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BACKGROUND: Cell wall integrity (CWI) is crucial for fungal growth, pathogenesis, and adaptation to extracellular environments. Calcofluor white (CFW) is a cell wall perturbant that inhibits fungal growth, yet little is known about how phytopathogenic fungi respond to the CFW-induced stress. RESULTS: In this study, we unveiled a significant discovery that CFW triggered the translocation of the transcription factor CgCrzA from the cytoplasm to the nucleus in Colletotrichum gloeosporioides. This translocation was regulated by an interacting protein, CgMkk1, a mitogen-activated protein kinase involved in the CWI pathway. Further analysis revealed that CgMkk1 facilitated nuclear translocation by phosphorylating CgCrzA at the Ser280 residue. Using chromatin immunoprecipitation sequencing, we identified two downstream targets of CgCrzA, namely CgCHS5 and CgCHS6, which are critical for growth, cell wall integrity, and pathogenicity as chitin synthase genes. CONCLUSIONS: These findings provide a novel insight into the regulatory mechanism of CgMkk1-CgCrzA-CgChs5/6, which enables response of the cell wall inhibitor CFW and facilitates infectious growth for C. gloeosporioides.
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Colletotrichum , Proteínas Fúngicas , Fatores de Transcrição , Virulência/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Colletotrichum/genética , Colletotrichum/patogenicidade , Parede Celular/metabolismo , Regulação Fúngica da Expressão Gênica , FosforilaçãoRESUMO
BACKGROUND: The genes involved in cephalopod development and their association with hatching and survival during early life stages have been extensively studied. However, few studies have investigated the paralarvae transcriptome of the East Asian common octopus (Octopus sinen sis). OBJECTIVE: This study aimed to identify the genes related to embryonic development and hatching in O. sinensis using RNA sequencing (RNA-seq) and verify the genes most relevant to different embryonic stages. METHODS: RNA samples from hatched and 25 days post-hatching (dph) O. sinensis paralarvae were used to construct cDNA libraries. Clean reads from individual samples were aligned to the reference O. sinensis database to identify the differentially expressed genes (DEGs) between the 0- and 25-dph paralarvae libraries. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to supplement the RNA-seq data for embryogenic developmental stages. RESULTS: A total of 12,597 transcripts were annotated and 5,468 DEGs were identified between the 0- and 25-dph O. sinensis paralarvae, including 2,715 upregulated and 2,753 downregulated transcripts in the 25-dph paralarvae. Several key DEGs were related to transmembrane transport, lipid biosynthesis, monooxygenase activity, lipid transport, neuropeptide signaling, transcription regulation, and protein-cysteine S-palmitoyltransferase activity during the post-hatching development of O. sinensis paralarvae. RT-qPCR analysis further revealed that SLC5A3A, ABCC12, and NPC1 transcripts in 20 and/or 30 days post-fertilization (dpf) embryos were significantly higher (p < 0.05) than those in 10-dpf embryos. CONCLUSION: Transcriptome profiles provide molecular targets to understand the embryonic development, hatching, and survival of O. sinensis paralarvae, and enhance octopus production.
