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1.
J Mol Biol ; 436(12): 168603, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38729259

RESUMEN

OXA-66 is a member of the OXA-51 subfamily of class D ß-lactamases native to the Acinetobacter genus that includes Acinetobacter baumannii, one of the ESKAPE pathogens and a major cause of drug-resistant nosocomial infections. Although both wild type OXA-66 and OXA-51 have low catalytic activity, they are ubiquitous in the Acinetobacter genomes. OXA-51 is also remarkably thermostable. In addition, newly emerging, single and double amino acid variants show increased activity against carbapenems, indicating that the OXA-51 subfamily is growing and gaining clinical significance. In this study, we used molecular dynamics simulations, X-ray crystallography, and thermal denaturation data to examine and compare the dynamics of OXA-66 wt and its gain-of-function variants: I129L (OXA-83), L167V (OXA-82), P130Q (OXA-109), P130A, and W222L (OXA-234). Our data indicate that OXA-66 wt also has a high melting temperature, and its remarkable stability is due to an extensive and rigid hydrophobic bridge formed by a number of residues around the active site and harbored by the three loops, P, Ω, and ß5-ß6. Compared to the WT enzyme, the mutants exhibit higher flexibility only in the loop regions, and are more stable than other robust carbapenemases, such as OXA-23 and OXA-24/40. All the mutants show increased rotational flexibility of residues I129 and W222, which allows carbapenems to bind. Overall, our data support the hypothesis that structural features in OXA-51 and OXA-66 promote evolution of multiple highly stable variants with increased clinical relevance in A. baumannii.


Asunto(s)
Acinetobacter baumannii , Simulación de Dinámica Molecular , beta-Lactamasas , Acinetobacter baumannii/genética , Acinetobacter baumannii/enzimología , beta-Lactamasas/química , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Cristalografía por Rayos X , Estabilidad de Enzimas , Conformación Proteica , Carbapenémicos/farmacología , Carbapenémicos/metabolismo , Evolución Molecular , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico
2.
J Neurodev Disord ; 16(1): 1, 2024 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-38166648

RESUMEN

BACKGROUND: Sensory processing dysfunction (SPD) is linked to altered white matter (WM) microstructure in school-age children. Sensory over-responsivity (SOR), a form of SPD, affects at least 2.5% of all children and has substantial deleterious impact on learning and mental health. However, SOR has not been well studied using microstructural imaging such as diffusion MRI (dMRI). Since SOR involves hypersensitivity to external stimuli, we test the hypothesis that children with SOR require compensatory neuroplasticity in the form of superior WM microstructural integrity to protect against internalizing behavior, leaving those with impaired WM microstructure vulnerable to somatization and depression. METHODS: Children ages 8-12 years old with neurodevelopmental concerns were assessed for SOR using a comprehensive structured clinical evaluation, the Sensory Processing 3 Dimensions Assessment, and underwent 3 Tesla MRI with multishell multiband dMRI. Tract-based spatial statistics was used to measure diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI) metrics from global WM and nineteen selected WM tracts. Correlations of DTI and NODDI measures with measures of somatization and emotional disturbance from the Behavioral Assessment System for Children, 3rd edition (BASC-3), were computed in the SOR group and in matched children with neurodevelopmental concerns but not SOR. RESULTS: Global WM fractional anisotropy (FA) is negatively correlated with somatization and with emotional disturbance in the SOR group but not the non-SOR group. Also observed in children with SOR are positive correlations of radial diffusivity (RD) and free water fraction (FISO) with somatization and, in most cases, emotional disturbance. These effects are significant in boys with SOR, whereas the study is underpowered for girls. The most affected white matter are medial lemniscus and internal capsule sensory tracts, although effects of SOR are observed in many cerebral, cerebellar, and brainstem tracts. CONCLUSION: White matter microstructure is related to affective behavior in children with SOR.


