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Lung group 2 innate lymphoid cells (ILC2s) control the nature of immune responses to airway allergens. Some microbial products, including those that stimulate interferons, block ILC2 activation, but whether this occurs after natural infections or causes durable ILC2 inhibition is unclear. In the present study, we cohoused laboratory and pet store mice as a model of physiological microbial exposure. Laboratory mice cohoused for 2 weeks had impaired ILC2 responses and reduced lung eosinophilia to intranasal allergens, whereas these responses were restored in mice cohoused for ≥2 months. ILC2 inhibition at 2 weeks correlated with increased interferon receptor signaling, which waned by 2 months of cohousing. Reinduction of interferons in 2-month cohoused mice blocked ILC2 activation. These findings suggest that ILC2s respond dynamically to environmental cues and that microbial exposures do not control long-term desensitization of innate type 2 responses to allergens.
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Alérgenos , Inmunidad Innata , Ratones , Animales , Linfocitos , Citocinas , Pulmón , Interferones , Interleucina-33RESUMEN
Bacterial flagella and type IV pili (TFP) are surface appendages that enable motility and mechanosensing through distinct mechanisms. These structures were previously thought to have no components in common. Here, we report that TFP and some flagella share proteins PilO, PilN, and PilM, which we identified as part of the Helicobacter pylori flagellar motor. H. pylori mutants lacking PilO or PilN migrated better than wild type in semisolid agar because they continued swimming rather than aggregated into microcolonies, mimicking the TFP-regulated surface response. Like their TFP homologs, flagellar PilO/PilN heterodimers formed a peripheral cage that encircled the flagellar motor. These results indicate that PilO and PilN act similarly in flagella and TFP by differentially regulating motility and microcolony formation when bacteria encounter surfaces.
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Proteínas Bacterianas , Fimbrias Bacterianas , Proteínas Bacterianas/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Bacterias , Flagelos/fisiologíaRESUMEN
BACKGROUND: The SPAN trial (Stroke Preclinical Assessment Network) is the largest preclinical study testing acute stroke interventions in experimental focal cerebral ischemia using endovascular filament middle cerebral artery occlusion (MCAo). Besides testing interventions against controls, the prospective design captured numerous biological and procedural variables, highlighting the enormous heterogeneity introduced by the multicenter structure that might influence stroke outcomes. Here, we leveraged the unprecedented sample size achieved by the SPAN trial and the prospective design to identify the biological and procedural variables that affect experimental stroke outcomes in transient endovascular filament MCAo. METHODS: The study cohort included all mice enrolled and randomized in the SPAN trial (N=1789). Mice were subjected to 60-minute MCAo and followed for a month. Thirteen biological and procedural independent variables and 4 functional (weight loss and 4-point neuroscore on days 1 and 2, corner test on days 7 and 28, and mortality) and 3 tissue (day 2, magnetic resonance imaging infarct volumes and swelling; day 30, magnetic resonance imaging tissue loss) outcome variables were prospectively captured. Multivariable regression with stepwise elimination was used to identify the predictors and their effect sizes. RESULTS: Older age, active circadian stage at MCAo, and thinner and longer filament silicone tips predicted higher mortality. Older age, larger body weight, longer anesthesia duration, and longer filament tips predicted worse neuroscores, while high-fat diet and blood flow monitoring predicted milder neuroscores. Older age and a high-fat diet predicted worse corner test performance. While shorter filament tips predicted more ipsiversive turning, longer filament tips appeared to predict contraversive turning. Age, sex, and weight interacted when predicting the infarct volume. Older age was associated with smaller infarcts on day 2 magnetic resonance imaging, especially in animals with larger body weights; this association was most conspicuous in females. High-fat diet also predicted smaller infarcts. In contrast, the use of cerebral blood flow monitoring and more severe cerebral blood flow drop during MCAo, longer anesthesia, and longer filament tips all predicted larger infarcts. Bivariate analyses among the dependent variables highlighted a disconnect between tissue and functional outcomes. CONCLUSIONS: Our analyses identified variables affecting endovascular filament MCAo outcome, an experimental stroke model used worldwide. Multiple regression refuted some commonly reported predictors and revealed previously unrecognized associations. Given the multicenter prospective design that represents a sampling of real-world conditions, the degree of heterogeneity mimicking clinical trials, the large number of predictors adjusted for in the multivariable model, and the large sample size, we think this is the most definitive analysis of the predictors of preclinical stroke outcome to date. Future multicenter experimental stroke trials should standardize or at least ensure a balanced representation of the biological and procedural variables identified herein as potential confounders.
