Modulation of m6A RNA modification by DAP3 in cancer cells.

N6-methyladenosine (m6A) RNA methylation is a prevalent RNA modification that significantly impacts RNA metabolism and cancer development. Maintaining the global m6A levels in cancer cells relies on RNA accessibility to methyltransferases and the availability of the methyl donor S-adenosylmethionine (SAM). Here, we reveal that death associated protein 3 (DAP3) plays a crucial role in preserving m6A levels through two distinct mechanisms. First, although DAP3 is not a component of the m6A writer complex, it directly binds to m6A target regions, thereby facilitating METTL3 binding. Second, DAP3 promotes MAT2A's last intron splicing, increasing MAT2A protein, cellular SAM, and m6A levels. Silencing DAP3 hinders tumorigenesis, which can be rescued by MAT2A overexpression. This evidence suggests DAP3's role in tumorigenesis, partly through m6A regulation. Our findings unveil DAP3's complex role as an RNA-binding protein and tumor promoter, impacting RNA processing, splicing, and m6A modification in cancer transcriptomes.

Modeling the structure and DAP5-binding site of the FGF-9 5'-UTR RNA utilized in cap-independent translation.

Cap-independent or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5' untranslated regions (5' UTRs) that directly bind DAP5 or eIF4GI. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem-loops, and the RNA-helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5' UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. However, the DAP5-binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which require an unpaired, accessible 5' end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186 nt FGF-9 5'-UTR RNA's complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV cross-linking experiments identify DAP5-RNA interactions. Modeling of FGF-9 5'-UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5-binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF-binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.

Architecture and dynamics of the abscisic acid gene regulatory network.

The plant hormone abscisic acid (ABA) regulates essential processes in plant development and responsiveness to abiotic and biotic stresses. ABA perception triggers a post-translational signaling cascade that elicits the ABA gene regulatory network (GRN), encompassing hundreds of transcription factors (TFs) and thousands of transcribed genes. To further our knowledge of this GRN, we performed an RNA-seq time series experiment consisting of 14 time points in the 16 h following a one-time ABA treatment of 5-week-old Arabidopsis rosettes. During this time course, ABA rapidly changed transcription levels of 7151 genes, which were partitioned into 44 coexpressed modules that carry out diverse biological functions. We integrated our time-series data with publicly available TF-binding site data, motif data, and RNA-seq data of plants inhibited in translation, and predicted (i) which TFs regulate the different coexpression clusters, (ii) which TFs contribute the most to target gene amplitude, (iii) timing of engagement of different TFs in the ABA GRN, and (iv) hierarchical position of TFs and their targets in the multi-tiered ABA GRN. The ABA GRN was found to be highly interconnected and regulated at different amplitudes and timing by a wide variety of TFs, of which the bZIP family was most prominent, and upregulation of genes encompassed more TFs than downregulation. We validated our network models in silico with additional public TF-binding site data and transcription data of selected TF mutants. Finally, using a drought assay we found that the Trihelix TF GT3a is likely an ABA-induced positive regulator of drought tolerance.

Identification and functional analysis of the LEAFY gene in longan flower induction.

BACKGROUND: Flowering at the right time is a very important factor affecting the stable annual yield of longan. However, a lack of knowledge of the regulatory mechanism and key genes of longan flowering restricts healthy development of the longan industry. Therefore, identifying relevant genes and analysing their regulatory mechanism are essential for scientific research and longan industry development. RESULTS: DlLFY (Dimocarpus longan LEAFY) contains a 1167 bp open reading frame and encodes 388 amino acids. The amino acid sequence has a typical LFY/FLO family domain. DlLFY was expressed in all tissues tested, except for the leaf, pericarp, and pulp, with the highest expression occurring in flower buds. Expression of DlLFY was significantly upregulated at the early flower induction stage in "SX" ("Shixia"). The results of subcellular localization and transactivation analysis showed that DlLFY is a typical transcription factor acting as a transcriptional activator. Moreover, overexpression of DlLFY in Arabidopsis promoted early flowering and restrained growth, resulting in reduced plant height and rosette leaf number and area in transgenic plants. DNA affinity purification sequencing (DAP-Seq) analysis showed that 13 flower-related genes corresponding to five homologous genes of Arabidopsis may have binding sites and be putative target genes. Among these five flower-related genes, only AtTFL1 (terminal flower 1) was strongly inhibited in transgenic lines. CONCLUSION: Taken together, these results indicate that DlLFY plays a pivotal role in controlling longan flowering, possibly by interacting with TFL1.