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Octopodiformes , Transcriptoma , Animais , Octopodiformes/genética , Octopodiformes/crescimento & desenvolvimento , Transcriptoma/genética , Regulação da Expressão Gênica no Desenvolvimento , Perfilação da Expressão Gênica/métodos , População do Leste AsiáticoRESUMO
Clinical bone-morphogenetic protein 2 (BMP2) treatment for bone regeneration, often resulting in complications like soft tissue inflammation and ectopic ossification due to high dosages and non-specific delivery systems, necessitates research into improved biomaterials for better BMP2 stability and retention. To tackle this challenge, we introduced a groundbreaking bone-targeted, lipoplex-loaded, three-dimensional bioprinted bilayer scaffold, termed the polycaprolactone-bioink-nanoparticle (PBN) scaffold, aimed at boosting bone regeneration. We encapsulated BMP2 within the fibroin nanoparticle based lipoplex (Fibroplex) and functionalized it with DSS6 for bone tissue-specific targeting. 3D printing technology enables customized, porous PCL scaffolds for bone healing and soft tissue growth, with a two-step bioprinting process creating a cellular lattice structure and a bioink grid using gelatin-alginate hydrogel and DSS6-Fibroplex, shown to support effective nutrient exchange and cell growth at specific pore sizes. The PBN scaffold is predicted through in silico analysis to exhibit biased BMP2 release between bone and soft tissue, a finding validated by in vitro osteogenic differentiation assays. The PBN scaffold was evaluated for critical calvarial defects, focusing on sustained BMP2 delivery, prevention of soft tissue cell infiltration and controlled fiber membrane pore size in vivo. The PBN scaffold demonstrated a more than eight times longer BMP2 release time than that of the collagen sponge, promoting osteogenic differentiation and bone regeneration in a calvarial defect animal. Our findings suggest that the PBN scaffold enhanced the local concentration of BMP2 in bone defects through sustained release and improved the spatial arrangement of bone formation, thereby reducing the risk of heterotopic ossification.
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We have developed a fluorescent probe DBT-Cl ((E)-2-(2-(4-(diphenylamino)benzylidene) hydrazinyl)-N,N,N-trimethyl-2-oxoethan-1-aminium chloride) for ClO- with an aggregation-induced emission (AIE) strategy depending on solvent polarity. DBT-Cl possessed a prominent solvatochromic emission property with intramolecular charge transfer (ICT) from the TPA (triphenylamine) to the amide group, which was studied by spectroscopic analysis and DFT calculations. These unique AIE properties of DBT-Cl led to the recognition of ClO- with high fluorescent selectivity. DBT-Cl quickly detected ClO- in less than 1 sec with a fluorescent color change from green to cyan. DBT-Cl had a low detection limit of 9.67 µM to ClO-. Detection mechanism of DBT-Cl toward ClO- was illustrated to be oxidative cleavage of DBT-Cl by 1H NMR titrations, ESI-mass, and DFT calculations. We established the viability for dependable detection of ClO- in actual water samples, as well as zebrafish and plant imaging. In particular, DBT-Cl was capable of easily monitoring ClO- through a smartphone application. Therefore, DBT-Cl assured a promising approach for a fast-responsive and multi-applicable ClO- probe in environmental and living organism systems.
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Corantes Fluorescentes , Ácido Hipocloroso , Smartphone , Espectrometria de Fluorescência , Peixe-Zebra , Ácido Hipocloroso/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Animais , Espectrometria de Fluorescência/métodos , Água/química , Limite de Detecção , Poluentes Químicos da Água/análise , Teoria da Densidade FuncionalRESUMO
Heat-treated Lactiplantibacillus plantarum nF1 (HT-nF1) increases immune cell activation and the production of various immunomodulators (e.g., interleukin (IL)-12) as well as immunoglobulin (Ig) G, which plays an important role in humoral immunity, and IgA, which activates mucosal immunity. To determine the effect of HT-nF1 intake on improving immune function, a randomized, double-blind, placebo-controlled study was conducted on 100 subjects with normal white blood cell counts. The HT-nF1 group was administered capsules containing 5 × 1011 cells of HT-nF1 once a day for 8 weeks. After 8 weeks of HT-nF1 intake, significant changes in IL-12 were observed in the HT-nF1 group (p = 0.045). In particular, the change in natural killer (NK) cell activity significantly increased in subjects with low secretory (s) IgA (≤49.61 µg/mL) and low NK activity (E:T = 10:1) (≤3.59%). These results suggest that HT-nF1 has no safety issues and improves the innate immune function by regulating T helper (Th)1-related immune factors. Therefore, we confirmed that HT-nF1 not only has a positive effect on regulating the body's immunity, but it is also a safe material for the human body, which confirms its potential as a functional health food ingredient.