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Infarto de la Arteria Cerebral Media , Animales , Masculino , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Infarto de la Arteria Cerebral Media/patología , Ratones , Femenino , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Accidente Cerebrovascular/diagnóstico por imagen , Imagen por Resonancia Magnética , Estudios Prospectivos , Accidente Cerebrovascular Isquémico/diagnóstico por imagenRESUMEN
MYB transcription factors are encoded by a large family of highly conserved genes from plants to vertebrates. There are three members of the MYB gene family in human, namely, MYB, MYBL1, and MYBL2 that encode MYB/c-MYB, MYBL1/A-MYB, and MYBL2/B-MYB, respectively. MYB was the first member to be identified as a cellular homolog of the v-myb oncogene carried by the avian myeloblastosis virus (AMV) causing leukemia in chickens. Under the normal scenario, MYB is predominantly expressed in hematopoietic tissues, colonic crypts, and neural stem cells and plays a role in maintaining the undifferentiated state of the cells. Over the years, aberrant expression of MYB genes has been reported in several malignancies and recent years have witnessed tremendous progress in understanding of their roles in processes associated with cancer development. Here, we review various MYB alterations reported in cancer along with the roles of MYB family proteins in tumor cell plasticity, therapy resistance, and other hallmarks of cancer. We also discuss studies that provide mechanistic insights into the oncogenic functions of MYB transcription factors to identify potential therapeutic vulnerabilities.
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Neoplasias , Factores de Transcripción , Animales , Humanos , Plasticidad de la Célula/genética , Pollos , Resistencia a Antineoplásicos/genética , Neoplasias/genética , Factores de Transcripción/genéticaRESUMEN
MOTIVATION: Molecular dynamics (MD) is a computational experiment that is crucial for understanding the structure of biological macro and micro molecules, their folding, and the inter-molecular interactions. Accurate knowledge of these structural features is the cornerstone in drug development and elucidating macromolecules functions. The open-source GROMACS biomolecular MD simulation program is recognized as a reliable and frequently used simulation program for its precision. However, the user requires expertise, and scripting skills to carrying out MD simulations. RESULTS: We have developed an end-to-end interactive MD simulation application, MolDy for Gromacs. This front-end application provides a customizable user interface integrated with the Python and Perl-based logical backend connecting the Linux shell and Gromacs software. The tool performs analysis and provides the user with simulation trajectories and graphical representations of relevant biophysical parameters. The advantages of MolDy are (i) user-friendly, does not requiring the researcher to have prior knowledge of Linux; (ii) easy installation by a single command; (iii) freely available for academic research; (iv) can run with minimum configuration of operating systems; (v) has valid default prefilled parameters for beginners, and at the same time provides scope for modifications for expert users. AVAILABILITY AND IMPLEMENTATION: MolDy is available freely as compressed source code files with user manual for installation and operation on GitHub: https://github.com/AIBResearchMolDy/Moldyv01.git and on https://aibresearch.com/innovations.
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Simulación de Dinámica Molecular , Programas Informáticos , Interfaz Usuario-ComputadorRESUMEN
BACKGROUND: Coronary artery disease (CAD) is the leading cause of death worldwide. Recent meta-analyses of genome-wide association studies have identified over 175 loci associated with CAD. The majority of these loci are in noncoding regions and are predicted to regulate gene expression. Given that vascular smooth muscle cells (SMCs) play critical roles in the development and progression of CAD, we aimed to identify the subset of the CAD loci associated with the regulation of transcription in distinct SMC phenotypes. METHODS: We measured gene expression in SMCs isolated from the ascending aortas of 151 heart transplant donors of various genetic ancestries in quiescent or proliferative conditions and calculated the association of their expression and splicing with ~6.3 million imputed single-nucleotide polymorphism markers across the genome. RESULTS: We identified 4910 expression and 4412 splicing quantitative trait loci (sQTLs) representing regions of the genome associated with transcript abundance and splicing. A total of 3660 expression quantitative trait loci (eQTLs) had not been observed in the publicly available Genotype-Tissue Expression dataset. Further, 29 and 880 eQTLs were SMC-specific and sex-biased, respectively. We made these results available for public query on a user-friendly website. To identify the effector transcript(s) regulated by CAD loci, we used 4 distinct colocalization approaches. We identified 84 eQTL and 164 sQTL that colocalized with CAD loci, highlighting the importance of genetic regulation of mRNA splicing as a molecular mechanism for CAD genetic risk. Notably, 20% and 35% of the eQTLs were unique to quiescent or proliferative SMCs, respectively. One CAD locus colocalized with a sex-specific eQTL (TERF2IP), and another locus colocalized with SMC-specific eQTL (ALKBH8). The most significantly associated CAD locus, 9p21, was an sQTL for the long noncoding RNA CDKN2B-AS1, also known as ANRIL, in proliferative SMCs. CONCLUSIONS: Collectively, our results provide evidence for the molecular mechanisms of genetic susceptibility to CAD in distinct SMC phenotypes.
