A comparative analysis of SARS-CoV-2 antivirals characterizes 3CLpro inhibitor PF-00835231 as a potential new treatment for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231's early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231's efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models.Importance:The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

Type I Interferon Susceptibility Distinguishes SARS-CoV-2 from SARS-CoV.

SARS-CoV-2, a novel coronavirus (CoV) that causes COVID-19, has recently emerged causing an ongoing outbreak of viral pneumonia around the world. While distinct from SARS-CoV, both group 2B CoVs share similar genome organization, origins to bat CoVs, and an arsenal of immune antagonists. In this report, we evaluate type I interferon (IFN-I) sensitivity of SARS-CoV-2 relative to the original SARS-CoV. Our results indicate that while SARS-CoV-2 maintains similar viral replication to SARS-CoV, the novel CoV is much more sensitive to IFN-I. In Vero E6 and in Calu3 cells, SARS-CoV-2 is substantially attenuated in the context of IFN-I pretreatment, whereas SARS-CoV is not. In line with these findings, SARS-CoV-2 fails to counteract phosphorylation of STAT1 and expression of ISG proteins, while SARS-CoV is able to suppress both. Comparing SARS-CoV-2 and influenza A virus in human airway epithelial cultures, we observe the absence of IFN-I stimulation by SARS-CoV-2 alone but detect the failure to counteract STAT1 phosphorylation upon IFN-I pretreatment, resulting in near ablation of SARS-CoV-2 infection. Next, we evaluated IFN-I treatment postinfection and found that SARS-CoV-2 was sensitive even after establishing infection. Finally, we examined homology between SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonists. The absence of an equivalent open reading frame 3b (ORF3b) and genetic differences versus ORF6 suggest that the two key IFN-I antagonists may not maintain equivalent function in SARS-CoV-2. Together, the results identify key differences in susceptibility to IFN-I responses between SARS-CoV and SARS-CoV-2 that may help inform disease progression, treatment options, and animal model development.IMPORTANCE With the ongoing outbreak of COVID-19, differences between SARS-CoV-2 and the original SARS-CoV could be leveraged to inform disease progression and eventual treatment options. In addition, these findings could have key implications for animal model development as well as further research into how SARS-CoV-2 modulates the type I IFN response early during infection.

The ETS transcription factor ELF1 regulates a broadly antiviral program distinct from the type I interferon response.

Induction of vast transcriptional programs is a central event of innate host responses to viral infections. Here we report a transcriptional program with potent antiviral activity, driven by E74-like ETS transcription factor 1 (ELF1). Using microscopy to quantify viral infection over time, we found that ELF1 inhibits eight diverse RNA and DNA viruses after multi-cycle replication. Elf1 deficiency results in enhanced susceptibility to influenza A virus infections in mice. ELF1 does not feed-forward to induce interferons, and ELF1's antiviral effect is not abolished by the absence of STAT1 or by inhibition of JAK phosphorylation. Accordingly, comparative expression analyses by RNA-seq revealed that the ELF1 transcriptional program is distinct from interferon signatures. Thus, ELF1 provides an additional layer of the innate host response, independent from the action of type I interferons.

Characteristics and laboratory findings on admission to the emergency department among 2873 hospitalized patients with COVID-19: the impact of adjusted laboratory tests in multicenter studies. A multicenter study in Spain (BIOCOVID-Spain study).

Identification of predictors for severe disease progression is key for risk stratification in COVID-19 patients. We aimed to describe the main characteristics and identify the early predictors for severe outcomes among hospitalized patients with COVID-19 in Spain. This was an observational, retrospective cohort study (BIOCOVID-Spain study) including COVID-19 patients admitted to 32 Spanish hospitals. Demographics, comorbidities and laboratory tests were collected. Outcome was in-hospital mortality. For analysis, laboratory tests values were previously adjusted to assure the comparability of results among participants. Cox regression was performed to identify predictors. Study population included 2873 hospitalized COVID-19 patients. Nine variables were independent predictors for in-hospital mortality, including creatinine (Hazard ratio [HR]:1.327; 95% Confidence Interval [CI]: 1.040-1.695, p = .023), troponin (HR: 2.150; 95% CI: 1.155-4.001; p = .016), platelet count (HR: 0.994; 95% CI: 0.989-0.998; p = .004) and C-reactive protein (HR: 1.037; 95% CI: 1.006-1.068; p = .019). This is the first multicenter study in which an effort was carried out to adjust the results of laboratory tests measured with different methodologies to guarantee their comparability. We reported a comprehensive information about characteristics in a large cohort of hospitalized COVID-19 patients, focusing on the analytical features. Our findings may help to identify patients early at a higher risk for an adverse outcome.