TREM1/DAP12 based novel multiple chain CAR-T cells targeting DLL3 show robust anti-tumour efficacy for small cell lung cancer.

Small cell lung cancer (SCLC), recognized as the most aggressive subtype of lung cancer, presents an extremely poor prognosis. Currently, patients with small cell lung cancer face a significant dearth of effective alternative treatment options once they experience recurrence and progression after first-line therapy. Despite the promising efficacy of immunotherapy, particularly immune checkpoint inhibitors in non-small cell lung cancer (NSCLC) and various other tumours, its impact on significantly enhancing the prognosis of SCLC patients remains elusive. DLL3 has emerged as a compelling target for targeted therapy in SCLC due to its high expression on the membranes of SCLC and other neuroendocrine carcinoma cells, with minimal to no expression in normal cells. Our previous work led to the development of a novel multiple chain chimeric antigen receptor (CAR) leveraging the TREM1 receptor and DAP12, which efficiently activated T cells and conferred potent cell cytotoxicity. In this study, we have developed a DLL3-TREM1/DAP12 CAR-T (DLL3-DT CAR-T) therapy, demonstrating comparable anti-tumour efficacy against SCLC cells in vitro. In murine xenograft and patient-derived xenograft models, DLL3-DT CAR-T cells exhibited a more robust tumour eradication efficiency than second-generation DLL3-BBZ CAR-T cells. Furthermore, we observed elevated memory phenotypes, induced durable responses, and activation under antigen-presenting cells in DLL3-DT CAR-T cells. Collectively, these findings suggest that DLL3-DT CAR-T cells may offer a novel and potentially effective therapeutic strategy for treating DLL3-expressing SCLC and other solid tumours.

Construction of Fe Nanoparticles Interfacial Layer on Micron Al Surface: Boosting the Efficient Energy Release of High-Energy DAP-4 as a Gradient Catalyst.

The high-energy (H2dabco)[NH4(ClO4)3] (DAP-4) with excellent energetic performance attracts wide attention from researchers. The investigation of its interaction with the Aluminum (Al) is of great importance. However, the higher ignition threshold of DAP-4 and the dense oxide layer (Al2O3) of Al severely limit the energy release efficiency of Al/DAP-4. In this study, a new idea to is first proposed to improve and adjust the thermal decomposition and combustion performance of Al/DAP-4 by constructing a highly dispersed iron (Fe) nanoparticle interfacial layer. It acts as a gradient catalyst to promote the thermal decomposition and combustion of DAP-4 and Al, and it also act as an oxygen transport channel to promote the contact and reaction of oxidizing gases with the internal reactive Al powder. It reduces the thermal decomposition temperature of Al@Fe-3/DAP-4 from 386.30 °C (Al/DAP-4) to 349.48 °C and leads to the vigorous combustion. Theoretical calculations show that Fe nanoparticle interfacial layer can facilitate the transport of oxygen through the established oxygen transport channels, and it can also significantly improve the energetic properties of Al@Fe-3/DAP-4 composites. In conclusion, the new approach is proposed to improve the performance of metal fuel/oxidizer composites by constructing interfacial layers, which is expected to promote their practical applications.

Untargeted mutagenesis of brassinosteroid receptor SbBRI1 confers drought tolerance by altering phenylpropanoid metabolism in Sorghum bicolor.

