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High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 ("Second-Strand Synthesis"), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inflamación/genética , ARN Citoplasmático Pequeño/genética , Piel/patología , Animales , Línea Celular , ADN Complementario/genética , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcripción Genética/genética , Transcriptoma/genéticaRESUMEN
Hydrodynamics accurately describe relativistic heavy-ion collision experiments well before local thermal equilibrium is established1. This unexpectedly rapid onset of hydrodynamics-which takes place on the fastest available timescale-is called hydrodynamization2-4. It occurs when an interacting quantum system is quenched with an energy density that is much greater than its ground-state energy density5,6. During hydrodynamization, energy gets redistributed across very different energy scales. Hydrodynamization precedes local equilibration among momentum modes5, which is local prethermalization to a generalized Gibbs ensemble7,8 in nearly integrable systems or local thermalization in non-integrable systems9. Although many theories of quantum dynamics postulate local prethermalization10,11, the associated timescale has not been studied experimentally. Here we use an array of one-dimensional Bose gases to directly observe both hydrodynamization and local prethermalization. After we apply a Bragg scattering pulse, hydrodynamization is evident in the fast redistribution of energy among distant momentum modes, which occurs on timescales associated with the Bragg peak energies. Local prethermalization can be seen in the slower redistribution of occupation among nearby momentum modes. We find that the timescale for local prethermalization in our system is inversely proportional to the momenta involved. During hydrodynamization and local prethermalization, existing theories cannot quantitatively model our experiment. Exact theoretical calculations in the Tonks-Girardeau limit12 show qualitatively similar features.
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In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.
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Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Francisella/genética , Virulencia/genética , ADN/genética , División del ADN , Regulación Bacteriana de la Expresión Génica/genética , Lipoproteínas/biosíntesis , Lipoproteínas/genética , ARN/genética , Transcripción GenéticaRESUMEN
Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.
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Proteínas Bacterianas , Pared Celular , Clostridioides difficile , Peptidoglicano , Clostridioides difficile/enzimología , Clostridioides difficile/metabolismo , Peptidoglicano/metabolismo , Pared Celular/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Peptidoglicano Glicosiltransferasa/metabolismo , Peptidoglicano Glicosiltransferasa/química , Peptidoglicano Glicosiltransferasa/genéticaRESUMEN
Antibiotics are considered one of the most important contributions to clinical medicine in the last century. Due to the use and overuse of these drugs, there have been increasing frequencies of infections with resistant pathogens. One form of resistance, heteroresistance, is particularly problematic; pathogens appear sensitive to a drug by common susceptibility tests. However, upon exposure to the antibiotic, resistance rapidly ascends, and treatment fails. To quantitatively explore the processes contributing to the emergence and ascent of resistance during treatment and the waning of resistance following cessation of treatment, we develop two distinct mathematical and computer-simulation models of heteroresistance. In our analysis of the properties of these models, we consider the factors that determine the response to antibiotic-mediated selection. In one model, heteroresistance is progressive, with each resistant state sequentially generating a higher resistance level. In the other model, heteroresistance is non-progressive, with a susceptible population directly generating populations with different resistance levels. The conditions where resistance will ascend in the progressive model are narrower than those of the non-progressive model. The rates of reversion from the resistant to the sensitive states are critically dependent on the transition rates and the fitness cost of resistance. Our results demonstrate that the standard test used to identify heteroresistance is insufficient. The predictions of our models are consistent with empirical results. Our results demand a reevaluation of the definition and criteria employed to identify heteroresistance. We recommend that the definition of heteroresistance should include a consideration of the rate of return to susceptibility.
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Antibacterianos , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Dinámica Poblacional , Pruebas de Sensibilidad MicrobianaRESUMEN
Change history: We could not replicate the results in Fig. 2a and g of this Letter, and new information has revealed a flaw in the interpretation of Fig. 2h. As a result, we do not have evidence to support RNA degradation as the mechanism that underlies Cas9-mediated regulation of FTN_1103 mRNA expression; see accompanying Amendment. This has not been corrected online.