Asunto(s)
Sustancia Blanca , Masculino , Niño , Femenino , Humanos , Sustancia Blanca/diagnóstico por imagen , Imagen de Difusión Tensora/métodos , Imagen de Difusión por Resonancia Magnética , Imagen por Resonancia Magnética , Cerebelo
3.
J Gen Virol ; 105(1)2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-38180085

RESUMEN

Host tissues represent diverse resources or barriers for pathogen replicative fitness. We tested whether viruses in specialist, generalist, and non-specialist interactions replicate differently in local entry tissue (fin), and systemic target tissue (kidney) using infectious hematopoietic necrosis virus (IHNV) and three salmonid fish hosts. Virus tissue replication was host specific, but one feature was shared by specialists and the generalist which was uncommon in the non-specialist interactions: high host entry and replication capacity in the local tissue after contact. Moreover, specialists showed increased replication in systemic target tissues early after host contact. By comparing ancestral and derived IHNV viruses, we also characterized replication tradeoffs associated with specialist and generalist evolution. Compared with the ancestral virus, a derived specialist gained early local replicative fitness in the new host but lost replicative fitness in the ancestral host. By contrast, a derived generalist showed small replication losses relative to the ancestral virus in the ancestral host but increased early replication in the local tissue of novel hosts. This study shows that the mechanisms of specialism and generalism are host specific and that local and systemic replication can contribute differently to overall within host replicative fitness for specialist and generalist viruses.


Asunto(s)
Salmonidae , Animales , Especialización , Riñón , Replicación Viral
4.
JAMA Netw Open ; 7(1): e2352440, 2024 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-38277148

RESUMEN

Importance: The number of active health care professionals has not kept pace with the increasing number of minoritized individuals in the US. The Summer Health Professions Education Program (SHPEP) was developed to alleviate this underrepresentation in the health workforce. Objective: To evaluate students' changes in perceived barriers and motivators for entering and succeeding in professional school after SHPEP participation. Design, Setting, and Participants: For this cohort study, anonymous electronic surveys were sent to the 2017 to 2021 SHPEP participants at an academic health center at a large university in the southern US. Participants were first- and second-year undergraduates interested in the health professions and enrolled in the SHPEP. Program participants were invited to complete the study. Exposures: An anonymous electronic survey was administered before and immediately after program completion. Main Outcomes and Measures: The SHPEP Career Barriers Survey (SCBS) is composed of 22 questions on motivators and 20 questions on barriers to entering and succeeding in health professional school. Students responded using a 5-point Likert scale, with 1 indicating strongly disagree and 5 indicating strongly agree. Mixed analysis of variance was used to analyze the program's latent factors. Results: Of all 402 SHPEP participants (mean [SD] age, 19.32 [0.88] years) from 2017 to 2021, 325 completed the preprogram survey and 259 also completed the postprogram survey. Of the 325 initial participants, 4 identified as American Indian or Alaska Native, Native Hawaiian, or Pacific Islander (1.2%); 12 as Asian (3.7%); 188 as Black (57.8%); 95 as Hispanic or Latino (29.2%); 7 as White (2.2%); and 16 as multiracial (4.9%). Two hundred twelve participants were female (65.2%), and 226 were first-generation college students (69.5%). Results of the SCBS indicate that the SHPEP had a significant small to moderate association on perceived motivators (mean [SD] x̅ = 84.60 [9.67] vs 80.95 [8.93]; P = .001) and decreases in perceived barriers (mean [SD] x̅ = 48.02 [13.20] vs 51.72 [11.39]; P = .008). There was no significant difference in program success between studied years. Conclusions and Relevance: In this cohort study, the SHPEP appeared to provide essential support for underrepresented students as measured by improved perceived motivators and reduced perceived barriers to entering professional education. Knowledge from this study can assist educators and health care professionals who wish to implement similar enrichment programs.


Asunto(s)
Personal de Salud , Estudiantes , Femenino , Humanos , Masculino , Adulto Joven , Estudios de Cohortes , Empleos en Salud , Personal de Salud/educación
5.
Mediastinum ; 7: 32, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38090036

RESUMEN

Malignant central airway obstruction (MCAO) impacts many patients with advanced primary lung cancers and metastatic disease to the thorax and may cause substantial symptoms and functional limitations in those affected. Making the diagnosis may be challenging as symptoms are often non-specific but identification is improved with a heightened level of suspicion and newer thoracic imaging modalities. Bronchoscopy plays a crucial role in the diagnosis and management of MCAO and therapeutic interventions may be lifesaving and result in palliation of symptoms. This may ultimately improve a patient's candidacy to receive additional systemic or local cancer therapies or potential tumor resection. After initial stabilization, it is important that patients with MCAO undergo prompt evaluation and treatment. Multiple bronchoscopic instruments are available for management depending on tumor characteristics, location of the obstruction, and viability of distal airways, and may be utilized in combination during therapeutic procedures. These modalities include dilation, endobronchial stent placement, thermal and non-thermal ablation, mechanical debulking, and novel endobronchial therapies. While these procedures are not without risk, there is ample evidence showing improvements in patient symptoms, quality of life, and survival following therapeutic bronchoscopy. This review article provides a general overview of the diagnosis and management of MCAO with a focus on bronchoscopic interventions.

