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Type III-A CRISPR-Cas surveillance complexes containing multi-subunit Csm effector, guide, and target RNAs exhibit multiple activities, including formation of cyclic-oligoadenylates (cAn) from ATP and subsequent cAn-mediated cleavage of single-strand RNA (ssRNA) by the trans-acting Csm6 RNase. Our structure-function studies have focused on Thermococcus onnurineus Csm6 to deduce mechanistic insights into how cA4 binding to the Csm6 CARF domain triggers the RNase activity of the Csm6 HEPN domain and what factors contribute to regulation of RNA cleavage activity. We demonstrate that the Csm6 CARF domain is a ring nuclease, whereby bound cA4 is stepwise cleaved initially to ApApApA>p and subsequently to ApA>p in its CARF domain-binding pocket, with such cleavage bursts using a timer mechanism to regulate the RNase activity of the Csm6 HEPN domain. In addition, we establish T. onnurineus Csm6 as an adenosine-specific RNase and identify a histidine in the cA4 CARF-binding pocket involved in autoinhibitory regulation of RNase activity.
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Nucleótidos de Adenina/química , Proteínas Arqueales/química , Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas , Oligorribonucleótidos/química , Ribonucleasas/química , Thermococcus/química , Sitios de Unión , Dominios ProteicosRESUMEN
AIOLOS, a vital member of the IKAROS protein family, plays a significant role in lymphocyte development and function through DNA binding and protein-protein interactions. Mutations in the IKZF3 gene, which encodes AIOLOS, lead to a rare combined immunodeficiency often linked with infections and malignancy. In this study, we evaluated a 1-year-4-month-old female patient presenting with recurrent infections, diarrhea, and failure to thrive. Laboratory investigations revealed decreased T lymphocyte and immunoglobulin levels. Through whole-exome and Sanger sequencing, we discovered a de novo mutation in IKZF3 (NM_012481; exon 5 c.571G > C, p.Gly191Arg), corresponding to the third DNA-binding zinc finger region of the encoded protein AIOLOS. Notably, the patient with the AIOLOS G191R mutation showed reduced recent thymic emigrants in naïve CD4+T cells compared to healthy counterparts of the same age, while maintaining normal levels of Th1, Th2, Th17, Treg, and Tfh cells. This mutation also resulted in decreased switched memory B cells and lower CD23 and IgM expression. In vitro studies revealed that AIOLOS G191R does not impact the expression of AIOLOS but compromises its stability, DNA binding and pericentromeric targeting. Furthermore, AIOLOS G191R demonstrated a dominant-negative effect over the wild-type protein. This case represents the first reported instance of a mutation in the third DNA-binding zinc finger region of AIOLOS highlighting its pivotal role in immune cell functionality.
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Factor de Transcripción Ikaros , Mutación , Humanos , Factor de Transcripción Ikaros/genética , Femenino , Mutación/genética , Lactante , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/diagnóstico , Secuenciación del Exoma , Linfocitos B/inmunologíaRESUMEN
Actinobacterial 2-hydroxyacyl-CoA lyase reversibly catalyzes the thiamine diphosphate-dependent cleavage of 2-hydroxyisobutyryl-CoA to formyl-CoA and acetone. This enzyme has great potential for use in synthetic one-carbon assimilation pathways for sustainable production of chemicals, but lacks details of substrate binding and reaction mechanism for biochemical reengineering. We determined crystal structures of the tetrameric enzyme in the closed conformation with bound substrate, covalent postcleavage intermediate, and products, shedding light on active site architecture and substrate interactions. Together with molecular dynamics simulations of the covalent precleavage complex, the complete catalytic cycle is structurally portrayed, revealing a proton transfer from the substrate acyl Cß hydroxyl to residue E493 that returns it subsequently to the postcleavage Cα-carbanion intermediate. Kinetic parameters obtained for mutants E493A, E493Q, and E493K confirm the catalytic role of E493 in the WT enzyme. However, the 10- and 50-fold reduction in lyase activity in the E493A and E493Q mutants, respectively, compared with WT suggests that water molecules may contribute to proton transfer. The putative catalytic glutamate is located on a short α-helix close to the active site. This structural feature appears to be conserved in related lyases, such as human 2-hydroxyacyl-CoA lyase 2. Interestingly, a unique feature of the actinobacterial 2-hydroxyacyl-CoA lyase is a large C-terminal lid domain that, together with active site residues L127 and I492, restricts substrate size to ≤C5 2-hydroxyacyl residues. These details about the catalytic mechanism and determinants of substrate specificity pave the ground for designing tailored catalysts for acyloin condensations for one-carbon and short-chain substrates in biotechnological applications.