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Enfermedad de la Arteria Coronaria , Masculino , Femenino , Humanos , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Estudio de Asociación del Genoma Completo/métodos , Regulación de la Expresión Génica , Sitios de Carácter Cuantitativo , Predisposición Genética a la Enfermedad , Expresión Génica , Polimorfismo de Nucleótido Simple , Homólogo 8 de AlkB ARNt Metiltransferasa/genética , Homólogo 8 de AlkB ARNt Metiltransferasa/metabolismoRESUMEN
BACKGROUND: Genome-wide association studies have identified hundreds of loci associated with common vascular diseases, such as coronary artery disease, myocardial infarction, and hypertension. However, the lack of mechanistic insights for many GWAS loci limits their translation into the clinic. Among these loci with unknown functions is UFL1-four-and-a-half LIM (LIN-11, Isl-1, MEC-3) domain 5 (FHL5; chr6q16.1), which reached genome-wide significance in a recent coronary artery disease/ myocardial infarction GWAS meta-analysis. UFL1-FHL5 is also associated with several vascular diseases, consistent with the widespread pleiotropy observed for GWAS loci. METHODS: We apply a multimodal approach leveraging statistical fine-mapping, epigenomic profiling, and ex vivo analysis of human coronary artery tissues to implicate FHL5 as the top candidate causal gene. We unravel the molecular mechanisms of the cross-phenotype genetic associations through in vitro functional analyses and epigenomic profiling experiments in coronary artery smooth muscle cells. RESULTS: We prioritized FHL5 as the top candidate causal gene at the UFL1-FHL5 locus through expression quantitative trait locus colocalization methods. FHL5 gene expression was enriched in the smooth muscle cells and pericyte population in human artery tissues with coexpression network analyses supporting a functional role in regulating smooth muscle cell contraction. Unexpectedly, under procalcifying conditions, FHL5 overexpression promoted vascular calcification and dysregulated processes related to extracellular matrix organization and calcium handling. Lastly, by mapping FHL5 binding sites and inferring FHL5 target gene function using artery tissue gene regulatory network analyses, we highlight regulatory interactions between FHL5 and downstream coronary artery disease/myocardial infarction loci, such as FOXL1 and FN1 that have roles in vascular remodeling. CONCLUSIONS: Taken together, these studies provide mechanistic insights into the pleiotropic genetic associations of UFL1-FHL5. We show that FHL5 mediates vascular disease risk through transcriptional regulation of downstream vascular remodeling gene programs. These transacting mechanisms may explain a portion of the heritable risk for complex vascular diseases.
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Enfermedad de la Arteria Coronaria , Hipertensión , Infarto del Miocardio , Humanos , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Estudio de Asociación del Genoma Completo , Remodelación Vascular , Infarto del Miocardio/metabolismo , Hipertensión/metabolismo , Miocitos del Músculo Liso/metabolismo , Polimorfismo de Nucleótido Simple , Predisposición Genética a la Enfermedad , Factores de Transcripción/metabolismo , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismoRESUMEN
Extensive desmoplasia and poor vasculature renders pancreatic tumors severely hypoxic, contributing to their aggressiveness and therapy resistance. Here, we identify the HuR/MYB/HIF1α axis as a critical regulator of the metabolic plasticity and hypoxic survival of pancreatic cancer cells. HuR undergoes nuclear-to-cytoplasmic translocation under hypoxia and stabilizes MYB transcripts, while MYB transcriptionally upregulates HIF1α. Upon MYB silencing, pancreatic cancer cells fail to survive and adapt metabolically under hypoxia, despite forced overexpression of HIF1α. MYB induces the transcription of several HIF1α-regulated glycolytic genes by directly binding to their promoters, thus enhancing the recruitment of HIF1α to hypoxia-responsive elements through its interaction with p300-dependent histone acetylation. MYB-depleted pancreatic cancer cells exhibit a dramatic reduction in tumorigenic ability, glucose-uptake and metabolism in orthotopic mouse model, even after HIF1α restoration. Together, our findings reveal an essential role of MYB in metabolic reprogramming that supports pancreatic cancer cell survival under hypoxia.