6.
J Med Chem ; 66(13): 8510-8525, 2023 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-37358467

RESUMEN

Class C Acinetobacter-derived cephalosporinases (ADCs) represent an important target for inhibition in the multidrug-resistant pathogen Acinetobacter baumannii. Many ADC variants have emerged, and characterization of their structural and functional differences is essential. Equally as important is the development of compounds that inhibit all prevalent ADCs despite these differences. The boronic acid transition state inhibitor, MB076, a novel heterocyclic triazole with improved plasma stability, was synthesized and inhibits seven different ADC ß-lactamase variants with Ki values <1 µM. MB076 acted synergistically in combination with multiple cephalosporins to restore susceptibility. ADC variants containing an alanine duplication in the Ω-loop, specifically ADC-33, exhibited increased activity for larger cephalosporins, such as ceftazidime, cefiderocol, and ceftolozane. X-ray crystal structures of ADC variants in this study provide a structural context for substrate profile differences and show that the inhibitor adopts a similar conformation in all ADC variants, despite small changes near their active sites.


Asunto(s)
Acinetobacter baumannii , Cefalosporinasa , Cefalosporinasa/genética , Cefalosporinasa/química , Cefalosporinasa/farmacología , Ácidos Borónicos/farmacología , Ácidos Borónicos/química , Cefalosporinas/farmacología , beta-Lactamasas/genética , beta-Lactamasas/química , Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana
7.
Front Neurosci ; 17: 1088052, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37139524

RESUMEN

Diffusion tensor imaging (DTI) studies have demonstrated white matter microstructural differences between the left and right hemispheres of the brain. However, the basis of these hemispheric asymmetries is not yet understood in terms of the biophysical properties of white matter microstructure, especially in children. There are reports of altered hemispheric white matter lateralization in ASD; however, this has not been studied in other related neurodevelopmental disorders such as sensory processing disorder (SPD). Firstly, we postulate that biophysical compartment modeling of diffusion MRI (dMRI), such as Neurite Orientation Dispersion and Density Imaging (NODDI), can elucidate the hemispheric microstructural asymmetries observed from DTI in children with neurodevelopmental concerns. Secondly, we hypothesize that sensory over-responsivity (SOR), a common type of SPD, will show altered hemispheric lateralization relative to children without SOR. Eighty-seven children (29 females, 58 males), ages 8-12 years, presenting at a community-based neurodevelopmental clinic were enrolled, 48 with SOR and 39 without. Participants were evaluated using the Sensory Processing 3 Dimensions (SP3D). Whole brain 3 T multi-shell multiband dMRI (b = 0, 1,000, 2,500 s/mm2) was performed. Tract Based Spatial Statistics were used to extract DTI and NODDI metrics from 20 bilateral tracts of the Johns Hopkins University White-Matter Tractography Atlas and the lateralization Index (LI) was calculated for each left-right tract pair. With DTI metrics, 12 of 20 tracts were left lateralized for fractional anisotropy and 17/20 tracts were right lateralized for axial diffusivity. These hemispheric asymmetries could be explained by NODDI metrics, including neurite density index (18/20 tracts left lateralized), orientation dispersion index (15/20 tracts left lateralized) and free water fraction (16/20 tracts lateralized). Children with SOR served as a test case of the utility of studying LI in neurodevelopmental disorders. Our data demonstrated increased lateralization in several tracts for both DTI and NODDI metrics in children with SOR, which were distinct for males versus females, when compared to children without SOR. Biophysical properties from NODDI can explain the hemispheric lateralization of white matter microstructure in children. As a patient-specific ratio, the lateralization index can eliminate scanner-related and inter-individual sources of variability and thus potentially serve as a clinically useful imaging biomarker for neurodevelopmental disorders.