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Acilcoenzima A , Liasas , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Carbono , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Liasas/química , Liasas/metabolismo , Protones , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The intestinal microbiota plays significant role in the physiology and functioning of host organisms. However, there is limited knowledge of the composition and evolution of microbiota-host relationships from wild ancestors to modern domesticated species. In this study, the 16S rRNA gene V3-V4 in the intestinal contents of different pig breeds was analyzed and was compared using high-throughput sequencing. This identified 18 323 amplicon sequence variants, of which the Firmicutes and Actinobacteria phyla and Bifidobacterium and Allobaculum genera were most prevalent in wild pigs (WP). In contrast, Proteobacteria and Firmicutes predominated in Chinese Shanxi Black pigs (CSB), while Firmicutes were the most prevalent phylum in Large White pigs (LW) and Iberian pigs (IB), followed by Bacteroidetes in IB and Proteobacteria in LW. At the genus level, Shigella and Lactobacillus were most prevalent in CSB and LW, while Actinobacillus and Sarcina predominated in IB. Differential gene expression together with phylogenetic and functional analyses indicated significant differences in the relative abundance of microbial taxa between different pig breeds. Although many microbial taxa were common to both wild and domestic pigs, significant diversification was observed in bacterial genes that potentially influence host phenotypic traits. Overall, these findings suggested that both the composition and functions of the microbiota were closely associated with domestication and the evolutionary changes in the host. The members of the microbial communities were vertically transmitted in pigs, with evidence of co-evolution of both the hosts and their intestinal microbial communities. These results enhance our understanding and appreciation of the complex interactions between intestinal microbes and hosts and highlight the importance of applying this knowledge in agricultural and microbiological research.
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Organic aerosol (OA) generally accounts for a large fraction of fine particulate matter (PM2.5) in the urban atmosphere. Despite significant advances in the understanding their emission sources, transformation processes and optical properties in the submicron aerosol fraction (PM1), larger size fractions - e.g., PM2.5 - still deserve complementary investigations. In this study, we conducted a comprehensive analysis on sources, formation process and optical properties of OA in PM1 and PM2.5 under haze and foggy environments in the Yangtze River Delta (eastern China), using two aerosol chemical speciation monitors, as well as a photoacoustic extinctiometer at 870 nm. Positive matrix factorization analysis - using multilinear engine (ME2) algorithm - was conducted on PM1 and PM2.5 organic mass spectra. Four OA factors were identified, including three primary OA (POA) factors, i.e., hydrocarbon-like OA (HOA), cooking OA (COA), and biomass burning OA (BBOA), and a secondary OA (SOA) factor, i.e., oxidized oxygenated OA (OOA). An enhanced PM1-2.5 COA concentration was clearly observed during cooking peak hours, suggesting important contribution of fresh cooking emissions on large-sized particles (i.e., PM1-2.5). The oxidation state and concentration of PM2.5 HOA were higher than that in PM1, suggesting that large-sized HOA particles might be linked to oxidized POA. High contribution (44%) of large-sized OOA to non-refractory PM2.5 mass was observed during haze episodes. During foggy episodes, PM1 and PM2.5 OOA concentrations increased as a positive relationship over time, along with an exponential increase in the PM2.5-OOA to PM1-OOA ratio. Meanwhile, OOA loadings increased with the aerosol liquid water content (ALWC) during foggy episodes. Random forest cross-validation analysis also supported the important influence of ALWC on OOA variations, supporting substantial impact of aqueous process on SOA formation during haze and/or foggy episodes. Obtained results also indicated high OOA contributions (21%-36%) and low POA contributions (6%-14%) to the PM2.5 scattering coefficient during haze and foggy episodes, respectively. Finally, we could illustrate that atmospheric vertical diffusion and horizontal transport have important but different effects on the concentrations of different primary and secondary OA factors in different particle size fractions.