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Neoplasias Pancreáticas , Ratones , Animales , Neoplasias Pancreáticas/genética , Hipoxia , Regiones Promotoras Genéticas , Hipoxia de la Célula/genética , Línea Celular Tumoral , Subunidad alfa del Factor 1 Inducible por Hipoxia/genéticaRESUMEN
The flagellar motor stator is an ion channel nanomachine that assembles as a ring of the MotA5MotB2 units at the flagellar base. The role of accessory proteins required for stator assembly and activation remains largely enigmatic. Here, we show that one such assembly factor, the conserved protein FliL, forms an integral part of the Helicobacter pylori flagellar motor in a position that colocalizes with the stator. Cryogenic electron tomography reconstructions of the intact motor in whole wild-type cells and cells lacking FliL revealed that the periplasmic domain of FliL (FliL-C) forms 18 circumferentially positioned rings integrated with the 18 MotAB units. FliL-C formed partial rings in the crystal, and the crystal structure-based full ring model was consistent with the shape of the rings observed in situ. Our data suggest that each FliL ring is coaxially sandwiched between the MotA ring and the dimeric periplasmic MotB moiety of the stator unit and that the central hole of the FliL ring has density that is consistent with the plug/linker region of MotB in its extended, active conformation. Significant structural similarities were found between FliL-C and stomatin/prohibitin/flotillin/HflK/C domains of scaffolding proteins, suggesting that FliL acts as a scaffold. The binding energy released upon association of FliL with the stator units could be used to power the release of the plug helices. The finding that isolated FliL-C forms stable partial rings provides an insight into the putative mechanism by which the FliL rings assemble around the stator units.
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Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Helicobacter pylori/fisiología , Proteínas de la Membrana/genética , Modelos Moleculares , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/metabolismo , Complejos Multiproteicos/química , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Relación Estructura-ActividadRESUMEN
Transcription of the ~200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15.7 kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAFI factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell. We find that conditional deletion of the TAF1B subunit of SL1 causes a striking depletion of UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and -2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation.
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Proteínas del Complejo de Iniciación de Transcripción Pol1 , ARN Polimerasa I , Animales , Niño , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Humanos , Ratones , Nucleosomas , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa I/genética , ARN Ribosómico/genética , Transcripción GenéticaRESUMEN
The genome's guardian, p53, is a master regulatory transcription factor that occupies sequence-specific response elements in many genes and modulates their expression. The target genes transcribe both coding RNA and non-coding RNA involved in regulating several biological processes such as cell division, differentiation, and cell death. Besides, p53 also regulates tumor immunology via regulating the molecules related to the immune response either directly or via regulating other molecules, including microRNAs (miRNAs). At the post-transcriptional level, the regulations of genes by miRNAs have been an emerging mechanism. Interestingly, p53 and various miRNAs cross-talk at different regulation levels. The cross-talk between p53 and miRNAs creates loops, turns, and networks that can influence cell metabolism, cell fate, cellular homeostasis, and tumor formation. Further, p53-miRNAs circuit has also been insinuated in the regulation of immune surveillance machinery. There are several examples of p53-miRNAs circuitry where p53 regulates immunomodulatory miRNA expression, such as miR-34a and miR-17-92. Similarly, a reverse process occurs in which miRNAs such as miR-125b and miR-let-7 regulate the expression of p53. Thus, the p53-miRNAs circuitry connects the immunomodulatory pathways and may shift the pro-inflammatory balance towards the pro-tumorigenic condition. In this review, we discuss the influence of p53-miRNAs circuitry in modulating the immune response in cancer development. We assume that thorough studies on the p53-miRNAs circuitry in various cancers may prove useful in developing effective new cancer therapeutics for successfully combating this disease.