8.
Antibiotics (Basel) ; 12(4)2023 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-37107006

RESUMEN

Acinetobacter baumannii is a Gram-negative organism listed as an urgent threat pathogen by the World Health Organization (WHO). Carbapenem-resistant A. baumannii (CRAB), especially, present therapeutic challenges due to complex mechanisms of resistance to ß-lactams. One of the most important mechanisms is the production of ß-lactamase enzymes capable of hydrolyzing ß-lactam antibiotics. Co-expression of multiple classes of ß-lactamases is present in CRAB; therefore, the design and synthesis of "cross-class" inhibitors is an important strategy to preserve the efficacy of currently available antibiotics. To identify new, nonclassical ß-lactamase inhibitors, we previously identified a sulfonamidomethaneboronic acid CR167 active against Acinetobacter-derived class C ß-lactamases (ADC-7). The compound demonstrated affinity for ADC-7 with a Ki = 160 nM and proved to be able to decrease MIC values of ceftazidime and cefotaxime in different bacterial strains. Herein, we describe the activity of CR167 against other ß-lactamases in A. baumannii: the cefepime-hydrolysing class C extended-spectrum ß-lactamase (ESAC) ADC-33 and the carbapenem-hydrolyzing OXA-24/40 (class D). These investigations demonstrate CR167 as a valuable cross-class (C and D) inhibitor, and the paper describes our attempts to further improve its activity. Five chiral analogues of CR167 were rationally designed and synthesized. The structures of OXA-24/40 and ADC-33 in complex with CR167 and select chiral analogues were obtained. The structure activity relationships (SARs) are highlighted, offering insights into the main determinants for cross-class C/D inhibitors and impetus for novel drug design.

9.
Animals (Basel) ; 12(14)2022 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-35883308

RESUMEN

Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonids in North America, Europe, and Asia that is phylogenetically classified into five major virus genogroups (U, M, L, E, and J). The geographic range of the U and M genogroup isolates overlap in the North American Columbia River Basin and Washington Coast region, where these genogroups pose different risks depending on the species of Pacific salmon (Oncorhynchus spp.). For certain management decisions, there is a need to both test for IHNV presence and rapidly determine the genogroup. Herein, we report the development and validation of a U/M multiplex reverse transcription, real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) protein gene. The new U/M RT-rPCR is a rapid, sensitive, and repeatable assay capable of specifically discriminating between North American U and M genogroup IHNV isolates. However, one M genogroup isolate obtained from commercially cultured Idaho rainbow trout (O. mykiss) showed reduced sensitivity with the RT-rPCR test, suggesting caution may be warranted before applying RT-rPCR as the sole surveillance test in areas associated with the Idaho trout industry. The new U/M assay had high diagnostic sensitivity (DSe > 94%) and specificity (DSp > 97%) in free-ranging adult Pacific salmon, when assessed relative to cell culture, the widely accepted reference standard, as well as the previously validated universal N RT-rPCR test. The high diagnostic performance of the new U/M assay indicates the test is suitable for surveillance, diagnosis, and confirmation of IHNV in Pacific salmon from the Pacific Northwest regions where the U and M genogroups overlap.

10.
Dis Aquat Organ ; 150: 61-67, 2022 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-35833545

RESUMEN

In recent decades, evidence has accumulated to suggest that the widespread and highly variable parasite Ichthyophonus hoferi is actually a species complex. Highly plastic morphology and a general lack of defining structures has contributed to the likely underestimate of biodiversity within this group. Molecular methods are a logical next step in the description of these parasites, but markers used to date have been too conserved to resolve species boundaries. Here we use mitochondrial encoded cytochrome-c oxidase (MTCO1) gene sequences and phylogenic analysis to compare Ichthyophonus spp. isolates from several marine and anadromous fish hosts. The resulting phylogeny displays lineage separation among isolates and possible host/niche segregation not previously described. The parasite type that infects Pacific herring Clupea pallasii, Atlantic herring C. harengus, Atlantic salmon Salmo salar, and Pacific staghorn sculpin Oligocottus maculosus (Clade A) is different from that which infects Chinook salmon Oncorhynchus tshawytscha, walleye pollock Gadus chalcogrammus, Greenland halibut Reinhardtius hippoglossoides, and Pacific halibut Hippoglossus stenolepsis (Clade B). MTCO1 sequences confirmed the presence of a more divergent Ichthyophonus sp. isolated from American shad Alosa sapidissima in rivers of eastern North America (Clade C), while American shad introduced to the Pacific Ocean are infected with the same parasite that infects Pacific herring (Clade A). Currently there are no consensus criteria for delimiting species within Ichthyophonidae, but MTCO1 sequences hold promise as a potential species identifying marker and useful epizootiological tool.


