Étude transcriptionnelle de l'actinophage JHJ-3

Dans le but d'établir un modèle de régulation génétique chez les actinomycètes, le bactériophage JHJ-3 a été choisi. L'actinophage tempéré JHJ-3 a été isolé suite à une induction à la mytomycine C de la souche Saccharopolyspora hirsuta 367 UC8106. Ce bactériophage a la même morphologie que le bactériophage $\lambda$ et possède un répresseur puisqu'il peut lysogéniser certaines souches de Saccharopolyspora. Ce répresseur a été localisé, caractérisé et nommé JrpI. De plus, un second gène transcrit de façon divergente a été localisé et nommé ORF II. Entre ces deux gènes, c'est-à-dire dans la région intergénique, 2 promoteurs ont été détectés: pr1 le promoteur du gène jrpI et pr2 le promoteur de l'ORF II. Ainsi, la région intergénique a été étudiée pour déterminer les nucléotides représentant ces deux promoteurs, et l'interaction entre JrpI et la région intergénique a été mise en évidence. Dans un premier temps, la région intergénique a subi des délétions par PCR et par exonuclease III - nuclease S1, pour donner plusieurs fragments de différentes longueurs de cette région. Ceci a permis de réduire les deux promoteurs d'environ 75%. En utilisant le plasmide rapporteur xylE-pOK12, la séquence représentant pr2 a été localisée dans un fragment d'ADN de 57 pb, tandis que pr1 n'a pu être localisé à cause d'un artéfact causé par le plasmide rapporteur. Ensuite, l'interaction entre JrpI et la région intergénique a été mise en évidence en établissant les conditions de liaison in vitro permettant de faire des gels de retardement. Ainsi, JrpI régulerait l'expression de l'ORF II et de son propre gène. [Résumé abrégé par UMI]

Étude transcriptionnelle de l'actinophage JHJ-3

Dans le but d'établir un modèle de régulation génétique chez les actinomycètes, le bactériophage JHJ-3 a été choisi. L'actinophage tempéré JHJ-3 a été isolé suite à une induction à la mytomycine C de la souche Saccharopolyspora hirsuta 367 UC8106. Ce bactériophage a la même morphologie que le bactériophage $\lambda$ et possède un répresseur puisqu'il peut lysogéniser certaines souches de Saccharopolyspora. Ce répresseur a été localisé, caractérisé et nommé JrpI. De plus, un second gène transcrit de façon divergente a été localisé et nommé ORF II. Entre ces deux gènes, c'est-à-dire dans la région intergénique, 2 promoteurs ont été détectés: pr1 le promoteur du gène jrpI et pr2 le promoteur de l'ORF II. Ainsi, la région intergénique a été étudiée pour déterminer les nucléotides représentant ces deux promoteurs, et l'interaction entre JrpI et la région intergénique a été mise en évidence. Dans un premier temps, la région intergénique a subi des délétions par PCR et par exonuclease III - nuclease S1, pour donner plusieurs fragments de différentes longueurs de cette région. Ceci a permis de réduire les deux promoteurs d'environ 75%. En utilisant le plasmide rapporteur xylE-pOK12, la séquence représentant pr2 a été localisée dans un fragment d'ADN de 57 pb, tandis que pr1 n'a pu être localisé à cause d'un artéfact causé par le plasmide rapporteur. Ensuite, l'interaction entre JrpI et la région intergénique a été mise en évidence en établissant les conditions de liaison in vitro permettant de faire des gels de retardement. Ainsi, JrpI régulerait l'expression de l'ORF II et de son propre gène. [Résumé abrégé par UMI]

In vitro activity of exebacase against methicillin-resistant Staphylococcus aureus biofilms on orthopedic Kirschner wires

