Heteroaryl urea inhibitors of fatty acid amide hydrolase: Structure–mutagenicity relationships for arylamine metabolites

The structure–activity relationships for a series of heteroaryl urea inhibitors of fatty acid amide hydrolase (FAAH) are described. Members of this class of inhibitors have been shown to inactivate FAAH by covalent modification of an active site serine with subsequent release of an aromatic amine from the urea electrophile. Systematic Ames II testing guided the optimization of urea substituents by defining the structure–mutagenicity relationships for the released aromatic amine metabolites. Potent FAAH inhibitors were identified having heteroaryl amine leaving groups that were non-mutagenic in the Ames II assay.     

Defined media optimization for growth of recombinant Escherichia coli X90

An optimized, defined minimal medium was developed to support balanced growth of Escherichia coli X90 harboring a recombinant plasmid. Foreign protein expression was repressed in these studies. A pulse injection technique was used to identify the growth responses to nutrients in a chemostat. Once the nutrients essential for growth had been identified, the yield coefficients for individual medium components. These yield coefficients were used to develop an optimized, glucose-limited defined minimal medium that supports balanced cell growth in chemostat culture. The biomass and substrate concentrations follow the Monod chemostat model. The maximum specific growth rate determined in a washout experiment is 0.87 h(-1) for this strain in the optimized medium. the glucose yield factor is 0.42 g DCW/g glucose and the maintenance coefficient is zero in the glucose-limited chemostat culture. (c) 1993 John Wiley & Sons, Inc.     

Role of PCIF1‐mediated 5′‐cap N6‐methyladeonsine mRNA methylation in colorectal cancer and anti‐PD‐1 immunotherapy

Adenosine N6‐methylation (m6A) and N6,2′‐O‐dimethylation (m6Am) are regulatory modifications of eukaryotic mRNAs. m6Am formation is catalyzed by the methyl transferase phosphorylated CTD‐interacting factor 1 (PCIF1); however, the pathophysiological functions of this RNA modification and PCIF1 in cancers are unclear. Here, we show that PCIF1 expression is upregulated in colorectal cancer (CRC) and negatively correlates with patient survival. CRISPR/Cas9‐mediated depletion of PCIF1 in human CRC cells leads to loss of cell migration, invasion, and colony formation in vitro and loss of tumor growth in athymic mice. Pcif1 knockout in murine CRC cells inhibits tumor growth in immunocompetent mice and enhances the effects of anti‐PD‐1 antibody treatment by decreasing intratumoral TGF‐β levels and increasing intratumoral IFN‐γ, TNF‐α levels, and tumor‐infiltrating natural killer cells. We further show that PCIF1 modulates CRC growth and response to anti‐PD‐1 in a context‐dependent mechanism with PCIF1 directly targeting FOS, IFITM3, and STAT1 via m6Am modifications. PCIF1 stabilizes FOS mRNA, which in turn leads to FOS‐dependent TGF‐β regulation and tumor growth. While during immunotherapy, Pcif1‐Fos‐TGF‐β, as well as Pcif1‐Stat1/Ifitm3‐IFN‐γ axes, contributes to the resistance of anti‐PD‐1 therapy. Collectively, our findings reveal a role of PCIF1 in promoting CRC tumorigenesis and resistance to anti‐PD‐1 therapy, supporting that the combination of PCIF1 inhibition with anti‐PD‐1 treatment is a potential therapeutic strategy to enhance CRC response to immunotherapy. Finally, we developed a lipid nanoparticles (LNPs) and chemically modified small interfering RNAs (CMsiRNAs)‐based strategy to silence PCIF1 in vivo and found that this treatment significantly reduced tumor growth in mice. Our results therefore provide a proof‐of‐concept for tumor growth suppression using LNP‐CMsiRNA to silence target genes in cancer.     

Empiric Antibiotic Therapy for Staphylococcus aureus Bacteremia May Not Reduce In-Hospital Mortality: A Retrospective Cohort Study

Background

Appropriate empiric therapy, antibiotic therapy with in vitro activity to the infecting organism given prior to confirmed culture results, may improve Staphylococcus aureus outcomes. We aimed to measure the clinical impact of appropriate empiric antibiotic therapy on mortality, while statistically adjusting for comorbidities, severity of illness and presence of virulence factors in the infecting strain.