Asunto(s)
Enfermedades de los Peces , Gadiformes , Mesomycetozoea , Animales , Complejo IV de Transporte de Electrones/genética , Enfermedades de los Peces/epidemiología , Enfermedades de los Peces/parasitología , Peces , Genotipo , Mesomycetozoea/genética , Océano Pacífico , Filogenia , Salmón
11.
J Biol Chem ; 298(7): 102127, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35709986

RESUMEN

The evolution of multidrug resistance in Acinetobacter spp. increases the risk of our best antibiotics losing their efficacy. From a clinical perspective, the carbapenem-hydrolyzing class D ß-lactamase subfamily present in Acinetobacter spp. is particularly concerning because of its ability to confer resistance to carbapenems. The kinetic profiles of class D ß-lactamases exhibit variability in carbapenem hydrolysis, suggesting functional differences. To better understand the structure-function relationship between the carbapenem-hydrolyzing class D ß-lactamase OXA-24/40 found in Acinetobacter baumannii and carbapenem substrates, we analyzed steady-state kinetics with the carbapenem antibiotics meropenem and ertapenem and determined the structures of complexes of OXA-24/40 bound to imipenem, meropenem, doripenem, and ertapenem, as well as the expanded-spectrum cephalosporin cefotaxime, using X-ray crystallography. We show that OXA-24/40 exhibits a preference for ertapenem compared with meropenem, imipenem, and doripenem, with an increase in catalytic efficiency of up to fourfold. We suggest that superposition of the nine OXA-24/40 complexes will better inform future inhibitor design efforts by providing insight into the complicated and varying ways in which carbapenems are selected and bound by class D ß-lactamases.


Asunto(s)
Proteínas Bacterianas , Carbapenémicos , beta-Lactamasas , Acinetobacter baumannii/enzimología , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Carbapenémicos/química , Carbapenémicos/metabolismo , Hidrólisis , Pruebas de Sensibilidad Microbiana , Conformación Proteica , Especificidad por Sustrato , beta-Lactamasas/química , beta-Lactamasas/metabolismo
12.
Pathogens ; 10(7)2021 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-34358005

RESUMEN

Environmental variation has important effects on host-pathogen interactions, affecting large-scale ecological processes such as the severity and frequency of epidemics. However, less is known about how the environment interacts with host immunity to modulate virus fitness within hosts. Here, we studied the interaction between host immune responses and water temperature on the long-term persistence of a model vertebrate virus, infectious hematopoietic necrosis virus (IHNV) in steelhead trout (Oncorhynchus mykiss). We first used cell culture methods to factor out strong host immune responses, allowing us to test the effect of temperature on viral replication. We found that 15 ∘C water temperature accelerated IHNV replication compared to the colder 10 and 8 ∘C temperatures. We then conducted in vivo experiments to quantify the effect of 6, 10, and 15 ∘C water temperatures on IHNV persistence over 8 months. Fish held at 15 and 10 ∘C were found to have higher prevalence of neutralizing antibodies compared to fish held at 6 ∘C. We found that IHNV persisted for a shorter time at warmer temperatures and resulted in an overall lower fish mortality compared to colder temperatures. These results support the hypothesis that temperature and host immune responses interact to modulate virus persistence within hosts. When immune responses were minimized (i.e., in vitro) virus replication was higher at warmer temperatures. However, with a full potential for host immune responses (i.e., in vivo experiments) longer virus persistence and higher long-term virulence was favored in colder temperatures. We also found that the viral RNA that persisted at later time points (179 and 270 days post-exposure) was mostly localized in the kidney and spleen tissues. These tissues are composed of hematopoietic cells that are favored targets of the virus. By partitioning the effect of temperature on host and pathogen responses, our results help to better understand environmental drivers of host-pathogen interactions within hosts, providing insights into potential host-pathogen responses to climate change.