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Contaminantes Atmosféricos , Aerosoles/análisis , Contaminantes Atmosféricos/análisis , China , Monitoreo del Ambiente/métodos , Material Particulado/análisis , RíosRESUMEN
Wind profile radar systems require antennas with multiple radiation beams for detecting wind velocity, as well as with a low sidelobe and dual polarization for enhancing the sensitivity for the weak signal reflected from the turbulence. This paper proposes a lens antenna operating at 24 GHz with four reconfigurable beams for wind profile radars. This lens antenna includes 2 × 2 corrugated horn antennas for radiating 24 GHz waves in two polarizations, and the dielectric lens for modulating four radiation beams with a high gain and low sidelobe. Experiments demonstrate that this lens antenna can realize reconfigurable beams with deflections of ±15° in dual polarizations, meanwhile with the gain of 30.58 dBi and the sidelobe of -20 dB. This proposed lens antenna can be applied to mmWave wind profile radars of wind turbines for enhancing wind power efficiency.
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A dual-polarized continuous transverse stub (CTS) K-band antenna with reconfigurable four beams and low profile is proposed based on substrate-integrated-waveguide (SIW) design. It consists of a line source generator (LSG) on the bottom surface, a spherical-wave to plane-wave transforming part on the middle layer, and CTS radiators on the top surface. Particularly, the LSG has four SIW-based H-plane horns, and a chip is integrated to switch among the two pairs of horns, so as to transfer the quasi-TEM waves on the bottom surface by a ±10° deflection angle to the middle layer for the CTS radiators on the top surface, resulting in four reconfigurable scanning beams with 10° for two polarizations. The measurements show that it realizes four reconfigurable beams with a 25.8 dBi gain at 24 GHz, verifying the design. The proposed antenna takes into account the advantages of reconfigurable multi-beam, dual polarization, low side lobes, low profile, and high gain, which can be applied to K-band sensing, especially for wind profile radars.
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Metamaterial antennas consisting of periodical units are suitable for achieving tunable properties by employing active elements to each unit. However, for compact metamaterials with a very limited number of periodical units, resonance blindness exists. In this paper, we introduce a method to achieve continuous tuning without resonance blindness by exploring hence, taking advantage of nonlinear properties of PIN diodes. First, we obtain the equivalent impedance of the PIN diode through measurements, then fit these nonlinear curves with mathematical expressions. Afterwards, we build the PIN diode model with these mathematical equations, making it compatible with implementing co-simulation between the passive electromagnetic model and the active element of PIN diodes and, particularly, the nonlinear effects can be considered. Next, we design a compact two-unit metamaterial antenna as an example to illustrate the electromagnetic co-simulation. Finally, we implement the experiments with a micro-control unit to validate this method. In addition, the nonlinear stability and the supplying voltage tolerance of nonlinear states for both two kinds of PIN diodes are investigated as well. This method of obtaining smooth tuning with nonlinear properties of PIN diodes can be applied to other active devices, if only PIN diodes are utilized.