The relationship with the voices as a dialogical experience: The role of self-focused attention and dissociation.

OBJECTIVE: The purpose of this article was to study the relationship of self-focused attention and dissociation with the dialogical relationship persons diagnosed with psychosis have with their voices. METHOD: The DAIMON Scale was applied to 62 persons diagnosed with psychosis to measure the dialogical relationship with their voices, and the Cambridge depersonalization scale, the Tellegen absorption scale, and the self-focused attention scale. RESULTS: The results showed that the dialogical relationship with the voices was associated with high levels of self-focused attention (private and public), depersonalization, and absorption. It was also found that absorption mediated significantly between public self-focused attention and the dialogical relationship with the voices. CONCLUSIONS: The role of dissociation and self-focused attention in forming the dialogical relationship a person with psychosis has with the voices is discussed and approaches to treatment are suggested.

Al2 Pt for Oxygen Evolution in Water Splitting: A Strategy for Creating Multifunctionality in Electrocatalysis.

The production of hydrogen via water electrolysis is feasible only if effective and stable catalysts for the oxygen evolution reaction (OER) are available. Intermetallic compounds with well-defined crystal and electronic structures as well as particular chemical bonding features are suggested here to act as precursors for new composite materials with attractive catalytic properties. Al2 Pt combines a characteristic inorganic crystal structure (anti-fluorite type) and a strongly polar chemical bonding with the advantage of elemental platinum in terms of stability against dissolution under OER conditions. We describe here the unforeseen performance of a surface nanocomposite architecture resulting from the self-organized transformation of the bulk intermetallic precursor Al2 Pt in OER.

A neonatal mouse model characterizes transmissibility of SARS-CoV-2 variants and reveals a role for ORF8.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets 1, 2 . Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not robustly transmit SARS-CoV-2 3 . Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron BA.1 and Omicron BQ.1.1. We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission in our model. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing for the first time a role for an accessory protein in this context.

A neonatal mouse model characterizes transmissibility of SARS-CoV-2 variants and reveals a role for ORF8.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets. Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not robustly transmit SARS-CoV-2. Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron BA.1 and Omicron BQ.1.1. We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission in our model. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing a role for an accessory protein in this context.

Gut microbiome dysbiosis during COVID-19 is associated with increased risk for bacteremia and microbial translocation.

The microbial populations in the gut microbiome have recently been associated with COVID-19 disease severity. However, a causal impact of the gut microbiome on COVID-19 patient health has not been established. Here we provide evidence that gut microbiome dysbiosis is associated with translocation of bacteria into the blood during COVID-19, causing life-threatening secondary infections. Antibiotics and other treatments during COVID-19 can potentially confound microbiome associations. We therefore first demonstrate in a mouse model that SARS-CoV-2 infection can induce gut microbiome dysbiosis, which correlated with alterations to Paneth cells and goblet cells, and markers of barrier permeability. Comparison with stool samples collected from 96 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, paralleling our observations in the animal model. Specifically, we observed blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species in hospitalized COVID-19 patients. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data obtained from these patients indicates that bacteria may translocate from the gut into the systemic circulation of COVID-19 patients. These results are consistent with a direct role for gut microbiome dysbiosis in enabling dangerous secondary infections during COVID-19.

Gut microbiome dysbiosis in antibiotic-treated COVID-19 patients is associated with microbial translocation and bacteremia.

Although microbial populations in the gut microbiome are associated with COVID-19 severity, a causal impact on patient health has not been established. Here we provide evidence that gut microbiome dysbiosis is associated with translocation of bacteria into the blood during COVID-19, causing life-threatening secondary infections. We first demonstrate SARS-CoV-2 infection induces gut microbiome dysbiosis in mice, which correlated with alterations to Paneth cells and goblet cells, and markers of barrier permeability. Samples collected from 96 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, including blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data indicates that bacteria may translocate from the gut into the systemic circulation of COVID-19 patients. These results are consistent with a direct role for gut microbiome dysbiosis in enabling dangerous secondary infections during COVID-19.

SARS-CoV-2 Portrayed against HIV: Contrary Viral Strategies in Similar Disguise.