13.
Diagn Microbiol Infect Dis ; 99(2): 115242, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33248392

RESUMEN

Successful treatment of Acinetobacter baumannii infections require early and appropriate antimicrobial therapy. One of the first steps in this process is understanding which ß-lactamase (bla) alleles are present and in what combinations. Thus, we performed WGS on 98 carbapenem-resistant A. baumannii (CR Ab). In most isolates, an acquired blaOXA carbapenemase was found in addition to the intrinsic blaOXA allele. The most commonly found allele was blaOXA-23 (n = 78/98). In some isolates, blaOXA-23 was found in addition to other carbapenemase alleles: blaOXA-82 (n = 12/78), blaOXA-72 (n = 2/78) and blaOXA-24/40 (n = 1/78). Surprisingly, 20% of isolates carried carbapenemases not routinely assayed for by rapid molecular diagnostic platforms, i.e., blaOXA-82 and blaOXA-172; all had ISAba1 elements. In 8 CR Ab, blaOXA-82 or blaOXA-172 was the only carbapenemase. Both blaOXA-24/40 and its variant blaOXA-72 were each found in 6/98 isolates. The most prevalent ADC variants were blaADC-30 (21%), blaADC-162 (21%), and blaADC-212 (26%). Complete combinations are reported.


Asunto(s)
Acinetobacter baumannii/genética , Proteínas Bacterianas/genética , Carbapenémicos/farmacología , Resistencia betalactámica/genética , beta-Lactamasas/genética , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/enzimología , Acinetobacter baumannii/aislamiento & purificación , Genoma Bacteriano/genética , Humanos
14.
Antimicrob Agents Chemother ; 64(12)2020 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-32988830

RESUMEN

Extended-spectrum class C ß-lactamases have evolved to rapidly inactivate expanded-spectrum cephalosporins, a class of antibiotics designed to be resistant to hydrolysis by ß-lactamase enzymes. To better understand the mechanism by which Acinetobacter-derived cephalosporinase-7 (ADC-7), a chromosomal AmpC enzyme, hydrolyzes these molecules, we determined the X-ray crystal structure of ADC-7 in an acyl-enzyme complex with the cephalosporin ceftazidime (2.40 Å) as well as in complex with a boronic acid transition state analog inhibitor that contains the R1 side chain of ceftazidime (1.67 Å). In the acyl-enzyme complex, the carbonyl oxygen is situated in the oxyanion hole where it makes key stabilizing interactions with the main chain nitrogens of Ser64 and Ser315. The boronic acid O1 hydroxyl group is similarly positioned in this area. Conserved residues Gln120 and Asn152 form hydrogen bonds with the amide group of the R1 side chain in both complexes. These complexes represent two steps in the hydrolysis of expanded-spectrum cephalosporins by ADC-7 and offer insight into the inhibition of ADC-7 by ceftazidime through displacement of the deacylating water molecule as well as blocking its trajectory to the acyl carbonyl carbon. In addition, the transition state analog inhibitor, LP06, was shown to bind with high affinity to ADC-7 (Ki , 50 nM) and was able to restore ceftazidime susceptibility, offering the potential for optimization efforts of this type of inhibitor.


Asunto(s)
Acinetobacter , Ácidos Borónicos , Ceftazidima , Cefalosporinasa , Antibacterianos/farmacología , Ácidos Borónicos/farmacología , Ceftazidima/farmacología , Cefalosporinasa/efectos de los fármacos , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas
15.
J Fish Dis ; 43(7): 719-728, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32476167

RESUMEN

Piscine orthoreovirus genotype 1 (PRV-1) is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar L.). The virus has also been found in Pacific salmonids in western North America, raising concerns about the risk to native salmon and trout. Here, we report the results of laboratory challenges using juvenile Chinook salmon, coho salmon and rainbow trout injected with tissue homogenates from Atlantic salmon testing positive for PRV-1 or with control material. Fish were sampled at intervals to assess viral RNA transcript levels, haematocrit, erythrocytic inclusions and histopathology. While PRV-1 replicated in all species, there was negligible mortality in any group. We observed a few erythrocytic inclusion bodies in fish from the PRV-1-infected groups. At a few time points, haematocrits were significantly lower in the PRV-1-infected groups relative to controls, but in no case was anaemia noted. The most common histopathological finding was mild, focal myocarditis in both the non-infected controls and PRV-1-infected fish. All cardiac lesions were judged mild, and none were consistent with those of HSMI. Together, these results suggest all three species are susceptible to PRV-1 infection, but in no case did infection cause notable disease in these experiments.