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Commensal microorganisms are essential to the normal development and function of many aspects of animal biology. However, the dynamic shift patterns of the microbiota of different gut segments in sheep and the correlation between fat type large-tailed phenotype and microbiota remain poorly unknown. This study therefore sought to assess the composition and distribution of the intestinal microbiome, and compared the difference of gut microbiota from different gastrointestinal segments within breeds and same intestinal sections between breeds. For these analyses, 16S rRNA V4 regions from 4 gut sections prepared from each of six individuals (3 from each breed) were sequenced to detect the microbiome composition in these samples. These analyses revealed the presence of 51,173 operational taxonomic units distributed across 24 phyla and 420 genera in these samples, with Firmicutes and Bacteroidetes being the most prevalent phyla of microbes present in these samples. Moreover, the bacterial composition showed distinct microbial communities in different gastrointestinal segments within breed, but showed similar and relative fixed bacterial abundance in the same intestinal segments from individuals of different breeds. We also found that only a few bacterial species (Lachnospiraceae, Akkermansia) were needed to distinguish between Small-tailed Han sheep (STH) and Large-tailed Han sheep (LTH) and their metabolic process maybe influence the fat type large-tailed phenotype formation in sheep. The functional profile analysis revealed that the environment information processing, genetic information processing, and metabolic pathways were enriched in all samples. The main functional roles of the gut microbiota were amino acid metabolism, replication and repair, carbohydrate metabolism, and membrane transport. Finally, our findings suggested that distinguished gut species between STH and LTH have relative fixed and the potential correlation is existing between the intestinal microorganisms and the large-tailed phenotype trait formation of sheep, which may offer clues for further investigation to detect the roles of intestinal microbiota in the metabolism and fat deposition in the tail of sheep.
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Brucellae are intracellular bacterial pathogens that cause Brucellosis, bringing great economic burdens to developing countries. The pathogenic mechanisms of Brucella are still poorly understood. Earlier immune response plays an important role in the Brucella infection. Phosphoglyceromutase (PGM) and dihydrodipicolinate reductase (DapB) were cloned, expressed, purified, and their immunocompetence was analyzed. Cytokines were detected by murine macrophages (RAW 264.7) and splenocytes that stimulated with the two recombinant proteins. The immune responses were analyzed by ELISA from mice with the two recombinant proteins immunized. TNF-α, IL-6 and IL-8 were produced in stimulated RAW 264.7 cells and splenocytes. Th1-type cytokines, IFN-γ and IL-2, induced in RAW 264.7 cells and splenocytes were higher then Th2-type cytokines, IL-4 and IL-5. Th2-related immune response was induced in splenocytes obtained 35 days after mice immunized with the two proteins. The production of IgG1 was higher than IgG2a in immunized mice. Taken together, our results demonstrated that the two proteins could induce Th1 and Th2-type immune responses in vivo and in vitro.
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Brucella abortus/enzimología , Brucella abortus/inmunología , Brucelosis/inmunología , Dihidrodipicolinato-Reductasa/farmacología , Fosfoglicerato Mutasa/farmacología , Células TH1/efectos de los fármacos , Células Th2/efectos de los fármacos , Animales , Brucella abortus/genética , Brucelosis/microbiología , China , Clonación Molecular , Citocinas/inmunología , Citocinas/metabolismo , Dihidrodipicolinato-Reductasa/genética , Femenino , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Inmunización , Inmunoglobulina G , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Fosfoglicerato Mutasa/genética , Células RAW 264.7/efectos de los fármacos , Proteínas Recombinantes/inmunología , Células TH1/inmunología , Células Th2/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Brucellosis is an important zoonotic disease of worldwide distribution, which causes animal and human disease. However, the current Brucella abortus (B. abortus) vaccines (S19 and RB51) have several drawbacks, including residual virulence for animals and humans. Moreover, S19 cannot allow serological differentiation between infected and vaccinated animals. We constructed double deletion (ΔNodVΔNodW) mutant from virulent B. abortus 2308 (S2308) by deleting the genes encoding two-component regulatory system (TCS) in chromosome II in S2308.2308ΔNodVΔNodW was significantly reduced survival in murine macrophages (RAW 264.7) and BALB/c mice. Moreover, the inoculated mice showed no splenomegaly. The mutant induced high protective immunity in BALB/c mice against challenge with S2308, and elicited an anti-Brucella-specific immunoglobulin G (IgG) response and induced the secretion of gamma interferon (IFN-γ) and interleukin-4 (IL-4). Moreover, NODV and NODW antigens would allow the serological differentiation between infected and vaccinated animals. These results suggest that 2308ΔNodVΔNodW mutant is a potential live attenuated vaccine candidate and can be used effectively against bovine brucellosis.