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Daptomycin is a cyclic lipopeptide antibiotic used to treat infections caused by some Gram-positive bacteria. Daptomycin disrupts synthesis of the peptidoglycan (PG) cell wall by inserting into the cytoplasmic membrane and binding multiple forms of the undecaprenyl carrier lipid required for PG synthesis. Membrane insertion requires phosphatidylglycerol, so studies of daptomycin can provide insight into assembly and maintenance of the cytoplasmic membrane. Here, we studied the effects of daptomycin on Clostridioides difficile, the leading cause of healthcare-associated diarrhea. We observed that growth of C. difficile strain R20291 in the presence of sub-MIC levels of daptomycin resulted in a chaining phenotype, minicell formation, and lysis-phenotypes broadly consistent with perturbation of membranes and PG synthesis. We also selected for and characterized eight mutants with elevated daptomycin resistance. The mutations in these mutants were mapped to four genes: cdsA (cdr20291_2041), ftsH2 (cdr20291_3396), esrR (cdr20291_1187), and draS (cdr20291_2456). Of these four genes, only draS has been characterized previously. Follow-up studies indicate these mutations confer daptomycin resistance by two general mechanisms: reducing the amount of phosphatidylglycerol in the cytoplasmic membrane (cdsA) or altering the regulation of membrane processes (ftsH2, esrR, and draS). Thus, the mutants described here provide insights into phospholipid synthesis and identify signal transduction systems involved in cell envelope biogenesis and stress response in C. difficile. IMPORTANCE: C. difficile is the leading cause of healthcare-associated diarrhea and is a threat to public health due to the risk of recurrent infections. Understanding biosynthesis of the atypical cell envelope of C. difficile may provide insight into novel drug targets to selectively inhibit C. difficile. Here, we identified mutations that increased daptomycin resistance and allowed us to better understand phospholipid synthesis, cell envelope biogenesis, and stress response in C. difficile.
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Clostridioides difficile , Daptomicina , Humanos , Daptomicina/farmacología , Daptomicina/química , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Farmacorresistencia Bacteriana/genética , Antibacterianos/farmacología , Antibacterianos/química , Fosfatidilgliceroles , DiarreaRESUMEN
The plant hormone abscisic acid (ABA) plays a central role in the regulation of seed maturation and dormancy. ABA also restrains germination under abiotic-stress conditions. Here, we show in tomato (Solanum lycopersicum) that the ABA importer ABA-IMPORTING TRANSPORTER 1.1 (AIT1.1/NPF4.6) has a role in radicle emergence under salinity conditions. AIT1.1 expression was upregulated following seed imbibition, and CRISPR/Cas9-derived ait1.1 mutants exhibited faster radicle emergence, increased germination and partial resistance to ABA. AIT1.1 was highly expressed in the endosperm, but not in the embryo, and ait1.1 isolated embryos did not show resistance to ABA. On the other hand, loss of AIT1.1 activity promoted the expression of endosperm-weakening-related genes, and seed-coat scarification eliminated the promoting effect of ait1.1 on radicle emergence. Therefore, we propose that imbibition-induced AIT1.1 expression in the micropylar endosperm mediates ABA-uptake into micropylar cells to restrain endosperm weakening. While salinity conditions strongly inhibited wild-type M82 seed germination, high salinity had a much weaker effect on ait1.1 germination. We suggest that AIT1.1 evolved to inhibit germination under unfavorable conditions, such as salinity. Unlike other ABA mutants, ait1.1 exhibited normal seed longevity, and therefore, the ait1.1 allele may be exploited to improve seed germination in crops.