Asunto(s)
Enfermedades de los Peces/virología , Genotipo , Hematócrito/veterinaria , Cuerpos de Inclusión Viral/fisiología , Oncorhynchus , Orthoreovirus/fisiología , Infecciones por Reoviridae/veterinaria , Animales , Oncorhynchus kisutch , Oncorhynchus mykiss , Orthoreovirus/genética , ARN Viral/análisis , Infecciones por Reoviridae/virología
16.
ACS Infect Dis ; 6(7): 1965-1975, 2020 07 10.
Artículo en Inglés | MEDLINE | ID: mdl-32502340

RESUMEN

Boronic acid transition state inhibitors (BATSIs) are known reversible covalent inhibitors of serine ß-lactamases. The selectivity and high potency of specific BATSIs bearing an amide side chain mimicking the ß-lactam's amide side chain are an established and recognized synthetic strategy. Herein, we describe a new class of BATSIs where the amide group is replaced by a bioisostere triazole; these compounds were designed as molecular probes. To this end, a library of 26 α-triazolylmethaneboronic acids was synthesized and tested against the clinically concerning Acinetobacter-derived cephalosporinase, ADC-7. In steady state analyses, these compounds demonstrated Ki values ranging from 90 nM to 38 µM (±10%). Five compounds were crystallized in complex with ADC-7 ß-lactamase, and all the crystal structures reveal the triazole is in the putative amide binding site, thus confirming the triazole-amide bioisosterism. The easy synthetic access of these new inhibitors as prototype scaffolds allows the insertion of a wide range of chemical groups able to explore the enzyme binding site and provides insights on the importance of specific residues in recognition and catalysis. The best inhibitor identified, compound 6q (Ki 90 nM), places a tolyl group near Arg340, making favorable cation-π interactions. Notably, the structure of 6q does not resemble the natural substrate of the ß-lactamase yet displays a pronounced inhibition activity, in addition to lowering the minimum inhibitory concentration (MIC) of ceftazidime against three bacterial strains expressing class C ß-lactamases. In summary, these observations validate the α-triazolylboronic acids as a promising template for further inhibitor design.


Asunto(s)
Acinetobacter baumannii , Inhibidores de beta-Lactamasas , Acinetobacter baumannii/metabolismo , Cefalosporinasa/genética , Cefalosporinasa/metabolismo , Relación Estructura-Actividad , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/metabolismo
17.
Artículo en Inglés | MEDLINE | ID: mdl-30863823

RESUMEN

We report here the genome sequences of two index strains of Pacific salmon paramyxovirus isolated in 1982 and 1983 from adult salmon in Oregon. The isolates are most closely related to Atlantic salmon paramyxovirus, the type species of the genus Aquaparamyxovirus, but are sufficiently distinct to be considered two genotypes of a novel species.

18.
Virology ; 525: 143-149, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30278384

RESUMEN

The aquaculture industry is growing rapidly to meet the needs for global protein consumption. Viral diseases in aquaculture are quite challenging due to lack of treatment options as well as limited injection-delivery vaccines, which are costly. Thus, water-immersion antiviral treatments are highly desirable. This study focused on broad-spectrum, light-activated antivirals that target the viral membrane (envelope) of viruses to prevent viral-cell membrane fusion, ultimately blocking viral entry into cells. Of the tested small-molecules, JL122, a new broad-spectrum antiviral previously unexplored against aquatic viruses, blocked infection of three aquatic rhabdoviruses (IHNV, VHSV and SVCV) in cell culture and in two live fish challenge models. Importantly, JL122 inhibited transmission of IHNV from infected to uninfected rainbow trout. Further, the effective antiviral concentrations were not toxic to cells or susceptible fish. These results show promise for JL122 to become an immersion treatment option for outbreaks of aquatic enveloped viral infections.