Drought is a critical issue in modern agriculture; therefore, there is a need to create crops with drought resilience. The complexity of plant responses to abiotic stresses, particularly in the field of brassinosteroid (BR) signalling, has been the subject of extensive research. In this study, we unveil compelling insights indicating that the BRASSINOSTEROID-INSENSITIVE 1 (BRI1) receptor in Arabidopsis and Sorghum plays a critical role as a negative regulator of drought responses. Introducing untargeted mutation in the sorghum BRI1 receptor (SbBRI1) effectively enhances the plant's ability to withstand osmotic and drought stress. Through DNA Affinity Purification sequencing (DAP-seq), we show that the sorghum BRI1-EMS-SUPPRESSOR 1 (SbBES1) transcription factor, a downstream player of the BR signalling, binds to a conserved G-box binding motif, and it is responsible for regulating BR homeostasis, as its Arabidopsis ortholog AtBES1. We further characterized the drought tolerance of sorghum bri1 mutants and decipher SbBES1-mediated regulation of phenylpropanoid pathway. Our findings suggest that SbBRI1 signalling serves a dual purpose: under normal conditions, it regulates lignin biosynthesis by SbBES1, but during drought conditions, BES1 becomes less active, allowing the activation of the flavonoid pathway. This adaptive shift improves the photosynthetic rate and photoprotection, reinforcing crop adaptation to drought.

DAP5 drives translation of specific mRNA targets with upstream ORFs in human embryonic stem cells.

Death associated protein 5 (DAP5/eIF4G2/NAT1) is a member of the eIF4G translation initiation factors that has been shown to mediate noncanonical and/or cap-independent translation. It is essential for embryonic development and for differentiation of embryonic stem cells (ESCs), specifically its ability to drive translation of specific target mRNAs. In order to expand the repertoire of DAP5 target mRNAs, we compared ribosome profiles in control and DAP5 knockdown (KD) human ESCs (hESCs) to identify mRNAs with decreased ribosomal occupancy upon DAP5 silencing. A cohort of 68 genes showed decreased translation efficiency in DAP5 KD cells. Mass spectrometry confirmed decreased protein abundance of a significant portion of these targets. Among these was KMT2D, a histone methylase previously shown to be essential for ESC differentiation and embryonic development. We found that nearly half of the cohort of DAP5 target mRNAs displaying reduced translation efficiency of their main coding sequences upon DAP5 KD contained upstream open reading frames (uORFs) that are actively translated independently of DAP5. This is consistent with previously suggested mechanisms by which DAP5 mediates leaky scanning through uORFs and/or reinitiation at the main coding sequence. Crosslinking protein-RNA immunoprecipitation experiments indicated that a significant subset of DAP5 mRNA targets bound DAP5, indicating that direct binding between DAP5 protein and its target mRNAs is a frequent but not absolute requirement for DAP5-dependent translation of the main coding sequence. Thus, we have extended DAP5's function in translation of specific mRNAs in hESCs by a mechanism allowing translation of the main coding sequence following upstream translation of short ORFs.

Ribosome profiling reveals novel regulation of C9ORF72 GGGGCC repeat-containing RNA translation.

GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-containing RNA is translated into dipeptide repeat (DPR) proteins, some of which are neurotoxic. Using dynamic ribosome profiling, we identified three translation initiation sites in the intron upstream of (G4C2) repeats; these sites are detected irrespective of the presence or absence of the repeats. During translocation, ribosomes appear to be stalled on the repeats. An AUG in the preceding C9ORF72 exon initiates a uORF that inhibits downstream translation. Polysome isolation indicates that unspliced (G4C2) repeat-containing RNA is a substrate for DPR protein synthesis. (G4C2) repeat-containing RNA translation is 5' cap-independent but inhibited by the initiation factor DAP5, suggesting an interplay with uORF function. These results define novel translational mechanisms of expanded (G4C2) repeat-containing RNA in disease.

Cap-independent translation by DAP5 controls cell fate decisions in human embryonic stem cells.