Abstract Orthopedic foreign body-associated infection can be difficult to treat due to the formation of biofilms protecting microorganisms from both antimicrobials and the immune system. Exebacase is an antistaphylococcal lysin (cell wall hydrolase) under consideration for local treatment for biofilm-based infections caused by methicillin-resistant Staphylococcus aureus (MRSA). To determine the activity of exebacase, we formed MRSA biofilms on orthopedic Kirschner wires and exposed them to varying concentrations (0.098, 0.98, 9.8 mg/ml) of exebacase and/or daptomycin over 24 h. The biofilm consisted of 5.49 log10 colony forming units (cfu)/K-wire prior to treatment and remained steady throughout the experiment. Exebacase showed significant biofilm reduction at all timepoints (up to 5.78 log10 cfu/K-wire; P  3 log10 cfu/K-wire reduction) observed for up to 12 h for the 0.098 and 0.98 mg/ml concentrations and at 24 h for 9.8 mg/ml. Daptomycin showed significant biofilm reduction, although non-bactericidal, at all time points for 0.98 and 9.8 mg/ml and at 4 and 8 h with 0.098 mg/ml (P < 0.0495). This study supports further evaluation of local administration of exebacase as a potential treatment for orthopedic implant infections

Toxicologic and Inhibitory Receptor Actions of the Etomidate Analog ABP-700 and Its Metabolite CPM-Acid

Editor's PerspectiveWhat We Already Know about This Topic The investigational etomidate analog ABP-700 causes involuntary muscle movements in humans at anesthetic doses and seizures in dogs at 10-fold higher toxicologic doses The mechanism of seizures in dogs, and their relationship to involuntary muscle movements in humans, is unknown What This Article Tells Us That Is New Toxicologic studies in dogs using supratherapeutic ABP-700 doses caused involuntary muscle movements and seizures, but these were temporally and electroencephalographically distinct, suggesting different underlying mechanisms Events occurred at ABP-700 and metabolite concentrations one and two orders of magnitude higher, respectively, than those found in humans Electrophysiologic studies of the principal metabolite of ABP-700 in oocyte-expressed gamma-aminobutyric acid type A receptors showed inhibition at the high supratherapeutic concentrations achieved in the dogs, and such inhibition may explain seizure activity Proepileptiform effects of ABP-700 in dogs may not be relevant to humans at therapeutic doses Background: The etomidate analog ABP-700 produces involuntary muscle movements that could be manifestations of seizures. To define the relationship (if any) between involuntary muscle movements and seizures, electroencephalographic studies were performed in Beagle dogs receiving supra-therapeutic (similar to 10x clinical) ABP-700 doses. gamma-aminobutyric acid type A (GABA(A)) and glycine receptor studies were undertaken to test receptor inhibition as the potential mechanism for ABP-700 seizures. Methods: ABP-700 was administered to 14 dogs (6 mg/kg bolus followed by a 2-h infusion at 1 mg center dot kg(-1) center dot min(-1), 1.5 mg center dot kg(-1) center dot min(-1), or 2.3 mg center dot kg(-1) center dot min(-1)). Involuntary muscle movements were documented, electroencephalograph was recorded, and plasma ABP-700 and CPM-acid concentrations were measured during and after ABP-700 administration. The concentration-dependent modulatory actions of ABP-700 and CPM-acid were defined in oocyte-expressed alpha(1)beta(3)gamma(2L) GABA(A) and alpha(1)beta glycine receptors (n = 5 oocytes/concentration) using electrophysiologic techniques. Results: ABP-700 produced both involuntary muscle movements (14 of 14 dogs) and seizures (5 of 14 dogs). However, these phenomena were temporally and electroencephalographically distinct. Mean peak plasma concentrations were (from lowest to highest dosed groups) 35 mu M, 45 mu M, and 102 mu M (ABP-700) and 282 mu M, 478 mu M, and 1,110 mu M (CPM-acid). ABP-700 and CPM-acid concentration-GABA(A) receptor response curves defined using 6 mu M gamma-aminobutyric acid exhibited potentiation at low and/or intermediate concentrations and inhibition at high ones. The half-maximal inhibitory concentrations of ABP-700 and CPM-acid defined using 1 mM gamma-aminobutyric acid were 770 mu M (95% CI, 590 to 1,010 mu M) and 1,450 mu M (95% CI, 1,340 to 1,560 mu M), respectively. CPM-acid similarly inhibited glycine receptors activated by 1 mM glycine with a half-maximal inhibitory concentration of 1,290 mu M (95% CI, 1,240 to 1,330 mu M). Conclusions: High dose ABP-700 infusions produce involuntary muscle movements and seizures in Beagle dogs via distinct mechanisms. CPM-acid inhibits both GABA(A) and glycine receptors at the high (similar to 100x clinical) plasma concentrations achieved during the dog studies, providing a plausible mechanism for the seizures

Effect of the Lysin Exebacase on Cardiac Vegetation Progression in a Rabbit Model of Methicillin-Resistant Staphylococcus aureus Endocarditis as Determined by Echocardiography.