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Vacuna contra la Brucelosis/inmunología , Brucella abortus/genética , Brucella abortus/inmunología , Brucelosis/inmunología , Brucelosis/prevención & control , Vacunas Atenuadas/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Vacuna contra la Brucelosis/genética , Brucelosis/sangre , Brucelosis Bovina/prevención & control , Bovinos , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Mutación , Células RAW 264.7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Bazo/inmunología , Vacunas Atenuadas/genética , Virulencia , Factores de Virulencia/genéticaRESUMEN
Despite much attention, history of sheep (Ovis aries) evolution, including its dating, demographic trajectory and geographic spread, remains controversial. To address these questions, we generated 45 complete and 875 partial mitogenomic sequences, and performed a meta-analysis of these and published ovine mitochondrial DNA sequences (n = 3,229) across Eurasia. We inferred that O. orientalis and O. musimon share the most recent female ancestor with O. aries at approximately 0.790 Ma (95% CI: 0.637-0.934 Ma) during the Middle Pleistocene, substantially predating the domestication event (â¼8-11 ka). By reconstructing historical variations in effective population size, we found evidence of a rapid population increase approximately 20-60 ka, immediately before the Last Glacial Maximum. Analyses of lineage expansions showed two sheep migratory waves at approximately 4.5-6.8 ka (lineages A and B: â¼6.4-6.8 ka; C: â¼4.5 ka) across eastern Eurasia, which could have been influenced by prehistoric West-East commercial trade and deliberate mating of domestic and wild sheep, respectively. A continent-scale examination of lineage diversity and approximate Bayesian computation analyses indicated that the Mongolian Plateau region was a secondary center of dispersal, acting as a "transportation hub" in eastern Eurasia: Sheep from the Middle Eastern domestication center were inferred to have migrated through the Caucasus and Central Asia, and arrived in North and Southwest China (lineages A, B, and C) and the Indian subcontinent (lineages B and C) through this region. Our results provide new insights into sheep domestication, particularly with respect to origins and migrations to and from eastern Eurasia.
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Migración Animal/fisiología , Genómica , Mitocondrias/genética , Ovinos/genética , Animales , Animales Domésticos/genética , ADN Mitocondrial/genética , Femenino , Variación Genética , Geografía , Metaanálisis como Asunto , Modelos Genéticos , Filogenia , Selección Genética , Factores de TiempoRESUMEN
Variation in two SNPs and one microsatellite on the Y chromosome was analyzed in a total of 663 rams representing 59 breeds from a large geographic range in northern Eurasia. SNPA-oY1 showed the highest allele frequency (91.55%) across the breeds, whereas SNPG-oY1 was present in only 56 samples. Combined genotypes established seven haplotypes (H4, H5, H6, H7, H8, H12 and H19). H6 dominated in northern Eurasia, and H8 showed the second-highest frequency. H4, which had been earlier reported to be absent in European breeds, was detected in one European breed (Swiniarka), whereas H7, which had been previously identified to be unique to European breeds, was present in two Chinese breeds (Ninglang Black and Large-tailed Han), one Buryatian (Transbaikal Finewool) and two Russian breeds (North Caucasus Mutton-Wool and Kuibyshev). H12, which had been detected only in Turkish breeds, was also found in Chinese breeds in this work. An overall low level of haplotype diversity (median h = 0.1288) was observed across the breeds with relatively higher median values in breeds from the regions neighboring the Near Eastern domestication center of sheep. H6 is the dominant haplotype in northwestern and eastern China, in which the haplotype distribution could be explained by the historical translocations of the H4 and H8 Y chromosomes to China via the Mongol invasions followed by expansions to northwestern and eastern China. Our findings extend previous results of sheep Y chromosomal genetic variability and indicate probably recent paternal gene flows between sheep breeds from distinct major geographic regions.