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Ácido Abscísico , Solanum lycopersicum , Ácido Abscísico/farmacología , Ácido Abscísico/metabolismo , Solanum lycopersicum/genética , Germinación/genética , Semillas/genética , Semillas/metabolismo , Salinidad , Proteínas de Transporte de Membrana/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Antibiotic resistance is a growing crisis that threatens many aspects of modern healthcare. Dogma is that resistance often develops due to acquisition of a resistance gene or mutation and that when this occurs, all the cells in the bacterial population are phenotypically resistant. In contrast, heteroresistance (HR) is a form of antibiotic resistance where only a subset of cells within a bacterial population are resistant to a given drug. These resistant cells can rapidly replicate in the presence of the antibiotic and cause treatment failures. If and how HR and resistance are related is unclear. Using carbapenem-resistant Enterobacterales (CRE), we provide evidence that HR to beta-lactams develops over years of antibiotic usage and that it is gradually supplanted by resistance. This suggests the possibility that HR may often develop before resistance and frequently be a stage in its progression, potentially representing a major shift in our understanding of the evolution of antibiotic resistance.
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Antibacterianos/farmacología , Farmacorresistencia Bacteriana , beta-Lactamas/farmacología , Enterobacteriaceae/efectos de los fármacos , Pruebas de Sensibilidad MicrobianaRESUMEN
In 1872, Maxwell proposed his famous 'demon' thought experiment1. By discerning which particles in a gas are hot and which are cold, and then performing a series of reversible actions, Maxwell's demon could rearrange the particles into a manifestly lower-entropy state. This apparent violation of the second law of thermodynamics was resolved by twentieth-century theoretical work2: the entropy of the Universe is often increased while gathering information3, and there is an unavoidable entropy increase associated with the demon's memory4. The appeal of the thought experiment has led many real experiments to be framed as demon-like. However, past experiments had no intermediate information storage5, yielded only a small change in the system entropy6,7 or involved systems of four or fewer particles8-10. Here we present an experiment that captures the full essence of Maxwell's thought experiment. We start with a randomly half-filled three-dimensional optical lattice with about 60 atoms. We make the atoms sufficiently vibrationally cold so that the initial disorder is the dominant entropy. After determining where the atoms are, we execute a series of reversible operations to create a fully filled sublattice, which is a manifestly low-entropy state. Our sorting process lowers the total entropy of the system by a factor of 2.44. This highly filled ultracold array could be used as the starting point for a neutral-atom quantum computer.
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Color constancy denotes the ability to assign a particular and stable color percept to an object, irrespective of its surroundings and illumination. The light reaching the eye confounds illumination and spectral reflectance of the object, making the recovery of constant object color an ill-posed problem. How good the visual system is at accomplishing this task is still a matter of heated debate, despite more than a 100 years of research. Depending on the laboratory task and the specific cues available to observers, color constancy was found to be at levels ranging between 15% and 80%, which seems incompatible with the relatively stable color appearance of objects around us and the consistent usage of color names in real life. Here, we show close-to-perfect color constancy using real objects in a natural task and natural environmental conditions, chosen to mimic the role of color constancy in everyday life. Participants had to identify the color of a (non-present) item familiar to them in an office room under five different experimental illuminations. They mostly selected the same colored Munsell chip as their match to the absent object, even though the light reaching the eye in each case differed substantially. Our results demonstrate that color constancy under ideal conditions in the real world can indeed be exceptionally good. We found it to be as good as visual memory permits and not generally compromised by sensory uncertainty.
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Señales (Psicología) , Iluminación , Humanos , Memoria , IncertidumbreRESUMEN
OBJECTIVE: Electrical injury (EI) is a significant, multifaceted trauma often with multi-domain cognitive sequelae, even when the expected current path does not pass through the brain. Chronic pain (CP) research suggests pain may affect cognition directly and indirectly by influencing emotional distress which then impacts cognitive functioning. As chronic pain may be critical to understanding EI-related cognitive difficulties, the aims of the current study were: examine the direct and indirect effects of pain on cognition following EI and compare the relationship between pain and cognition in EI and CP populations. METHOD: This cross-sectional study used data from a clinical sample of 50 patients with EI (84.0% male; Mage = 43.7 years) administered standardized measures of pain (Pain Patient Profile), depression, and neurocognitive functioning. A CP comparison sample of 93 patients was also included. RESULTS: Higher pain levels were associated with poorer attention/processing speed and executive functioning performance among patients with EI. Depression was significantly correlated with pain and mediated the relationship between pain and attention/processing speed in patients with EI. When comparing the patients with EI and CP, the relationship between pain and cognition was similar for both clinical groups. CONCLUSIONS: Findings indicate that pain impacts mood and cognition in patients with EI, and the influence of pain and its effect on cognition should be considered in the assessment and treatment of patients who have experienced an electrical injury.