Multiple transcriptional and epigenetic changes drive differentiation of embryonic stem cells (ESCs). This study unveils an additional level of gene expression regulation involving noncanonical, cap-independent translation of a select group of mRNAs. This is driven by death-associated protein 5 (DAP5/eIF4G2/NAT1), a translation initiation factor mediating IRES-dependent translation. We found that the DAP5 knockdown from human ESCs (hESCs) resulted in persistence of pluripotent gene expression, delayed induction of differentiation-associated genes in different cell lineages, and defective embryoid body formation. The latter involved improper cellular organization, lack of cavitation, and enhanced mislocalized apoptosis. RNA sequencing of polysome-associated mRNAs identified candidates with reduced translation efficiency in DAP5-depleted hESCs. These were enriched in mitochondrial proteins involved in oxidative respiration, a pathway essential for differentiation, the significance of which was confirmed by the aberrant mitochondrial morphology and decreased oxidative respiratory activity in DAP5 knockdown cells. Further analysis identified the chromatin modifier HMGN3 as a cap-independent DAP5 translation target whose knockdown resulted in defective differentiation. Thus, DAP5-mediated translation of a specific set of proteins is critical for the transition from pluripotency to differentiation, highlighting the importance of cap-independent translation in stem cell fate decisions.

Highly Sensitive Measurement of Horseradish Peroxidase Using Surface-Enhanced Raman Scattering of 2,3-Diaminophenazine.

The development of various enzyme-linked immunosorbent assays (ELISAs) coupled with surface-enhanced Raman scattering (SERS) detection is a growing area in analytical chemistry due to their potentially high sensitivity. A SERS-based ELISA with horseradish peroxidase (HRP) as an enzymatic label, an o-phenylenediamine (oPD) substrate, and a 2,3-diaminophenazine (DAP) enzymatic product was one of the first examples of such a system. However, the full capabilities of this long-known approach have yet to be revealed. The current study addresses a previously unrecognized problem of SERS detection stage performance. Using silver nanoparticles and model mixtures of oPD and DAP, the effects of the pH, the concentration of the aggregating agent, and the particle surface chloride stabilizer were extensively evaluated. At the optimal mildly acidic pH of 3, a 0.93 to 1 M citrate buffer, and AgNPs stabilized with 20 mM chloride, a two orders of magnitude advantage in the limits of detection (LODs) for SERS compared to colorimetry was demonstrated for both DAP and HRP. The resulting LOD for HRP of 0.067 pmol/L (1.3 amol per assay) underscores that the developed approach is a highly sensitive technique. We suppose that this improved detection system could become a useful tool for the development of SERS-based ELISA protocols.

Exploring Camelina sativa lipid metabolism regulation by combining gene co-expression and DNA affinity purification analyses.

Camelina (Camelina sativa) is an annual oilseed plant that is gaining momentum as a biofuel cover crop. Understanding gene regulatory networks is essential to deciphering plant metabolic pathways, including lipid metabolism. Here, we take advantage of a growing collection of gene expression datasets to predict transcription factors (TFs) associated with the control of Camelina lipid metabolism. We identified approximately 350 TFs highly co-expressed with lipid-related genes (LRGs). These TFs are highly represented in the MYB, AP2/ERF, bZIP, and bHLH families, including a significant number of homologs of well-known Arabidopsis lipid and seed developmental regulators. After prioritizing the top 22 TFs for further validation, we identified DNA-binding sites and predicted target genes for 16 out of the 22 TFs tested using DNA affinity purification followed by sequencing (DAP-seq). Enrichment analyses of targets supported the co-expression prediction for most TF candidates, and the comparison to Arabidopsis revealed some common themes, but also aspects unique to Camelina. Within the top potential lipid regulators, we identified CsaMYB1, CsaABI3AVP1-2, CsaHB1, CsaNAC2, CsaMYB3, and CsaNAC1 as likely involved in the control of seed fatty acid elongation and CsaABI3AVP1-2 and CsabZIP1 as potential regulators of the synthesis and degradation of triacylglycerols (TAGs), respectively. Altogether, the integration of co-expression data and DNA-binding assays permitted us to generate a high-confidence and short list of Camelina TFs involved in the control of lipid metabolism during seed development.