SARS-CoV-2 and HIV are zoonotic viruses that rapidly reached pandemic scale, causing global losses and fear. The COVID-19 and AIDS pandemics ignited massive efforts worldwide to develop antiviral strategies and characterize viral architectures, biological and immunological properties, and clinical outcomes. Although both viruses have a comparable appearance as enveloped viruses with positive-stranded RNA and envelope spikes mediating cellular entry, the entry process, downstream biological and immunological pathways, clinical outcomes, and disease courses are strikingly different. This review provides a systemic comparison of both viruses' structural and functional characteristics, delineating their distinct strategies for efficient spread.

[Minimal important clinical difference in health-related quality of life.] / Diferencia mínima clínica importante en calidad de vida relacionada con la salud.

Una de las limitaciones para el uso de los Cuestionarios de Calidad de Vida Relacionada con la Salud y de los resultados reportados por pacientes es la dificultad de interpretar los valores obtenidos. La diferencia mínima clínica importante es una medida que ayuda a comprender los resultados de estos cuestionarios y valorar la relevancia clínica del efecto conseguido por la intervención realizada. En este trabajo revisamos el concepto de diferencia mínima clínica importante, describimos los métodos utilizados para su obtención y exponemos sus dificultades, limitaciones y aplicabilidad. Dentro de los resultados reportados por pacientes y, particularmente, en los de calidad de vida, la diferencia mínima clínica importante es una herramienta que ayuda a los clínicos a utilizar correctamente las escalas de medida e interpretar el efecto de las intervenciones. Con este artículo esperamos facilitar la implantación y uso de la diferencia mínima clínica importante y los Cuestionarios de Calidad de Vida Relacionada con la Salud en la práctica habitual con nuestros pacientes.

One of the limitations for the use of Health-related Quality of Life Questionnaires and the results reported by patients is the difficulty of interpreting the values obtained. The minimal important clinical difference is a measure that helps to understand the results of these questionnaires and assess the clinical relevance of the effect achieved by the intervention performed. In this paper, we review the concept of minimal important clinical difference, describe the methods used to obtain it, and expose its difficulties, limitations, and applicability. Within the results reported by patients, and particularly in quality of life, the minimal important clinical difference is a tool that helps clinicians to correctly use the measurement scales and interpret the effect of the interventions. With this article, we hope to facilitate the implementation and use of the minimal important clinical difference and the Health-related Quality of Life Questionnaires in routine practice with our patients.

A comparative analysis of SARS-CoV-2 antivirals in human airway models characterizes 3CLpro inhibitor PF-00835231 as a potential new treatment for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231's early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231's efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models.

Gut microbiome dysbiosis during COVID-19 is associated with increased risk for bacteremia and microbial translocation.

The microbial populations in the gut microbiome have recently been associated with COVID-19 disease severity. However, a causal impact of the gut microbiome on COVID-19 patient health has not been established. Here we provide evidence that gut microbiome dysbiosis is associated with translocation of bacteria into the blood during COVID-19, causing life-threatening secondary infections. Antibiotics and other treatments during COVID-19 can potentially confound microbiome associations. We therefore first demonstrate that the gut microbiome is directly affected by SARS-CoV-2 infection in a dose-dependent manner in a mouse model, causally linking viral infection and gut microbiome dysbiosis. Comparison with stool samples collected from 97 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, paralleling our observations in the animal model. Specifically, we observed blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species in hospitalized COVID-19 patients. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data obtained from these patients suggest that bacteria translocate from the gut into the systemic circulation of COVID-19 patients. These results are consistent with a direct role for gut microbiome dysbiosis in enabling dangerous secondary infections during COVID 19.

A shortened version of the Headache-Specific Locus of Control Scale in Spanish population.

BACKGROUND AND OBJECTIVE: Further questions need to be addressed in the evaluation of locus of control (LOC) in headaches, such as reducing scale length and adapting them to diverse cultural environments, as in the case of Spain. METHODS: We perform a confirmatory factor analysis of the most outstanding items contained in the Headache-Specific Locus of Control Scale in the responses of 118 patients suffering from headaches who received assistance at public health care centers in the province of Seville (Spain). RESULTS: The adjustment was positive, thus confirming the original structure of 3 factors: internal locus of control (LOC-I), health care professionals' LOC, and chance locus of control (LOC-C). Scale validation was performed by examining associations both with headache clinical parameters and psychological measures. The latter included self-efficacy, internal language, coping strategies, and pain behaviors. LOC-C results deserve special mention, supporting the idea that it seems more important to avoid that patients develop LOC-C rather than boosting LOC-I and LOC-P expectations. CONCLUSIONS: The so-called Headache-Specific Locus of Control Scale-Short Form 9 has turned out to be a parsimonious (9 items), valid, and reliable measure of headache LOC.