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Dolor Crónico , Traumatismos por Electricidad , Humanos , Masculino , Adulto , Femenino , Estudios Transversales , Traumatismos por Electricidad/psicología , Cognición , Función Ejecutiva , Pruebas NeuropsicológicasRESUMEN
PURPOSE: In the US, Black people diagnosed with schizophrenia experience worse psychosocial and clinical outcomes than their White counterparts. While racism-related factors contribute to these disparities, an additional understudied explanation may be that psychosocial treatments for psychotic disorders are less effective for Black than White individuals. The purpose of this study is to examine the extent to which best treatment practices for first-episode psychosis (FEP) are effective for Black and White participants. METHODS: We conducted a secondary data analysis of the Recovery After an Initial Schizophrenia Episode Early Treatment Program (RAISE-ETP), a two-year multisite trial that compared a coordinated specialty care intervention for FEP (NAVIGATE) to community care as usual (CC) in 34 sites across the US. Specifically, we compared interviewer-rated quality of life and symptoms, as well as self-reported mental health and stigma, between 139 Non-Latinx Black and 172 Non-Latinx White participants with FEP in NAVIGATE and CC. RESULTS: We found few differences between Black and White participants over two-year outcomes, either overall or in terms of benefit from NAVIGATE. Across both treatment conditions, Black participants improved less than White participants on positive symptoms, an effect driven primarily by suspiciousness/persecution. In NAVIGATE, self-reported mental health stigma decreased for both Black and White participants, while in CC stigma decreased for White participants but increased for Black participants. This effect was driven primarily by experienced stigma rather than self-stigma. CONCLUSION: NAVIGATE benefits both Black and White individuals diagnosed with FEP. Mental health stigma and positive symptoms may be particularly important aspects of treatment for Black individuals diagnosed with FEP.
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Trastornos Psicóticos , Esquizofrenia , Humanos , Negro o Afroamericano , Trastornos Psicóticos/psicología , Calidad de Vida , Esquizofrenia/terapiaRESUMEN
Engineering neutron diffraction can nondestructively and noninvasively probe stress, strain, temperature, and phase evolutions deep within bulk materials. In this work, we demonstrate operando lattice strain measurement of internal combustion engine components by neutron diffraction. A modified commercial generator engine was mounted in the VULCAN diffractometer at the Spallation Neutron Source, and the lattice strains in both the cylinder block and head were measured under static nonfiring conditions as well as steady state and cyclic transient operation. The dynamic temporal response of the lattice strain change during transient operation was resolved in two locations by asynchronous stroboscopic neutron diffraction. We demonstrated that operando neutron measurements can allow for understanding of how materials behave throughout operational engineering devices. This study opens a pathway for the industrial and academic communities to better understand the complexities of material behavior during the operation of internal combustion engines and other real-scale devices and systems and to leverage techniques developed here for future investigations of numerous new platforms and alloys.