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Interleucina-12 , Células Matadoras Naturais , Probióticos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Método Duplo-Cego , Temperatura Alta , Sistema Imunitário , Imunidade Inata , Imunoglobulina A/sangue , Células Matadoras Naturais/imunologia , Lactobacillus plantarum , Probióticos/administração & dosagemRESUMO
BACKGROUND: In previous studies, denosumab, a RANKL human monoclonal antibody used in osteoporosis treatment, has shown efficacy in tendon healing after rotator cuff repair. This prospective study investigated the effects of denosumab on tendon healing, re-tear rates, and clinical outcomes post rotator cuff repair in women with osteoporosis. METHOD: This was a prospective, observational study, employing propensity score matching for the control group. From March 2018 to March 2023, female patients over the age of 60 with normal bone density undergoing arthroscopic rotator cuff repair were selected as controls through propensity score matching (PSM) and compared with female patients of the same age group with osteoporosis who were receiving denosumab treatment. The control group was matched using 1-to-2 propensity score matching. Radiological examinations and functional outcomes were assessed preoperatively and at 6 months postoperatively. RESULTS: In the final analysis, the study comprised 34 patients in the denosumab treatment group (Group 1) and 68 patients in the control group (Group 2). The functional scores showed significant improvement at 6 months post-surgery in both groups. No significant difference in the functional scores was observed among the groups. The re-tear rate, defined according to Sugaya's classification (types IV and V) as re-tear, was slightly higher in Group 1 at 16.7% (6 of 34) compared to Group 2 at 11.7% (8 of 68), but the difference was not statistically significant (p = 0.469). The re-tear patterns, classified according to Rhee's classification, also showed no significant difference among the groups (Group 1: 2/4 of 6; Group 2: 4/4 of 8; p = 0.571). The occurrence of type I re-tear exhibited no significant difference between the two groups (5.9% vs. 5.9%; p = 1.000). CONCLUSIONS: The administration of denosumab following arthroscopic rotator cuff repair in women aged 60 and over with osteoporosis resulted in a re-tear rate that was similar to that observed in patients without osteoporosis. This result suggests that denosumab administration might be beneficial for rotator cuff healing, particularly in the context of osteoporosis, a known risk factor for increased retear rates. Therefore, comprehensive osteoporosis screening and treatment should be considered in conjunction with rotator cuff repair surgery in middle-aged women.
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Background: Large-to-massive rotator cuff tears (LMRCTs) present challenges in achieving successful repair due to factors such as muscle atrophy and tendon retraction. Arthroscopic rotator cuff repair (ARCR) with reinforcement techniques like superior capsule reconstruction (SCR) or patch graft augmentation (PGA) has emerged as a less invasive option to improve shoulder joint stability and prevent retear. This study aimed to compare the clinical and radiological outcomes of SCR and PGA as reinforcement techniques for the arthroscopic repair of LMRCTs. Methods: A single-center retrospective study was conducted on patients undergoing LMRCT repair between January 2019 and December 2021. Patients were divided into two groups: those receiving SCR (Group 1) and those receiving PGA (Group 2). Various clinical parameters including range of motion, functional scores, and radiological assessments were evaluated preoperatively and six months postoperatively. Results: Both SCR and PGA techniques demonstrated significant improvements in the range of motion and clinical scores postoperatively. However, Group 2 showed higher postoperative SST and UCLA scores compared to Group 1. Radiologically, there was a slightly higher retear rate in Group 2, although this was not statistically significant. Group 2 also had a shorter mean duration of surgery compared to Group 1. Conclusions: In the arthroscopic repair of LMRCTs, both SCR and PGA techniques exhibit favorable clinical and radiological outcomes. Despite the simplicity of PGA compared to SCR, it offers comparable results with a shorter surgical duration, making it a feasible reinforcement option for surgeons.