Type I interferon susceptibility distinguishes SARS-CoV-2 from SARS-CoV.

SARS-CoV-2, a novel coronavirus (CoV) that causes COVID-19, has recently emerged causing an ongoing outbreak of viral pneumonia around the world. While distinct from SARS-CoV, both group 2B CoVs share similar genome organization, origins to bat CoVs, and an arsenal of immune antagonists. In this report, we evaluate type-I interferon (IFN-I) sensitivity of SARS-CoV-2 relative to the original SARS-CoV. Our results indicate that while SARS-CoV-2 maintains similar viral replication to SARS-CoV, the novel CoV is much more sensitive to IFN-I. In Vero and in Calu3 cells, SARS-CoV-2 is substantially attenuated in the context of IFN-I pretreatment, while SARS-CoV is not. In line with these findings, SARS-CoV-2 fails to counteract phosphorylation of STAT1 and expression of ISG proteins, while SARS-CoV is able to suppress both. Comparing SARS-CoV-2 and influenza A virus in human airway epithelial cultures (HAEC), we observe the absence of IFN-I stimulation by SARS-CoV-2 alone, but detect failure to counteract STAT1 phosphorylation upon IFN-I pretreatment resulting in near ablation of SARS-CoV-2 infection. Next, we evaluated IFN-I treatment post infection and found SARS-CoV-2 was sensitive even after establishing infection. Finally, we examined homology between SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonists. The absence of an equivalent open reading frame (ORF) 3b and changes to ORF6 suggest the two key IFN-I antagonists may not maintain equivalent function in SARS-CoV-2. Together, the results identify key differences in susceptibility to IFN-I responses between SARS-CoV and SARS-CoV-2 that may help inform disease progression, treatment options, and animal model development.

The Rhizophagus irregularis Genome Encodes Two CTR Copper Transporters That Mediate Cu Import Into the Cytosol and a CTR-Like Protein Likely Involved in Copper Tolerance.

Arbuscular mycorrhizal fungi increase fitness of their host plants under Cu deficient and toxic conditions. In this study, we have characterized two Cu transporters of the CTR family (RiCTR1 and RiCTR2) and a CTR-like protein (RiCTR3A) of Rhizophagus irregularis. Functional analyses in yeast revealed that RiCTR1 encodes a plasma membrane Cu transporter, RiCTR2 a vacuolar Cu transporter and RiCTR3A a plasma membrane protein involved in Cu tolerance. RiCTR1 was more highly expressed in the extraradical mycelia (ERM) and RiCTR2 in the intraradical mycelia (IRM). In the ERM, RiCTR1 expression was up-regulated by Cu deficiency and down-regulated by Cu toxicity. RiCTR2 expression increased only in the ERM grown under severe Cu-deficient conditions. These data suggest that RiCTR1 is involved in Cu uptake by the ERM and RiCTR2 in mobilization of vacuolar Cu stores. Cu deficiency decreased mycorrhizal colonization and arbuscule frequency, but increased RiCTR1 and RiCTR2 expression in the IRM, which suggest that the IRM has a high Cu demand. The two alternatively spliced products of RiCTR3, RiCTR3A and RiCTR3B, were more highly expressed in the ERM. Up-regulation of RiCTR3A by Cu toxicity and the yeast complementation assays suggest that RiCTR3A might function as a Cu receptor involved in Cu tolerance.

Untargeted metabolomics reveals specific withanolides and fatty acyl glycoside as tentative metabolites to differentiate organic and conventional Physalis peruviana fruits.

The agronomic production systems may affect the levels of food metabolites. Metabolomics approaches have been applied as useful tool for the characterization of fruit metabolome. In this study, metabolomics techniques were used to assess the differences in phytochemical composition between goldenberry samples produced by organic and conventional systems. To verify that the organic samples were free of pesticides, individual pesticides were analyzed. Principal component analysis showed a clear separation of goldenberry samples from two different farming systems. Via targeted metabolomics assays, whereby carotenoids and ascorbic acid were analyzed, not statistical differences between both crops were found. Conversely, untargeted metabolomics allowed us to identify two withanolides and one fatty acyl glycoside as tentative metabolites to differentiate goldenberry fruits, recording organic fruits higher amounts of these compounds than conventional samples. Hence, untargeted metabolomics technology could be suitable to research differences on phytochemicals under different agricultural management practices and to authenticate organic products.