Functional investigation of five R2R3-MYB transcription factors associated with wood development in Eucalyptus using DAP-seq-ML.

A multi-tiered transcriptional network regulates xylem differentiation and secondary cell wall (SCW) formation in plants, with evidence of both conserved and lineage-specific SCW network architecture. We aimed to elucidate the roles of selected R2R3-MYB transcription factors (TFs) linked to Eucalyptus wood formation by identifying genome-wide TF binding sites and direct target genes through an improved DAP-seq protocol combined with machine learning for target gene assignment (DAP-seq-ML). We applied this to five TFs including a well-studied SCW master regulator (EgrMYB2; homolog of AtMYB83), a repressor of lignification (EgrMYB1; homolog of AtMYB4), a TF affecting SCW thickness and vessel density (EgrMYB137; homolog of PtrMYB074) and two TFs with unclear roles in SCW regulation (EgrMYB135 and EgrMYB122). Each DAP-seq TF peak set (average 12,613 peaks) was enriched for canonical R2R3-MYB binding motifs. To improve the reliability of target gene assignment to peaks, a random forest classifier was developed from Arabidopsis DAP-seq, RNA-seq, chromatin, and conserved noncoding sequence data which demonstrated significantly higher precision and recall to the baseline method of assigning genes to proximal peaks. EgrMYB1, EgrMYB2 and EgrMYB137 predicted targets showed clear enrichment for SCW-related biological processes. As validation, EgrMYB137 overexpression in transgenic Eucalyptus hairy roots increased xylem lignification, while its dominant repression in transgenic Arabidopsis and Populus reduced xylem lignification, stunted growth, and caused downregulation of SCW genes. EgrMYB137 targets overlapped significantly with those of EgrMYB2, suggesting partial functional redundancy. Our results show that DAP-seq-ML identified biologically relevant R2R3-MYB targets supported by the finding that EgrMYB137 promotes SCW lignification in planta.

Dissecting the roles of EIF4G homologs reveals DAP5 as a modifier of CGG repeat-associated toxicity in a Drosophila model of FXTAS.

Neurodegeneration in Fragile X-associated tremor/ataxia syndrome (FXTAS) is caused by a CGG trinucleotide repeat expansion in the 5' UTR of FMR1. Expanded CGG repeat RNAs form stable secondary structures, which in turn support repeat-associated non-AUG (RAN) translation to produce toxic peptides. The parameters that impact RAN translation initiation efficiency are not well understood. Here we used a Drosophila melanogaster model of FXTAS to evaluate the role of the eIF4G family of eukaryotic translation initiation factors (EIF4G1, EIF4GII and EIF4G2/DAP5) in modulating RAN translation and CGG repeat-associated toxicity. DAP5 knockdown robustly suppressed CGG repeat-associated toxicity and inhibited RAN translation. Furthermore, knockdown of initiation factors that preferentially associate with DAP5 (such as EIF2ß, EIF3F and EIF3G) also selectively suppressed CGG repeat-induced eye degeneration. In mammalian cellular reporter assays, DAP5 knockdown exhibited modest and cell-type specific effects on RAN translation. Taken together, these data support a role for DAP5 in CGG repeat associated toxicity possibly through modulation of RAN translation.

Picturing local adaptation: Spectral and structural traits from drone remote sensing reveal clinal responses to climate transfer in common-garden trials of interior spruce (Picea engelmannii × glauca).