Methicillin-resistant Staphylococcus aureus (MRSA) poses significant therapeutic challenges related to its frequency in clinical infections, innate virulence properties, and propensity for multiantibiotic resistance. MRSA is among the most common causes of endovascular infections, including infective endocarditis (IE). Our objective was to employ transthoracic echocardiography (TTE) to evaluate the effect of exebacase, a novel direct lytic agent, in experimental aortic valve MRSA IE. TTE was utilized to evaluate the in vivo effect of exebacase on MRSA-infected vegetation progression when combined with daptomycin (versus daptomycin alone). Primary intravegetation outcomes were maximum size, weights at sacrifice, and MRSA counts at infection baseline versus after 4 days of daptomycin treatment (alone or in addition to exebacase administered once on treatment day 1). A single dose of exebacase in addition to daptomycin cleared significantly more intravegetation MRSA than daptomycin alone. This was associated with a statistical trend toward reduced maximum vegetation size in the exebacase plus daptomycin versus the daptomycin alone therapy groups (P = 0.07). Also, mean vegetation weights in the exebacase-treated group were significantly lower than those of the daptomycin alone group (P &lt; 0.0001). Maximum vegetation size by TTE correlated with vegetation weight (P = 0.005). In addition, intravegetation MRSA counts in the combination group were significantly lower than those of untreated controls (P &lt; 0.0001) and the daptomycin alone group (P &lt; 0.0001). This study suggests that exebacase has a salutary impact on MRSA-infected vegetation progression when combined with daptomycin, especially in terms of vegetation MRSA burden, size, and weight. Moreover, TTE appears to be an efficient noninvasive tool to assess therapeutic efficacies in experimental MRSA IE

Mechanisms underlying the cardiometabolic protective effect of walnut consumption in obese people: A cross-over, randomized, double-blind, controlled inpatient physiology study

Aims: To assess the effects of walnuts on cardiometabolic outcomes in obese people and to explore the underlying mechanisms using novel methods including metabolomic, lipidomic, glycomic and microbiome analysis, integrated with lipid particle fractionation, appetite-regulating hormones and haemodynamic measurements. Materials and Methods: A total of 10 obese individuals were enrolled in this cross-over, randomized, double-blind, placebo-controlled clinical trial. The participants had two 5-day inpatient stays, during which they consumed a smoothie containing 48 g walnuts or a macronutrient-matched placebo smoothie without nuts, with a 1-month washout period between the two visits. Results: Walnut consumption improved aspects of the lipid profile; it reduced fasting small and dense LDL particles (P < 0.02) and increased postprandial large HDL particles (P < 0.01). Lipoprotein insulin resistance score, glucose and the insulin area under the curve (AUC) decreased significantly after walnut consumption (P < 0.01, P < 0.02 and P < 0.04, respectively). Consuming walnuts significantly increased 10 N-glycans, with eight of them carrying a fucose core. Lipidomic analysis showed a robust reduction in harmful ceramides, hexosylceramides and sphingomyelins, which have been shown to mediate effects on cardiometabolic risk. The peptide YY AUC significantly increased after walnut consumption (P < 0.03). No major significant changes in haemodynamic or metabolomic analysis or in microbiome host health-promoting bacteria such as Faecalibacterium were found. Conclusions: These data provide a more comprehensive mechanistic perspective of the effect of dietary walnut consumption on cardiometabolic variables. Lipidomic and lipid nuclear magnetic resonance spectroscopy analysis showed an early but significant reduction in ceramides and other atherogenic lipids with walnut consumption, which may explain the longer-term benefits of walnuts or other nuts on insulin resistance, cardiovascular risk and mortality