Comparison of distortion product otoacoustic emissions in 28 inbred strains of mice.

Cochlear function was evaluated in a longitudinal study of 28 inbred strains of mice at 3 and 5 mo of age using measures of distortion product otoacoustic emissions (DPOAEs) in response to a federal initiative to develop rapid mouse phenotyping methodologies. DP-grams at f(2) frequencies ranging from 6.3 to 54.2kHz were obtained in about 3min/ear by eliciting 2f(1)-f(2) DPOAEs in 0.1-octave steps of f(2) with primary tones at L(1)=L(2) =55, 65, and 75dB SPL. CBA/CaJ mice exhibited average levels of approximately 26dB SPL and this strain was selected as the normal reference strain against which the others were compared. Based upon the configurations of their DP-grams, the 28 mouse strains could be categorized into four distinct groups. That is, nine of the strains including the CBA were designated as the CBA-like group because these mice displayed robust DPOAE levels across frequency. In contrast, the remaining three groups all exhibited irregular DP-gram patterns. Specifically, eight of the remaining 19 strains showed a progressive high- to low-frequency reduction in DPOAE levels that was typical of age-related hearing loss (AHL) associated with mouse strains homozygous for the ahl allele and were labeled as AHL-like strains. Seven strains demonstrating relatively even patterns of reduced DPOAE levels across the frequency-test range were designated as Flat-loss strains. Finally, the remaining four strains exhibited no measurable DPOAEs at either 3 or 5 mo of age and thus were classified as Absent strains. Extending the f(2) test frequencies up to approximately 54kHz led to the detection of very early-onset reductions in cochlear function in non-CBA-like groups so that all strains could be categorized by 3 mo of age. Predictably, the AHL-like strains showed more pronounced DPOAE losses at 5 mo than at 3 mo. A similar deterioration in DPOAE levels was not apparent for the Flat-loss strains. Both the AHL-like and Flat-loss strains showed considerably more variability in DPOAE levels than did the CBA-like strains. Together, these findings indicate that DP-grams adequately reveal both frequency-specific loss patterns and details of inbred strain variability.

Aspirin prevents Escherichia coli lipopolysaccharide- and Staphylococcus aureus-induced downregulation of endothelial nitric oxide synthase expression in guinea pig pericardial tissue.

The aim was to analyze whether pericardial tissue expresses endothelial NO synthase (eNOS) protein and to determine the presence of cytosolic proteins that bind to eNOS mRNA. The effect of aspirin on the above-mentioned parameters was also analyzed. eNOS protein was expressed in pericardial tissue from male guinea pigs. Escherichia coli lipopolysaccharide (LPS, 10 microgram/mL) and Staphylococcus aureus endotoxin (SA, 10 microgram/mL) reduced eNOS protein expression and shortened the half-life of the eNOS messenger. Under basal conditions, cytosolic extracts from pericardial samples bound to the 3'-untranslated region (3'-UTR) of eNOS mRNA, which was enhanced by LPS and SA. Proteinase K fully prevented the binding of cytosolic pericardial extracts to 3'-UTR of eNOS mRNA, suggesting the involvement of proteins that were further characterized as 60- and 51-kDa proteins. Aspirin (1 to 10 mmol/L) restored eNOS expression in either LPS- and SA-stimulated pericardial samples and reduced the binding activity of the pericardial cytosolic proteins to 3'-UTR of eNOS mRNA. Indomethacin also reduced the downregulation of eNOS by LPS and diminished the binding activity of the cytosolic proteins, although higher doses of indomethacin than of aspirin were needed to improve these parameters. In conclusion, eNOS protein is expressed in guinea pig pericardial tissue. LPS and SA stimulate the binding activity of pericardial cytosolic proteins to 3'-UTR of eNOS mRNA and reduce eNOS protein expression. High doses of aspirin and indomethacin protect eNOS protein expression and reduce the binding activity of the cytosolic proteins to 3'-UTR of eNOS mRNA, suggesting an inverse association between the presence of these cytosolic proteins and eNOS expression.