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Haplotipos , Oveja Doméstica/genética , Cromosoma Y/genética , Animales , Asia , Europa (Continente) , Frecuencia de los Genes , Masculino , Repeticiones de Microsatélite , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , Oveja Doméstica/clasificaciónRESUMEN
Migrastatin is a biologically active natural product isolated from Streptomyces that has been shown to inhibit tumor cell migration. Upon completion of the first total synthesis of migrastatin, a number of structurally simplified analogs were prepared. Following extensive in vitro screening, a new generation of analogs was identified that demonstrates substantially higher levels of in vitro inhibitory activity, stability and synthetic accessibility when compared to the parent natural product. Herein, we describe two promising ether-derivative analogs, the migrastatin core ether (ME) and the carboxymethyl-ME (CME), which exhibit high efficacy in blocking tumor cell migration and metastasis in lung cancer. These compounds show an in vitro migration inhibition in the micromolar range (IC(50): ME 1.5 to 8.2 µM, CME 0.5 to 5 µM). In a human small-cell lung carcinoma (SCLC) primary xenograft model, ME and CME compounds were found to be highly potent in inhibiting overall metastasis even at the lowest dosage used (degree of inhibition: 96.2% and 99.3%, respectively). Together these very encouraging findings suggest that these analogs have promise as potent antimetastatic agents in lung cancer.
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Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Macrólidos/síntesis química , Macrólidos/uso terapéutico , Metástasis de la Neoplasia/tratamiento farmacológico , Piperidonas/síntesis química , Piperidonas/uso terapéutico , Animales , Línea Celular Tumoral , Movimiento Celular , Éteres/síntesis química , Éteres/química , Humanos , Macrólidos/química , Masculino , Ratones , Metástasis de la Neoplasia/patología , Piperidonas/química , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We previously described four small molecules that reduced the growth of lung adenocarcinoma cell lines with either epidermal growth factor receptor (EGFR) or KRAS mutations in a high-throughout chemical screen. By combining affinity proteomics and gene expression analysis, we now propose superoxide dismutase 1 (SOD1) as the most likely target of one of these small molecules, referred to as lung cancer screen 1 (LCS-1). siRNAs against SOD1 slowed the growth of LCS-1 sensitive cell lines; conversely, expression of a SOD1 cDNA increased proliferation of H358 cells and reduced sensitivity of these cells to LCS-1. In addition, SOD1 enzymatic activity was inhibited in vitro by LCS-1 and two closely related analogs. These results suggest that SOD1 is an LCS-1-binding protein that may act in concert with mutant proteins, such as EGFR and KRAS, to promote cell growth, providing a therapeutic target for compounds like LCS-1.
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Adenocarcinoma/patología , División Celular/efectos de los fármacos , Neoplasias Pulmonares/patología , Superóxido Dismutasa/metabolismo , Adenocarcinoma/enzimología , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Neoplasias Pulmonares/enzimología , ARN Interferente Pequeño/genética , Superóxido Dismutasa/efectos de los fármacos , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
Profiling antibiotic resistance genes (ARGs) in the Yellow River of China's Henan Province is essential for understanding the health risks of antibiotic resistance. The profiling of ARGs was investigated using high-throughput qPCR from water samples in seven representative regions of the Yellow River. The absolute and relative abundances of ARGs and moble genetic elements (MGEs) were higher in summer than in winter (ANOVA, p < 0.001). The diversity and abundance of ARGs were higher in the Yellow River samples from PY and KF than the other sites. Temperature (r = 0.470 ~ 0.805, p < 0.05) and precipitation (r = 0.492 ~ 0.815, p < 0.05) positively influenced the ARGs, while pH had a negative effect (r = - 0.462 ~ - 0.849, p < 0.05). Network analysis indicated that the pathogenic bacteria Rahnella, Bacillus, and Shewanella were the possible hub hosts of ARGs, and tnpA1 was the potential MGE hub. These findings provide insights into the factors influencing ARG dynamics and the complex interaction among the MGEs, pathogenic bacteria and environmental parameters in enriching ARGs in the Yellow River of Henan Province.