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Real-time quantitative PCR (RT-qPCR) is a widely used method for accurate quantitative gene expression analysis. For accurate quantitative verification of RT-qPCR, it is essential to select a reference gene with high expression stability depending on the experimental environment or the different tissues. In this study, we evaluated the stability of nine candidate reference genes, labeled elongation factor 1-alpha (EF1A), ERBB receptor feedback inhibitor 1-like isoform x2 (ERRFI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), integrin beta2 like (ITGB2), phosphatidylinositol-binding clathrin assembly protein-like isoform x3 (PICALM), 60 s ribosomal protein L5 (RPL5), 60 s ribosomal protein L7 (RPL7), tubulin beta chain (TUBB), and ubiquitin-conjugating enzyme E2A (UBE2A), in the brain (including pituitary gland) gonads and caudal fins of silvertip tetra (Hasemania nana) males and females. The stability evaluation of the reference gene was analyzed using a program based on the geNorm, NormFinder, BestKeeper, and RankAggreg algorithms. As a result, RPL5 (brain, caudal fin), EF1A (gonad), and PICALM (three tissue types) genes were evaluated as the most stable genes in silvertip tetra females. In males, TUBB (brain, caudal fin) and ITGB2 (gonads, three tissue types) genes were the most stable, and in both sexes, TUBB (brain), ITGB2 (caudal fin), RPL5 (gonads), and PICALM (three tissue types) genes are considered appropriate as reference genes for qRT-PCR analysis. However, the GAPDH gene was judged to be inappropriate for use as a reference gene because gene stability in the brain, caudal fin, and gonads was evaluated to be low in all males and females. As an introductory study on silvertip tetra, a new research model fish, the results of this study are expected to provide helpful information regarding sex differentiation and determination in fish.
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The multifunctional carbon catabolite repression negative on TATA-box-less complex (CCR4-NOT) is a multi-subunit complex present in all eukaryotes, including fungi. This complex plays an essential role in gene expression; however, a functional study of the CCR4-NOT complex in the rice blast fungus Magnaporthe oryzae has not been conducted. Seven genes encoding the putative CCR4-NOT complex were identified in the M. oryzae genome. Among these, a homologous gene, MoNOT3, was overexpressed during appressorium development in a previous study. Deletion of MoNOT3 in M. oryzae resulted in a significant reduction in hyphal growth, conidiation, abnormal septation in conidia, conidial germination, and appressorium formation compared to the wild-type. Transcriptional analyses suggest that the MoNOT3 gene affects conidiation and conidial morphology by regulating COS1 and COM1 in M. oryzae. Furthermore, Δmonot3 exhibited a lack of pathogenicity, both with and without wounding, which is attributable to deficiencies in the development of invasive growth in planta. This result was also observed in onion epidermal cells, which are non-host plants. In addition, the MoNOT3 gene was involved in cell wall stress responses and heat shock. Taken together, these observations suggest that the MoNOT3 gene is required for fungal infection-related cell development and stress responses in M. oryzae.
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Ascomicetos , Magnaporthe , Oryza , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ascomicetos/metabolismo , Esporos Fúngicos , Oryza/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Regulação Fúngica da Expressão GênicaRESUMO
Background: This study compared the outcomes of surgical aortic valve replacement (AVR) in patients aged 50 to 70 years based on the type of prosthetic valve used. Methods: We compared patients who underwent mechanical AVR to those who underwent bioprosthetic AVR at our institution between January 2000 and March 2019. Competing risk analysis and the inverse probability of treatment weighting (IPTW) method based on propensity score were employed for comparisons. Results: A total of 1,580 patients (984 patients with mechanical AVR; 596 patients with bioprosthetic AVR) were enrolled. There was no significant difference in early mortality between the mechanical AVR and bioprosthetic AVR groups (0.9% vs. 1.7%, p=0.177). After IPTW adjustment, the risk of all-cause mortality was significantly higher in the bioprosthetic AVR group than in the mechanical AVR group (hazard ratio [HR], 1.39; 95% confidence interval [CI], 1.07-1.80; p=0.014). Competing risk analysis revealed lower risks of stroke (sub-distributional hazard ratio [sHR], 0.44; 95% CI, 0.28-0.67; p<0.001) and anticoagulation- related bleeding (sHR, 0.35; 95% CI, 0.23-0.53; p<0.001) in the bioprosthetic AVR group. Conversely, the risk of aortic valve (AV) reintervention was higher in the bioprosthetic AVR group (sHR, 6.14; 95% CI, 3.17-11.93; p<0.001). Conclusion: Among patients aged 50 to 70 years who underwent surgical AVR, those receiving mechanical valves showed better survival than those with bioprosthetic valves. The mechanical AVR group exhibited a higher risk of stroke and anticoagulation-related bleeding, while the bioprosthetic AVR group showed a higher risk of AV reintervention.
