-ANCA-positive UC/PSC. Full article

Tuberculosis (TB) is an infection caused by Mycobacterium tuberculosis complex (MTBC), which can be exacerbated by fungal infections. This study evaluated the clinical characteristics and virulence of Candida spp. in patients with tuberculosis. Antifungal sensitivity, phospholipase and proteinase production, biofilm formation, phagocytic index, and reactive oxygen (ROS) and nitrogen (RNS) species were assessed. Candida spp. were isolated from 14 patients, 28.5% women and 71.4% men, mainly from sputum and tracheal secretions. Five (35.7%) patients were co-infected with Mycobacterium, Candida, and HIV. Candida albicans (78.6%) and Candida tropicalis (21.4%) were identified in all 14 patients. All isolates showed sensitivity to amphotericin B and dose-dependent responses to fluconazole (16 μg/mL). Phospholipase activity was detected in 35.7% of the isolates, whereas all isolates showed proteinase activity (100%). A significant difference in phospholipase activity, phagocytosis, and production of reactive oxygen species (ROS) and nitrogen species (RNS) was observed when Candida isolates from patients with TB, living with or without HIV, were compared to Candida isolates from healthy individuals. All isolates were biofilm producers. This study highlights the relevance of mycoses diagnosis in patients with TB, since Candida spp. may be more virulent and contribute to the deterioration of the clinical condition. Full article

This study investigated the effects of dietary Rhodotorula mucilaginosa supplementation with different concentrations (0.0 g/kg, 0.1 g/kg, 1.0 g/kg, 10.0 g/kg) on red claw crayfish (Cherax quadricarinatus). Four groups were established: control group (CK, 0.0 g/kg), low-dose group (HL, 0.1 g/kg), medium-dose group (HM, 1.0 g/kg), and high-dose group (HH, 10.0 g/kg). The feeding trial lasted for 56 days. The results showed that, compared with the control group, all supplementation groups exhibited significantly reduced feed conversion ratios (p < 0.05). The HM and HH groups demonstrated significant increases in body length growth rate, specific growth rate, weight gain rate, hepatosomatic index, and survival rate (p < 0.05). All supplemented groups showed significantly enhanced trypsin and lipase activities in intestines and trypsin activity in the hepatopancreas (p < 0.05). The HM and HH groups exhibited elevated α-amylase activity in the hepatopancreas (p < 0.05). Compared with the control group, marine red yeast supplementation reduced colonization of potential pathogens while increasing probiotic abundance, effectively improving intestinal microbiota structure. The HM group significantly improved intestinal villus length, width, and muscular thickness (p < 0.05). All supplemented groups showed considerable upregulation of hepatopancreatic genes related to immunity (heat shock protein 70, down syndrome cell adhesion molecule, crustacean antibacterial peptide, serine proteinase inhibitors, crustacean hyperglycemic hormone, anti-lipopolysaccharide factor, lysozyme, and alkaline phosphatase) and antioxidant defense (superoxide dismutase, glutathione peroxidase, glutathione, and catalase) (p < 0.05). These findings indicate that R. mucilaginosa can significantly enhance digestive enzyme activity, maintain intestinal health, improve antioxidant and immune-related gene expression, and promote growth performance in red claw crayfish, with the HM group (1.0 g/kg R. mucilaginosa) showing optimal promotion effects. Full article

Real-time nitrogen (N) management based on the leaf color chart (LCC) is considered a potential alternative to traditional farmer practices. However, its physiological mechanisms for enhancing rice N utilization and its effects on paddy field N balance remain unclear. We aimed to elucidate the potential enzymatic mechanisms underlying LCC’s influence on rice N use and quantify the impact of LCC on paddy field N balance. In 2022 and 2023, a single-factor randomized block design experiment was conducted during the rice planting season. Four N treatments: no N (ONF), farmers’ conventional practices + urea [FNR] as the control, LCC + urea [SSNM1], LCC + controlled-release urea [SSNM2] were administered. Rice yield and N uptake were positive correlations with nitrate reductase (NR), glutamine synthetase (GS), glutamate-pyruvate transaminase (GPT), glutamate-oxaloacetate transaminase (GOT) and glutamate dehydrogenase (GDH) activities, which were higher under SSNM1 and SSNM2 compared with FNR, but were negative correlation with proteinase activity. Moreover, SSNM1 and SSNM2 increased rice yield by 9.2% and 9.4%, N uptake by 15.4% and 15.3%, and N use efficiency by 46.9% and 65.0%, and reduced reactive N losses by 46.2% and 66.7%, respectively. The annual net soil N inputs under FNR, SSNM1, and SSNM2 were 12.6, 8.9, and 4.2 kg N ha⁻¹, respectively. LCC-based N management increased N uptake and rice yield by enhancing the activities of NR, GS, GPT, GOT, and GDH while reducing protease activity. Moreover, LCC maintained soil N supply capacity even with reduced nitrogen fertilizer application. Full article

The giant freshwater prawn (Macrobrachium rosenbergii) has economic significance in the aquatic industry, but production is impacted by infectious diseases induced by various pathogens. Herein, we investigated the impact of adding feed additives to the diet of M. rosenbergii to promote health. Diets were formulated to contain different levels of Centella asiatica (L.) Urb. crude extracts (1, 5, and 10 g kg⁻¹), with growth performance and innate immune parameters assessed after 28 days of feeding. Real-time quantitative PCR (RT-qPCR) was employed to determine the mRNA levels of serine proteinase inhibitor (SPI) and alpha-2 macroglobulin (Mr-2α2M) from 12 h to 28 days of feeding. Prawns feeding at 5 and 10 g kg⁻¹ showed statistically significant differences in specific growth rate, lysozyme assay, and phenoloxidase activity. The expression levels of all the immune-related genes studied were significantly upregulated in prawns fed with supplemented diets compared to the control group. Findings revealed that the observed upregulations varied in response to alterations in feeding time and concentration. Furthermore, 16S rRNA analysis showed that the supplemented diets at 10 g kg⁻¹ supplementation increased beneficial bacteria (Lactococcus sp.) and reduced pathogenic taxa (e.g., Candidatus Hepatoplasma, Flavobacteriaceae, Weeksellaceae, Thiothrix sp.). Full article