Asunto(s)
Antivirales/uso terapéutico , Enfermedades de los Peces/virología , Virus de la Necrosis Hematopoyética Infecciosa , Novirhabdovirus , Oncorhynchus mykiss , Infecciones por Rhabdoviridae/veterinaria , Animales , Antivirales/clasificación , Línea Celular Tumoral , Enfermedades de los Peces/tratamiento farmacológico , Estructura Molecular , Infecciones por Rhabdoviridae/prevención & control , Infecciones por Rhabdoviridae/virología , Relación Estructura-Actividad
19.
Biochem J ; 475(1): 273-288, 2018 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-29229762

RESUMEN

OXA-239 is a class D carbapenemase isolated from an Acinetobacter baumannii strain found in Mexico. This enzyme is a variant of OXA-23 with three amino acid substitutions in or near the active site. These substitutions cause OXA-239 to hydrolyze late-generation cephalosporins and the monobactam aztreonam with greater efficiency than OXA-23. OXA-239 activity against the carbapenems doripenem and imipenem is reduced ∼3-fold and 20-fold, respectively. Further analysis demonstrated that two of the substitutions (P225S and D222N) are largely responsible for the observed alteration of kinetic parameters, while the third (S109L) may serve to stabilize the protein. Structures of OXA-239 with cefotaxime, doripenem and imipenem bound as acyl-intermediates were determined. These structures reveal that OXA-239 has increased flexibility in a loop that contains P225S and D222N. When carbapenems are bound, the conformation of this loop is essentially identical with that observed previously for OXA-23, with a narrow active site that makes extensive contacts to the ligand. When cefotaxime is bound, the loop can adopt a different conformation that widens the active site to allow binding of that bulky drug. This alternate conformation is made possible by P225S and further stabilized by D222N. Taken together, these results suggest that the three substitutions were selected to expand the substrate specificity profile of OXA-23 to cephalosporins and monobactams. The loss of activity against imipenem, however, suggests that there may be limits to the plasticity of class D enzymes with regard to evolving active sites that can effectively bind multiple classes of ß-lactam drugs.


Asunto(s)
Acinetobacter baumannii/enzimología , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Carbapenémicos/química , Cefotaxima/química , Imipenem/química , beta-Lactamasas/química , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbapenémicos/metabolismo , Carbapenémicos/farmacología , Dominio Catalítico , Cefotaxima/metabolismo , Cefotaxima/farmacología , Clonación Molecular , Cristalografía por Rayos X , Doripenem , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Imipenem/metabolismo , Imipenem/farmacología , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , beta-Lactamasas/genética , beta-Lactamasas/metabolismo
20.
ACS Infect Dis ; 4(3): 325-336, 2018 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-29144724

RESUMEN

Acinetobacter baumannii is a multidrug resistant pathogen that infects more than 12 000 patients each year in the US. Much of the resistance to ß-lactam antibiotics in Acinetobacter spp. is mediated by class C ß-lactamases known as Acinetobacter-derived cephalosporinases (ADCs). ADCs are unaffected by clinically used ß-lactam-based ß-lactamase inhibitors. In this study, five boronic acid transition state analog inhibitors (BATSIs) were evaluated for inhibition of the class C cephalosporinase ADC-7. Our goal was to explore the properties of BATSIs designed to probe the R1 binding site. Ki values ranged from low micromolar to subnanomolar, and circular dichroism (CD) demonstrated that each inhibitor stabilizes the ß-lactamase-inhibitor complexes. Additionally, X-ray crystal structures of ADC-7 in complex with five inhibitors were determined (resolutions from 1.80 to 2.09 Å). In the ADC-7/CR192 complex, the BATSI with the lowest Ki (0.45 nM) and greatest Δ Tm (+9 °C), a trifluoromethyl substituent, interacts with Arg340. Arg340 is unique to ADCs and may play an important role in the inhibition of ADC-7. The ADC-7/BATSI complexes determined in this study shed light into the unique recognition sites in ADC enzymes and also offer insight into further structure-based optimization of these inhibitors.


Asunto(s)
Acinetobacter/enzimología , Ácidos Borónicos/química , Ácidos Borónicos/farmacología , Cefalosporinasa/química , Cefalosporinasa/metabolismo , Inhibidores de beta-Lactamasas/química , Inhibidores de beta-Lactamasas/farmacología , Sitios de Unión , Dicroismo Circular , Cristalografía por Rayos X , Modelos Moleculares , Unión Proteica , Conformación Proteica
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