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The purpose of this study was to investigate the fracture morphology of distal radius fractures (DRFs) with the status of triangular fibrocartilage complex (TFCC) foveal insertion in patients with or without osteoporosis and to identify the relationship between osteoporosis and foveal tear. Seventy-five patients who underwent surgery for DRF from January 2021 to September 2023 were included. All patients were evaluated by standard radiography and dual-energy X-ray absorptiometry and underwent a 3.0 T magnetic-resonance imaging examination of the involved wrist to identify TFCC foveal tear. Patients were allocated into two groups according to the presence of osteoporosis: patients with osteoporosis (group I) and those without osteoporosis (group II). Group I showed a significantly larger displacement of fractures compared to group II (radial inclination; 13.7 ± 5.4 vs. 17.9 ± 4.2; p < 0.001, dorsal angulation; 22.2 ± 12.1 vs. 16.5 ± 9.4; p = 0.024, ulnar variance; 4.15 ± 2.1 vs. 2.2 ± 1.9; p < 0.001). Dorsal angulation and ulnar variance were found to be independent prognostic factors for TFCC foveal tear in logistic regression analysis. Displacement of fractures was related to osteoporosis, and dorsal angulation and ulnar variance were independent prognostic factors for TFCC foveal tear. However, osteoporosis was not identified as a factor associated with TFCC foveal tears.
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Modulation of the plant defense response by bioactive molecules is of increasing interest. However, despite plant cell lipids being one of the major cellular components, their role in plant immunity remains elusive. We found that the exogenous application of the cell-membrane localized phospholipid lyso-phosphatidylethanolamine (LPE) reprograms the plant transcript profile in favor of defense-associated genes thereby priming the plant immune system. Exogenous LPE application to different Arabidopsis accessions increases resistance against the necrotrophic pathogens, Botrytis cinerea and Cochliobolus heterostrophus. We found that the immunity-promoting effect of LPE is repealed in the jasmonic acid (JA) receptor mutant coi1, but multiplied in the JA-hypersensitive mutant feronia (fer-4). The JA-signaling repressor JAZ1 is degraded following LPE administration, suggesting that JA-signaling is promoted by LPE. Following LPE-treatment, reactive oxygen species (ROS) accumulation is affected in coi1 and fer-4. Moreover, FER signaling inhibitors of the RALF family are strongly expressed after LPE application, and RALF23 is internalized in stress granules, suggesting the LPE-mediated repression of FER-signaling by promoting RALF function. The in-situ increase of LPE-abundance in the LPE-catabolic mutants lpeat1 and lpeat2 elevates plant resistance to B. cinerea, in contrast to the endogenous LPE-deficient mutant pla2-alpha. We show that LPE increases plant resistance against necrotrophs by promoting JA-signaling and ROS-homeostasis, thereby paving the way for the LPE-targeted genomic engineering of crops to raise their ability to resist biotic threats.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidiletanolaminas/farmacologia , Arabidopsis/metabolismo , Oxilipinas/metabolismo , Ciclopentanos/metabolismo , Homeostase , Doenças das Plantas/genética , Botrytis/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Studies comparing the ocular toxicity potential between legacy and alternative PFAS are lacking. To address this research gap, zebrafish larvae were exposed to both legacy PFAS (i.e., perfluorooctanesulfonic acid [PFOS] and perfluorooctanoic acid [PFOA]) and their corresponding alternatives (i.e., perfluorobutanesulfonic acid [PFBS] and perfluorobutanoic acid [PFBA]). Alterations in their visual behaviors, such as phototactic and optomotor responses (OMR), were assessed at sublethal concentrations. Gene expression variations in visual function-associated pathways were also measured. Visual behavioral assessment revealed that PFOS exposure resulted in concentration-dependent reductions in phototactic responses at 10-1000 µg/L, with PFOA exerting reduction effects only at 100 mg/L. However, their two alternatives had no effect at all tested concentrations. Following an improved contrast-OMR (C-OMR) assessment, PFOS decreased the OMR to a water flow stimulus at 10, 100, and 1000 µg/L. The gene expression analysis revealed that PFOS exposure markedly downregulated most genes involved in the opsins in the photoreceptor and phototransduction cascade, which explains the observed visual behavior changes well. Our findings indicate that PFOS is the most likely PFAS to cause visual toxicity, with PFOA present but less likely, and their substitutes, PFBS and PFBA, cannot be classified as visually toxic to zebrafish.