Methodology

We conducted a retrospective cohort study of adult patients admitted to a tertiary-care facility from January 1, 2003 to June 30, 2007, who had S. aureus bacteremia. Time to appropriate therapy was measured from blood culture collection to the receipt of antibiotics with in vitro activity to the infecting organism. Cox proportional hazard models were used to measure the association between receipt of appropriate empiric therapy and in-hospital mortality, statistically adjusting for patient and pathogen characteristics.

Principal Findings

Among 814 admissions, 537 (66%) received appropriate empiric therapy. Those who received appropriate empiric therapy had a higher hazard of 30-day in-hospital mortality (Hazard Ratio (HR): 1.52; 95% confidence interval (CI): 0.99, 2.34). A longer time to appropriate therapy was protective against mortality (HR: 0.79; 95% CI: 0.60, 1.03) except among the healthiest quartile of patients (HR: 1.44; 95% CI: 0.66, 3.15).

Conclusions/Significance

Appropriate empiric therapy was not associated with decreased mortality in patients with S. aureus bacteremia except in the least ill patients. Initial broad antibiotic selection may not be widely beneficial.     

Therapeutic antimicrobial peptides may compromise natural immunity

Antimicrobial peptides (AMPs) have been proposed as a promising new class of antimicrobials despite warnings that therapeutic use could drive the evolution of pathogens resistant to our own immunity peptides. Using experimental evolution, we demonstrate that Staphylococcus aureus rapidly evolved resistance to pexiganan, a drug-candidate for diabetic leg ulcer infections. Evolved resistance was costly in terms of impaired growth rate, but costs-of-resistance were completely ameliorated by compensatory adaptation. Crucially, we show that, in some populations, experimentally evolved resistance to pexiganan provided S. aureus with cross-resistance to human-neutrophil-defensin-1, a key component of the innate immune response to infection. This unintended consequence of therapeutic use could drastically undermine our innate immune system's ability to control and clear microbial infections. Our results therefore highlight grave potential risks of AMP therapies, with implications for their development.     

The Role of the N-Terminus of the Myosin Essential Light Chain in Cardiac Muscle Contraction

To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice by partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43-amino-acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle, and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force-generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of nonpathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.     

Adult Cardiac Progenitor Cell Aggregates Exhibit Survival Benefit Both In Vitro and In Vivo

Background

A major hurdle in the use of exogenous stems cells for therapeutic regeneration of injured myocardium remains the poor survival of implanted cells. To date, the delivery of stem cells into myocardium has largely focused on implantation of cell suspensions.

Methodology and Principal Findings

We hypothesize that delivering progenitor cells in an aggregate form would serve to mimic the endogenous state with proper cell-cell contact, and may aid the survival of implanted cells. Microwell methodologies allow for the culture of homogenous 3D cell aggregates, thereby allowing cell-cell contact. In this study, we find that the culture of cardiac progenitor cells in a 3D cell aggregate augments cell survival and protects against cellular toxins and stressors, including hydrogen peroxide and anoxia/reoxygenation induced cell death. Moreover, using a murine model of cardiac ischemia-reperfusion injury, we find that delivery of cardiac progenitor cells in the form of 3D aggregates improved in vivo survival of implanted cells.

Conclusion

Collectively, our data support the notion that growth in 3D cellular systems and maintenance of cell-cell contact improves exogenous cell survival following delivery into myocardium. These approaches may serve as a strategy to improve cardiovascular cell-based therapies.     

Viral and cellular kinases are potential antiviral targets and have a central role in varicella zoster virus pathogenesis

Herpesviruses utilize viral and cellular kinases for replication, and these mediate essential functions that are important for viral pathogenesis. Elucidating the roles of kinases in herpesvirus infections may highlight virus-host interactions that are possible targets for kinase inhibitors with antiviral activity. Varicella zoster virus (VZV) encodes two kinases that phosphorylate viral proteins involved in regulation, assembly, and virulence. VZV infection also induces the activity of host cell cyclin-dependent kinases (cdk4 and cdk2) in nondividing cells, causing a disregulation of the cell cycle. Roscovitine and Purvalanol, kinase inhibitors that target cdks, prevent VZV replication at concentrations with few cytotoxic effects. Cdk inhibitors therefore have potential as antivirals that may extend to a broad range of viruses and have the added advantage that resistance does not arise easily.