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MicroARNs , Neoplasias , Diferenciación Celular , Regulación de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias/genética , Neoplasias/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Atherosclerosis, a chronic inflammatory disease of aorta, remains the major cause of morbidity and mortality among cardiovascular disease patients. Macrophage foam cell formation and inflammation are critically involved in early stages of atherosclerosis, hence chemopreventive targeting of foam cell formation by nutraceuticals may be a promising approach to curbing the progression of atherosclerosis. However, many nutraceuticals including berberine and ginkgetin have low stability, tissue/cell penetration and bioavailability resulting in inadequate chemotherapeutic effects of these nutraceuticals. We have used avocado-derived extracellular vesicles (EV) isolated from avocado (EVAvo ) as a novel carrier of nutraceuticals, in a strategy to alleviate the build-up of macrophage foam cells and expression of inflammatory genes. Our key findings are: (i) Avocado is a natural source of plant-derived EVs as shown by the results from transmission electron microscopy, dynamic light scattering and NanoBrook Omni analysis and atomic force microscopy; (ii) EVAvo are taken up by macrophages, a critical cell type in atherosclerosis; (iii) EVAvo can be loaded with high amounts of ginkgetin and berberine; (iv) ginkgetin plus berberine-loaded EVAvo (EVAvo(B+G) ) suppress activation of NFκB and NLRP3, and inhibit expression of pro-inflammatory and atherogenic genes, specifically Cd36, Tnfα, Il1ß and Il6; (v) EVAvo(B+G) attenuate oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cell formation and (vi) EVAvo(B+G) inhibit oxLDL uptake but not its cell surface binding during foam cell formation. Overall, our results suggest that using EVAvo as a natural carrier of nutraceuticals may improve strategies to curb the progression of atherosclerosis by limiting inflammation and pro-atherogenic responses.
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Aterosclerosis , Berberina , Biflavonoides , Persea , Humanos , Células Espumosas , Berberina/farmacología , Macrófagos , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/prevención & control , Lipoproteínas LDLRESUMEN
MYB, a proto-oncogene, is overexpressed in prostate cancer (PCa) and promotes its growth, aggressiveness, and resistance to androgen-deprivation therapy. Here, we examined the effect of androgen signaling on MYB expression and delineated the underlying molecular mechanisms. Paralleling a dichotomous effect on growth, low-dose androgen induced MYB expression at both transcript and protein levels, whereas it was suppressed in high-dose androgen-treated PCa cells. Interestingly, treatment with both low- and high-dose androgen transcriptionally upregulated MYB by increasing the binding of androgen receptor to the MYB promoter. In a time-course assay, androgen induced MYB expression at early time points followed by a sharp decline in high-dose androgen-treated cells due to decreased stability of MYB mRNA. Additionally, profiling of MYB-targeted miRNAs demonstrated significant induction of miR-150 in high-dose androgen-treated PCa cells. We observed a differential binding of androgen receptor on miR-150 promoter with significantly greater occupancy recorded in high-dose androgen-treated cells than those treated with low-dose androgen. Functional inhibition of miR-150 relieved MYB suppression by high-dose androgen, while miR-150 mimic abolished MYB induction by low-dose androgen. Furthermore, MYB-silencing or miR-150 mimic transfection suppressed PCa cell growth induced by low-dose androgen, whereas miR-150 inhibition rescued PCa cells from growth repression by high-dose androgen. Similarly, we observed that MYB silencing suppressed the expression of androgen-responsive, cell cycle-related genes in low-dose androgen-treated cells, while miR-150 inhibition increased their expression in cells treated with high-dose androgen. Overall, these findings reveal novel androgen-mediated mechanisms of MYB regulation that support its biphasic growth control in PCa cells.