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We sequenced the genome of Westerdykella aurantiaca NNIBRFG27121 strain isolated from the wetland of Maehwamarum Habitat in Korea. The final assembly consisted of six scaffolds with a size of 31.96 Mb and an N50 of 8,770,400 bp. This genome will help in comparing species within the Westerdykella genus.
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Studies on the comparison of developmental (neuro) toxicity of parabens are currently limited, and unharmonized concentrations between phenotypic observations and transcriptome analysis hamper the understanding of their differential molecular mechanisms. Thus, developmental toxicity testing was conducted herein using the commonly used methyl- (MtP), ethyl- (EtP), and propyl-parabens (PrP) in zebrafish embryos. With a benchmark dose of 5%, embryonic-mortality-based point-of-departure (M-POD) values of the three parabens were determined, and changes in locomotor behavior were evaluated at concentrations of 0, M-POD/50, M-POD/10, and M-POD, where transcriptome analysis was conducted to explore the underlying neurotoxicity mechanism. Higher long-chained parabens were more toxic than short-chained parabens, as determined by the M-POD values of 154.1, 72.6, and 24.2 µM for MtP, EtP, and PrP, respectively. Meanwhile, exposure to EtP resulted in hyperactivity, whereas no behavioral effect was observed with MtP and PrP. Transcriptome analysis revealed that abnormal behaviors in the EtP-exposed group were associated with distinctly enriched pathways in signaling, transport, calcium ion binding, and metal binding. In contrast, exposure to MtP and PrP mainly disrupted membranes and transmembranes, which are closely linked to abnormal embryonic development rather than neurobehavioral changes. According to the changes in the expressions of signature mRNAs, tentative transcriptome-based POD values for each paraben were determined as MtP (2.68 µM), EtP (3.85 µM), and PrP (1.4 µM). This suggests that different molecular perturbations initiated at similar concentrations determined the extent and toxicity outcome differently. Our findings provide insight into better understanding the differential developmental neurotoxicity mechanisms of parabens.
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Poluentes Ambientais , Parabenos , Animais , Parabenos/análise , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Poluentes Ambientais/análise , Perfilação da Expressão GênicaRESUMO
The assay for transposase-accessible chromatin with sequencing (ATAC-seq) is the most widely used method for measuring chromatin accessibility. Researchers have included multi-sample replication in ATAC-seq experimental designs. In epigenomic analysis, researchers should measure subtle changes in the peak by considering the read depth of individual samples. It is important to determine whether the peaks of each replication have an integrative meaning for the region of interest observed during multi-sample integration. We developed multi-epigenome sample integration approach for precise peak calling (MESIA), which integrates replication with high representativeness and reproducibility in multi-sample replication and determines the optimal peak. After identifying the reproducibility between all replications, our method integrated multiple samples determined as representative replicates. MESIA detected 6.06 times more peaks, and the value of the peaks was 1.32 times higher than the previously used method. MESIA is a shell-script-based open-source code that provides researchers involved in the epigenome with comprehensive insights.