Common-garden trials of forest trees provide phenotype data used to assess growth and local adaptation; this information is foundational to tree breeding programs, genecology, and gene conservation. As jurisdictions consider assisted migration strategies to match populations to suitable climates, in situ progeny and provenance trials provide experimental evidence of adaptive responses to climate change. We used drone technology, multispectral imaging, and digital aerial photogrammetry to quantify spectral traits related to stress, photosynthesis, and carotenoids, and structural traits describing crown height, size, and complexity at six climatically disparate common-garden trials of interior spruce (Picea engelmannii × glauca) in western Canada. Through principal component analysis, we identified key components of climate related to temperature, moisture, and elevational gradients. Phenotypic clines in remotely sensed traits were analyzed as trait correlations with provenance climate transfer distances along principal components (PCs). We used traits showing clinal variation to model best linear unbiased predictions for tree height (R2 = .98-.99, root mean square error [RMSE] = 0.06-0.10 m) and diameter at breast height (DBH, R2 = .71-.97, RMSE = 2.57-3.80 mm) and generated multivariate climate transfer functions with the model predictions. Significant (p < .05) clines were present for spectral traits at all sites along all PCs. Spectral traits showed stronger clinal variation than structural traits along temperature and elevational gradients and along moisture gradients at wet, coastal sites, but not at dry, interior sites. Spectral traits may capture patterns of local adaptation to temperature and montane growing seasons which are distinct from moisture-limited patterns in stem growth. This work demonstrates that multispectral indices improve the assessment of local adaptation and that spectral and structural traits from drone remote sensing produce reliable proxies for ground-measured height and DBH. This phenotyping framework contributes to the analysis of common-garden trials towards a mechanistic understanding of local adaptation to climate.

The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus.

Filamentous fungi, such as Neurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.

Identification of crucial factors involved in Cynoglossus semilaevis sexual size dimorphism by GWAS and demonstration of zbed1 regulatory network by DAP-seq.

Sexual size dimorphism (SSD), whereby females and males exhibit different body sizes, are widely documented in animals. To explore crucial regulators implicated in female-biased SSD of Chinese tongue sole (Cynoglossus semilaevis), GWAS was conducted on 350 females and 59 males. Twenty SNPs and 25 genes including zbed1, nsd3, cdc45, klhl29, and smad4 with -log(p) > 7 were screened, mainly mapping to sex chromosome. The chromosome W-linked gene zbed1 attracted particular attention because it is a master key for cell proliferation. Thus, the regulatory network of zbed1 in C. semilaevis was explored by DAP-seq and 1352 peaks were discovered in the female brain. Moreover, zbed1 potentially regulated hippo signaling pathway, cell cycle, translation, and PI3k-Akt signaling pathway in C. semilaevis. These findings identify crucial SNPs and genes associated with female-biased SSD in C. semilaevis, also provide the first genome-wide survey for the zbed1 regulatory network in fish species.

iE-DAP Induced Inflammatory Response and Tight Junction Disruption in Bovine Mammary Epithelial Cells via NOD1-Dependent NF-κB and MLCK Signaling Pathway.

γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), a bacterial cell wall component, can trigger an inflammatory response. A mammary inflammatory response causes tight junction (TJ) dysfunction. This study aimed to explore the effects and involved mechanisms of iE-DAP-induced inflammatory response on the TJ integrity in bovine mammary epithelial cells (BMECs). The results showed that iE-DAP-induced inflammatory response and TJ disruption was associated with increased expression levels of inflammatory cytokines and decreased gene expression of ZO-1 and Occludin, as well as a reduction in transepithelial electrical resistance and elevation in paracellular dextran passage. While MLCK inhibitor ML-7 reversed the TJ disruption induced by iE-DAP. NF-κB inhibitor BAY 11-7085 hindered the activation of NF-κB and MLCK signaling pathways, the inflammatory response and TJ disruption induced by iE-DAP. NOD1-specific shRNA also inhibited the activation of the NOD1/NF-κB signaling pathway and reversed the inflammatory response and TJ injury in iE-DAP-treated BMECs. Above results suggest that iE-DAP activated the NF-κB and MLCK signaling pathway in NOD1-dependent manner, which promoted the transcription of inflammatory cytokines and altered the expression and distribution of tight junction proteins, finally caused inflammatory response and TJ disruption. This study might provide theoretical basis and scientific support for the prevention and treatment of mastitis.