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The WalR-WalK two-component regulatory system (TCS) is found in all Firmicutes, in which it regulates the expression of multiple genes required for remodeling the cell envelope during growth and division. Unlike most TCSs, WalRK is essential for viability, so it has attracted interest as a potential antibiotic target. In this study, we used overexpression of WalR and CRISPR interference to investigate the Wal system of Clostridioides difficile, a major cause of hospital-associated diarrhea in high-income countries. We confirmed that the wal operon is essential and identified morphological defects and cell lysis as the major terminal phenotypes of altered wal expression. We also used transcriptome sequencing (RNA-seq) to identify over 150 genes whose expression changes in response to WalR levels. This gene set is enriched in cell envelope genes and includes genes encoding several predicted PG hydrolases and proteins that could regulate PG hydrolase activity. A distinct feature of the C. difficile cell envelope is the presence of an S-layer, and we found that WalR affects expression of several genes which encode S-layer proteins. An unexpected finding was that some Wal-associated phenotypic defects were inverted in comparison to what has been reported for other Firmicutes. For example, downregulation of Wal signaling caused C. difficile cells to become longer rather than shorter, as in Bacillus subtilis. Likewise, downregulation of Wal rendered C. difficile more sensitive to vancomycin, whereas reduced Wal activity is linked to increased vancomycin resistance in Staphylococcus aureus. IMPORTANCE The WalRK two-component system (TCS) is essential for coordinating synthesis and turnover of peptidoglycan in Firmicutes. We investigated the WalRK TCS in Clostridioides difficile, an important bacterial pathogen with an atypical cell envelope. We confirmed that WalRK is essential and regulates cell envelope biogenesis, although several of the phenotypic changes we observed were opposite to what has been reported for other Firmicutes. We also identified over 150 genes whose expression is controlled either directly or indirectly by WalR. Overall, our findings provide a foundation for future investigations of an important regulatory system and potential antibiotic target in C. difficile.
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Clostridioides difficile , Antibacterianos/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Clostridioides , Clostridioides difficile/genética , Regulación Bacteriana de la Expresión GénicaRESUMEN
Bacterial SPOR domains target proteins to the divisome by binding septal peptidoglycan (PG) at sites where cell wall amidases have removed stem peptides. These PG structures are referred to as denuded glycans. Although all characterized SPOR domains bind denuded glycans, whether there are differences in affinity is not known. Here, we use isothermal titration calorimetry (ITC) to determine the relative PG glycan binding affinity (<i>K</i><sub>d</sub>) of four Escherichia coli SPOR domains and one Cytophaga hutchinsonii SPOR domain. We found that the <i>K</i><sub>d</sub> values ranged from approximately 1 µM for E. coli DamX<sup>SPOR</sup> and <i>C. hutchinsonii</i> CHU2221<sup>SPOR</sup> to about 10 µM for E. coli FtsN<sup>SPOR</sup>. To investigate whether these differences in PG binding affinity are important for SPOR domain protein function, we constructed and characterized a set of DamX and FtsN "swap" proteins. As expected, all SPOR domain swap proteins localized to the division site, and, in the case of FtsN, all of the heterologous SPOR domains supported cell division. However, for DamX, only the high-affinity SPOR domain from CHU2221 supported normal function in cell division. In summary, different SPOR domains bind denuded PG glycans with different affinities, which appears to be important for the functions of some SPOR domain proteins (e.g., DamX) but not for the functions of others (e.g., FtsN). <b>IMPORTANCE</b> SPOR domain proteins are prominent components of the cell division apparatus in a wide variety of bacteria. The primary function of SPOR domains is targeting proteins to the division site, which they accomplish by binding to septal peptidoglycan. However, whether SPOR domains have any functions beyond septal targeting is unknown. Here, we show that SPOR domains vary in their PG binding affinities and that, at least in the case of the E. coli cell division protein DamX, having a high-affinity SPOR domain contributes to proper function.
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Proteínas de Escherichia coli , Peptidoglicano , Amidohidrolasas/metabolismo , Bacterias/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Unión ProteicaRESUMEN
OBJECTIVES: To describe the prevalence of colistin heteroresistance in carbapenem-resistant Pseudomonas aeruginosa (CRPA) and evaluate the association with clinical outcomes. METHODS: Colistin heteroresistance was evaluated in CRPA isolates collected from patients without cystic fibrosis in Atlanta, Georgia, USA using two definitions: HR1, growth at 4 and 8 mg/L of colistin at a frequency ≥1â×â10-6 the main population; and HR2, growth at a colistin concentration ≥8× the MIC of the main population at a frequency ≥1â×â10-7. A modified population analysis profile (mPAP) technique was compared with reference PAP for detecting heteroresistance. For adults hospitalized at the time of or within 1 week of CRPA culture, multivariable logistic regression estimated the association between heteroresistance and 90 day mortality. RESULTS: Of 143 colistin-susceptible CRPA isolates, 8 (6%) met the HR1 definition and 37 (26%) met the HR2 definition. Compared with the reference PAP, mPAP had a sensitivity and specificity of 50% and 100% for HR1 and 32% and 99% for HR2. Of 82 hospitalized patients, 45 (56%) were male and the median age was 63 years (IQR 49-73). Heteroresistance was not associated with 90 day mortality using HR1 (0% in heteroresistant versus 22% in non-heteroresistant group; Pâ=â0.6) or HR2 (12% in heteroresistant versus 24% in non-heteroresistant group; Pâ=â0.4; adjusted OR 0.8; 95% CI 0.2-3.4). CONCLUSIONS: Colistin heteroresistance was identified in up to 26% of patients with CRPA in our sample, although the prevalence varied depending on the definition. We did not observe an apparent association between colistin heteroresistance and 90 day mortality.