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Epigenoma , Sequenciamento de Nucleotídeos em Larga Escala , Reprodutibilidade dos Testes , Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , Análise de Sequência de DNA/métodosRESUMO
In the nucleus, distinct, discrete spots or regions called "foci" have been identified, each harboring a specific molecular function. Accurate and efficient quantification of these foci is essential for understanding cellular dynamics and signaling pathways. In this study, we present an innovative automated image analysis method designed to precisely quantify subcellular foci within the cell nucleus. Manual foci counting methods can be tedious and time-consuming. To address these challenges, we developed an open-source software that automatically counts the number of foci from the indicated image files. We compared the foci counting efficiency, velocity, accuracy, and convenience of Foci-Xpress with those of other conventional methods in foci-induced models. We can adjust the brightness of foci to establish a threshold. The Foci-Xpress method was significantly faster than other conventional methods. Its accuracy was similar to that of conventional methods. The most significant strength of Foci-Xpress is automation, which eliminates the need for analyzing equipment while counting. This enhanced throughput facilitates comprehensive statistical analyses and supports robust conclusions from experiments. Furthermore, automation completely rules out biases caused by researchers, such as manual errors or daily variations. Thus, Foci-Xpress is a convincing, convenient, and easily accessible focus-counting tool for cell biologists.
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Processamento de Imagem Assistida por Computador , Software , Processamento de Imagem Assistida por Computador/métodos , AutomaçãoRESUMO
Peptidylarginine deiminase (PADI) 2 catalyzes the post-translational conversion of peptidyl-arginine to peptidyl-citrulline in a process called citrullination. However, the precise functions of PADI2 in bone formation and homeostasis remain unknown. In this study, our objective was to elucidate the function and regulatory mechanisms of PADI2 in bone formation employing global and osteoblast-specific Padi2 knockout mice. Our findings demonstrate that Padi2 deficiency leads to the loss of bone mass and results in a cleidocranial dysplasia (CCD) phenotype with delayed calvarial ossification and clavicular hypoplasia, due to impaired osteoblast differentiation. Mechanistically, Padi2 depletion significantly reduces RUNX2 levels, as PADI2-dependent stabilization of RUNX2 protected it from ubiquitin-proteasomal degradation. Furthermore, we discovered that PADI2 binds to RUNX2 and citrullinates it, and identified ten PADI2-induced citrullination sites on RUNX2 through high-resolution LC-MS/MS analysis. Among these ten citrullination sites, the R381 mutation in mouse RUNX2 isoform 1 considerably reduces RUNX2 levels, underscoring the critical role of citrullination at this residue in maintaining RUNX2 protein stability. In conclusion, these results indicate that PADI2 plays a distinct role in bone formation and osteoblast differentiation by safeguarding RUNX2 against proteasomal degradation. In addition, we demonstrate that the loss-of-function of PADI2 is associated with CCD, thereby providing a new target for the treatment of bone diseases.
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Citrulinação , Displasia Cleidocraniana , Animais , Camundongos , Osteogênese , Cromatografia Líquida , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Espectrometria de Massas em Tandem , Camundongos KnockoutRESUMO
Biomolecular operations, which involve the conversion of molecular signals or interactions into specific functional outputs, are fundamental to the field of biology and serve as the important foundation for the design of diagnostic and therapeutic systems. To maximize their functionalities and broaden their applicability, it is crucial to develop novel outputs and facile chemical transformation methods. With this aim, in this study, we present a straightforward method for converting nucleic acid signals into fluorescein outputs that exhibit a wide range of functionalities. This operation is designed through a DNA-templated reaction based on riboflavin-photocatalyzed oxidation of dihydrofluorescein, which is readily prepared by simple NaBH4 reduction of the fluorescein with no complicated chemical caging steps. The templated photooxidation exhibits high efficiency (kapp = 2.7 × 10-3/s), generating a clear fluorescein output signal distinguishable from a low background, originating from the high stability of the synthesized dihydrofluorescein. This facile and efficient operation allows the nucleic acid-initiated activation of various fluorescein functions, such as fluorescence and artificial oxidase activity, which are applied in the design of novel bioanalytical systems, including fluorescent and colorimetric DNA sensors. The operation presented herein would expand the scope of biomolecular circuit systems for diagnostic and therapeutic applications.