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Farmacorresistencia Microbiana , Ríos , Ríos/microbiología , China , Farmacorresistencia Microbiana/genética , Genes Bacterianos , Estaciones del Año , Farmacorresistencia Bacteriana/genética , Bacterias/genética , Bacterias/efectos de los fármacos , Bacterias/clasificación , Antibacterianos/farmacología , Microbiología del AguaRESUMEN
Four experiments were performed to investigate the role of the mitogen-activated protein kinase (MAPK) signaling pathway in intestinal absorption of phosphorus (P) and calcium (Ca) in broiler chickens. Experiment 1 assessed how dietary levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) influence the gene expression of intestinal P and Ca transporters in broilers. Experiment 2 evaluated the effects of 1,25(OH)2D3 administered via intraperitoneal injection on the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways. Experiments 3 and 4 investigated the effect of ERK and p38MAPK inhibitors on the expression of intestinal P and Ca transporters. The findings demonstrated that broilers (1-21 days old) fed a 1,25(OH)2D3-deficient diet (0.625 µg/kg) exhibited reduced body weight, tibia P and Ca levels, and mRNA levels of P transporters (NaPi-IIb, PiT-1, and PiT-2), Ca transporters (NCX1, PMCA1b, and CaBP-D28k), vitamin D receptors (VDR), ERK, and p38MAPK in the duodenum (Experiment 1) (P < 0.05). By comparison, the growth, bone quality, and mRNA levels of genes (except for duodenal NaPi-IIb) in broilers were similar to those in broilers fed the control diet when dietary 1,25(OH)2D3 was adequate (5 µg/kg) (Experiment 1) (P > 0.05). After intraperitoneal injection of 1,25(OH)2D3, the mRNA level of jejunal NaPi-IIb and the protein level of p-p38MAPK/t-p38MAPK in broilers (9-14 days old) decreased (P < 0.05), whereas the mRNA level of CaBP-D28k and the protein level of p-ERK/t-ERK increased (Experiment 2) (P < 0.05). The mRNA and protein expression of jejunal NaPi-IIb and the protein expression of CaBP-D28k in broilers (9-17 days old) treated with the ERK inhibitor PD98059 were greater than those in the control group (Experiment 3) (P < 0.05). Similarly, compared with control broilers, broilers (9-17 days old) treated with the p38MAPK inhibitor SB203580 showed elevated mRNA expression of jejunal NaPi-IIb and CaBP-D28k (Experiment 4) (P < 0.05). These results suggest that adequate supplementation with 1,25(OH)2D3 (5 µg/kg) can restore broiler growth and bone quality by upregulating the transcription of genes involved in intestinal P and Ca absorption. Additionally, the ERK and p38MAPK signaling pathways are implicated in the modulatory effect of 1,25(OH)2D3 on the absorption of P and Ca in broilers.
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Brucellosis is a zoonotic disease that affects wild and domestic animals. It is caused by members of the bacterial genus Brucella. Guanylate-binding protein 1 (GBP1) is associated with microbial infections. However, the role of GBP1 during Brucella infection remains unclear. This investigation aimed to identify the association of GBP1 with brucellosis. Results showed that Brucella infection induced GBP1 upregulation in RAW 264.7 murine macrophages. Small interfering GBP1 targeting RNAs were utilized to explore how GBP1 regulates the survival of Brucella intracellularly. Results revealed that GBP1 knockdown promoted Brucella's survival ability, activated Nod-like receptor (NLR) containing a pyrin domain 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammatory corpuscles, and induced pro-inflammatory cytokines IFN-γ and IL-1ß. Furthermore, Brucella stimulated the expression of GBP1 in bone marrow-derived macrophages (BMDMs) and mice. During the inhibition of GBP1 in BMDMs, the intracellular growth of Brucella increased. In comparison, GBP1 downregulation enhanced the accumulation of Brucella-induced reactive oxygen species (ROS) in macrophages. Overall, the data indicate a significant role of GBP1 in regulating brucellosis and suggest the function underlying its suppressive effect on the survival and growth of Brucella intracellularly.