Development and Validation of a New UFLC-MS/MS Method for the Detection of Organophosphate Pesticide Metabolites in Urine.

To monitor human exposure to pesticides, experts commonly measure their metabolites in urine, particularly dialkyl phosphates (DAPs), which include diethyl phosphate (DEP), Diethyl thiophosphate (DETP), diethyl dithiophosphate (DEDTP), dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP) and dimethyl dithiophosphate (DMDTP)to monitor the metabolites of organophosphates. These DAP metabolites are a urinary biomarker for assessing pesticide exposure and potential health risks. This study presented a new screening method combining ultrafast liquid chromatography with tandem mass spectrometry (UFLC-MS/MS) to detect six DAP metabolites in human urine. The study also compared standard sample extraction methods, namely, liquid-liquid extraction (LLE); quick, easy, cheap, effective, ruggedand safe (QuEChERS); and lyophilization. After a comprehensive analysis of the methods used to extract the analytes, including recovery rate, repeatability and reproducibility, the liquid-liquid extraction (LLE) method was found to be the best. It had a high recovery rate, was easy to handle, required less sample volume and had a short extraction time. Therefore, the LLE method was chosen for further analysis. The results showed excellent performance with high recoveries between 93% and 102%, precise repeatability (RSD) between 0.62% and 5.46% and acceptable reproducibility values (RSD) between 0.80% and 11.33%. The method also had limits of detection (LOD) ranging from 0.0201 ng/mL to 0.0697 ng/mL and limits of quantification (LOQ) ranging from 0.0609 ng/mL to 0.2112 ng/mL. Furthermore, the UFLC-MS/MS method was validated based on the SANTE guidance and successfully analyzed 150 urine samples from farmers and non-farmers. This validated method proved useful for biomonitoring studies focusing on OP pesticide exposure. It offers several advantages, such as a reduced need for samples, chemicals and materials, and a shorter analysis time. The method is sensitive and selective in detecting metabolites in human urine, making it a valuable approach for the practical and efficient assessment of pesticide exposure.

Use of secondary air kerma from dose area product and fluoroscopy time for preventive effective dose estimation in interventional radiology procedures for staff.

INTRODUCTION: interventional radiology workers are potentially exposed to high levels of ionizing radiation, therefore preventive dose estimation is mandatory for the correct risk classification of staff. Effective dose (ED) is a radiation protection quantity strictly related to the secondary air kerma (KS), using appropriate multiplicative conversion factors (ICRP 106). The aim of this work is to evaluate the accuracy ofKSestimated from physically measurable quantities such as dose-area product (DAP) or fluoroscopy time (FT). METHODS: radiological units (n= 4) were characterized in terms of primary beam air kerma and DAP-meter response, consequently defining a DAP-meter correction factor (CF) for each unit.KS, scattered from an anthropomorphic phantom and measured by a digital multimeter, was then compared with the value estimated from DAP and FT. Different combinations of tube voltages, field sizes, current and scattering angles were used to simulate the variation of working conditions. Further measurements were performed to estimate the couch transmission factor for different phantom placements on the operational couch, defining a CF as the mean transmission factor. RESULTS: when no CFs were applied, the measuredKSshowed a median percentage difference of between 33.8% and 115.7% with respect toKSevaluated from DAP, and between -46.3% and 101.8% forKSevaluated from FT. By contrast, when previously defined CFs were applied to the evaluatedKS, the median percentage difference between the measuredKSand the value evaluated from DAP ranged from between -7.94% and 15.0%, and between -66.2% and 17.2% for that evaluated from FT. CONCLUSION: when appropriate CF are applied, the preventive ED estimation from the median DAP value seems to be more conservative and easier to obtain with respect to the one obtained from the FT value. Further measurements should be performed with a personal dosimeter during routine activities to assess the properKSto ED conversion factor.