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Andrógenos , MicroARNs , Neoplasias de la Próstata , Proteínas Proto-Oncogénicas c-myb , Humanos , Masculino , Antagonistas de Andrógenos , Andrógenos/farmacología , Andrógenos/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas c-myb/metabolismo , Procesamiento Proteico-Postraduccional , Células Tumorales CultivadasRESUMEN
In situ profiling of single-nucleotide variations (SNVs) can elucidate drug-resistant genotypes with single-cell resolution. The capacity to directly "see" genetic information is crucial for investigating the relationship between mutated genes and phenotypes. Fluorescence in situ hybridization serves as a canonical tool for genetic imaging; however, it cannot detect subtle sequence alteration including SNVs. Herein, we develop an in situ Cas12a-based amplification refractory mutation system-PCR (ARMS-PCR) method that allows the visualization of SNVs related to quinolone resistance inside cells. The capacity of discriminating SNVs is enhanced by incorporating optimized mismatched bases in the allele-specific primers, thus allowing to specifically amplify quinolone-resistant related genes. After in situ ARMS-PCR, we employed a modified Cas12a/CRISPR RNA to tag the amplicon, thereby enabling specific binding of fluorophore-labeled DNA probes. The method allows to precisely quantify quinolone-resistant Salmonella enterica in the bacterial mixture. Utilizing this method, we investigated the survival competition capacity of quinolone-resistant and quinolone-sensitive bacteria toward antimicrobial peptides and indicated the enrichment of quinolone-resistant bacteria under colistin sulfate stress. The in situ Cas12a-based ARMS-PCR method holds the potential for profiling cellular phenotypes and gene regulation with single-nucleotide resolution at the single-cell level.
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Quinolonas , Salmonella enterica , Sistemas CRISPR-Cas/genética , Alelos , Hibridación Fluorescente in Situ , Quinolonas/farmacología , Salmonella enterica/genética , Reacción en Cadena de la PolimerasaRESUMEN
Generative adversarial networks (GANs) have gained significant attention in the field of image synthesis, particularly in computer vision. GANs consist of a generative model and a discriminative model trained in an adversarial setting to generate realistic and novel data. In the context of image synthesis, the generator produces synthetic images, whereas the discriminator determines their authenticity by comparing them with real examples. Through iterative training, the generator allows the creation of images that are indistinguishable from real ones, leading to high-quality image generation. Considering their success in computer vision, GANs hold great potential for medical diagnostic applications. In the medical field, GANs can generate images of rare diseases, aid in learning, and be used as visualization tools. GANs can leverage unlabeled medical images, which are large in size, numerous in quantity, and challenging to annotate manually. GANs have demonstrated remarkable capabilities in image synthesis and have the potential to significantly impact digital histopathology. This review article focuses on the emerging use of GANs in digital histopathology, examining their applications and potential challenges. Histopathology plays a crucial role in disease diagnosis, and GANs can contribute by generating realistic microscopic images. However, ethical considerations arise because of the reliance on synthetic or pseudogenerated images. Therefore, the manuscript also explores the current limitations and highlights the ethical considerations associated with the use of this technology. In conclusion, digital histopathology has seen an emerging use of GANs for image enhancement, such as color (stain) normalization, virtual staining, and ink/marker removal. GANs offer significant potential in transforming digital pathology when applied to specific and narrow tasks (preprocessing enhancements). Evaluating data quality, addressing biases, protecting privacy, ensuring accountability and transparency, and developing regulation are imperative to ensure the ethical application of GANs.
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Colorantes , Exactitud de los Datos , Humanos , Coloración y Etiquetado , Procesamiento de Imagen Asistido por ComputadorRESUMEN
The 2023 Joint International Congress of the International Liver Transplantation Society (ILTS), the European Liver and Intestine Transplant Association (ELITA), and the Liver Intensive Care Group of Europe (LICAGE) held in Rotterdam, the Netherlands, marked a significant recovery milestone for the liver transplant community after COVID-19. With 1159 participants and a surge in abstract submissions, the event focused on "Liver Disorders and Transplantation: Innovations and Evolving Indications." This conference report provides a comprehensive overview of the key themes discussed during the event, encompassing Hepatology, Anesthesia and Critical Care, Acute Liver Failure, Infectious Disease, Immunosuppression, Pediatric Liver Transplantation, Living Donor Liver Transplantation, Transplant Oncology, Surgical Approaches, and Machine Perfusion. The congress provided a platform for extensive discussions on a wide range of topics, reflecting the continuous advancements and collaborative efforts within the liver transplant community.
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Trasplante de Hígado , Niño , Humanos , Terapia de Inmunosupresión , Donadores VivosRESUMEN
Helicobacter pylori encodes homologues of PilM, PilN and PilO from bacteria with Type IV pili, where these proteins form a pilus alignment complex. Inactivation of pilO changes H. pylori motility in semi-solid media, suggesting a link to the chemosensory pathways or flagellar motor. Here, we showed that mutation of the pilO or pilN gene in H. pylori strain SS1 reduced the mean linear swimming speed in liquid media, implicating PilO and PilN in the function, or regulation of, the flagellar motor. We also demonstrated that the soluble variants of H. pylori PilN and PilO share common biochemical properties with their Type IV pili counterparts which suggests their adapted function in the bacterial flagellar motor may be similar to that in the Type IV pili.