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Colistina , Pseudomonas aeruginosa , Adulto , Antibacterianos/farmacología , Carbapenémicos/farmacología , Colistina/farmacología , Humanos , Masculino , Persona de Mediana Edad , PrevalenciaRESUMEN
Steroidal glycoalkaloids (SGAs) are protective metabolites constitutively produced by Solanaceae species. Genes and enzymes generating the vast structural diversity of SGAs have been largely identified. Yet, mechanisms of hormone pathways coordinating defence (jasmonate; JA) and growth (gibberellin; GA) controlling SGAs metabolism remain unclear. We used tomato to decipher the hormonal regulation of SGAs metabolism during growth vs defence tradeoff. This was performed by genetic and biochemical characterisation of different JA and GA pathways components, coupled with in vitro experiments to elucidate the crosstalk between these hormone pathways mediating SGAs metabolism. We discovered that reduced active JA results in decreased SGA production, while low levels of GA or its receptor led to elevated SGA accumulation. We showed that MYC1 and MYC2 transcription factors mediate the JA/GA crosstalk by transcriptional activation of SGA biosynthesis and GA catabolism genes. Furthermore, MYC1 and MYC2 transcriptionally regulate the GA signalling suppressor DELLA that by itself interferes in JA-mediated SGA control by modulating MYC activity through protein-protein interaction. Chemical and fungal pathogen treatments reinforced the concept of JA/GA crosstalk during SGA metabolism. These findings revealed the mechanism of JA/GA interplay in SGA biosynthesis to balance the cost of chemical defence with growth.
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Alcaloides , Solanum lycopersicum , Alcaloides/metabolismo , Ciclopentanos/metabolismo , Regulación de la Expresión Génica de las Plantas , Giberelinas/metabolismo , Solanum lycopersicum/metabolismo , Oxilipinas/metabolismoRESUMEN
The pleiotropic and complex gibberellin (GA) response relies on targeted proteolysis of DELLA proteins mediated by a GA-activated GIBBERELLIN-INSENSITIVE DWARF1 (GID1) receptor. The tomato (Solanum lycopersicum) genome encodes for a single DELLA protein, PROCERA (PRO), and three receptors, SlGID1a (GID1a), GID1b1, and GID1b2, that may guide specific GA responses. In this work, clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR associated protein 9-derived gid1 mutants were generated and their effect on GA responses was studied. The gid1 triple mutant was extremely dwarf and fully insensitive to GA. Under optimal growth conditions, the three receptors function redundantly and the single gid1 mutants exhibited very mild phenotypic changes. Among the three receptors, GID1a had the strongest effects on germination and growth. Yeast two-hybrid assays suggested that GID1a has the highest affinity to PRO. Analysis of lines with a single active receptor demonstrated a unique role for GID1a in protracted response to GA that was saturated only at high doses. When the gid1 mutants were grown in the field under ambient changing environments, they showed phenotypic instability, the high redundancy was lost, and gid1a exhibited dwarfism that was strongly exacerbated by the loss of another GID1b receptor gene. These results suggest that multiple GA receptors contribute to phenotypic stability under environmental extremes.