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INTRODUCTION: Previous bronchoalveolar lavage fluid (BALF) proteomic analysis has evaluated limited numbers of subjects for only a few proteins of interest, which may differ between asthma and normal controls. Our objective was to examine a more comprehensive inflammatory biomarker panel in quantitative proteomic analysis for a large asthma cohort to identify molecular phenotypes distinguishing severe from nonsevere asthma. METHODS: Bronchoalveolar lavage fluid from 48 severe and 77 nonsevere adult asthma subjects were assessed for 75 inflammatory proteins, normalized to BALF total protein concentration. Validation of BALF differences was sought through equivalent protein analysis of autologous sputum. Subjects' data, stratified by asthma severity, were analysed by standard statistical tests, principal component analysis and 5 machine learning algorithms. RESULTS: The severe group had lower lung function and greater health care utilization. Significantly increased BALF proteins for severe asthma compared to nonsevere asthma were fibroblast growth factor 2 (FGF2), TGFα, IL1Ra, IL2, IL4, CCL8, CCL13 and CXCL7 and significantly decreased were platelet-derived growth factor a-a dimer (PDGFaa), vascular endothelial growth factor (VEGF), interleukin 5 (IL5), CCL17, CCL22, CXCL9 and CXCL10. Four protein differences were replicated in sputum. FGF2, PDGFaa and CXCL7 were independently identified by 5 machine learning algorithms as the most important variables for discriminating severe and nonsevere asthma. Increased and decreased proteins identified for the severe cluster showed significant protein-protein interactions for chemokine and cytokine signalling, growth factor activity, and eosinophil and neutrophil chemotaxis differing between subjects with severe and nonsevere asthma. CONCLUSION: These inflammatory protein results confirm altered airway remodelling and cytokine/chemokine activity recruiting leukocytes into the airways of severe compared to nonsevere asthma as important processes even in stable status.
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Asma , Factor A de Crecimiento Endotelial Vascular , Adulto , Humanos , Proteómica , Factor 2 de Crecimiento de Fibroblastos , Citocinas/metabolismo , Lavado Broncoalveolar , Quimiocinas , Líquido del Lavado BronquioalveolarRESUMEN
Obesity is highly prevalent in hepatology clinics and has a significant impact on chronic liver disease and patient management. Hepatologists and gastroenterologists need to be actively engaged in the management of obesity. This review provides a detailed approach to this challenging comorbidity.
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PURPOSE: The purpose of this paper is to report on changes in overall survival, progression-free survival, and complete cytoreduction rates in the 5-year period after the implementation of a multidisciplinary surgical team (MDT). METHODS: Two cohorts were used. Cohort A was a retrospectively collated cohort from 2006 to 2015. Cohort B was a prospectively collated cohort of patients from January 2017 to September 2021. RESULTS: This study included 146 patients in cohort A (2006-2015) and 174 patients in cohort B (2017-2021) with FIGO stage III/IV ovarian cancer. Median follow-up in cohort A was 60 months and 48 months in cohort B. The rate of primary cytoreductive surgery increased from 38% (55/146) in cohort A to 46.5% (81/174) in cohort B. Complete macroscopic resection increased from 58.9% (86/146) in cohort A to 78.7% (137/174) in cohort B (p < 0.001). At 3 years, 75% (109/144) patients had disease progression in cohort A compared with 48.8% (85/174) in cohort B (log-rank, p < 0.001). Also at 3 years, 64.5% (93/144) of patients had died in cohort A compared with 24% (42/174) of cohort B (log-rank, p < 0.001). Cox multivariate analysis demonstrated that MDT input, residual disease, and age were independent predictors of overall (hazard ratio [HR] 0.29, 95% confidence interval [CI] 0.203-0.437, p < 0.001) and progression-free survival (HR 0.31, 95% CI 0.21-0.43, p < 0.001). Major morbidity remained stable throughout both study periods (2006-2021). CONCLUSIONS: Our data demonstrate that the implementation of multidisciplinary-team, intraoperative approach allowed for a change in surgical philosophy and has resulted in a significant improvement in overall survival, progression-free survival, and complete resection rates.