Etiological agents of acute, persistent, or relapsing clinical infections are often refractory to antibiotics due to multidrug resistance and/or antibiotic tolerance. Pseudomonas aeruginosa is an opportunistic Gram-negative bacterial pathogen that causes recalcitrant and severe acute chronic and persistent human infections. Here, we target the MvfR-regulated P. aeruginosa quorum sensing (QS) virulence pathway to isolate robust molecules that specifically inhibit infection without affecting bacterial growth or viability to mitigate selective resistance. Using a whole-cell high-throughput screen (HTS) and structure-activity relationship (SAR) analysis, we identify compounds that block the synthesis of both pro-persistence and pro-acute MvfR-dependent signaling molecules. These compounds, which share a benzamide-benzimidazole backbone and are unrelated to previous MvfR-regulon inhibitors, bind the global virulence QS transcriptional regulator, MvfR (PqsR); inhibit the MvfR regulon in multi-drug resistant isolates; are active against P. aeruginosa acute and persistent murine infections; and do not perturb bacterial growth. In addition, they are the first compounds identified to reduce the formation of antibiotic-tolerant persister cells. As such, these molecules provide for the development of next-generation clinical therapeutics to more effectively treat refractory and deleterious bacterial-human infections. Antibiotic resistant and tolerant bacterial pathogens are responsible for acute, chronic and persistent human infections recalcitrant to any current treatments. Therefore, there is an urgent need to identify new antimicrobial drugs that will help circumvent the current antibiotic resistance crisis. Bacterial pathogens often develop resistance to antibiotic drugs that target bacterial growth or viability. In contrast, strategies that specifically target virulence pathways non-essential for growth could limit selective resistance, and thus are candidates for the development of next-generation antimicrobial therapeutics. In this study we target the bacterial communication system MvfR (PqsR), which is known to control virulence of the opportunistic bacterial pathogen Pseudomonas aeruginosa. We identified and improved upon new small molecules that effectively silence the MvfR communication system, and as a result block P. aeruginosa virulence both in vitro and in vivo. Moreover, these new compounds are the first known to restrict the ability of bacteria to form antibiotic-tolerant cells and consequently proved to be very effective at preventing persistent infection in a mammalian infection model. Because of their ability to simultaneously block acute and persistent infections, these new molecules may provide a very strong basis for the development of next generation antimicrobials.

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A pilot clinical trial based on nutritional modulation was designed to assess the efficacy of a one-year low-protein diet in activating autophagy in skeletal muscle of patients affected by COL6/collagen VI-related myopathies. Ullrich congenital muscular dystrophy and Bethlem myopathy are rare inherited muscle disorders caused by mutations of COL6 genes and for which no cure is yet available. Studies in col6 null mice revealed that myofiber degeneration involves autophagy defects and that forced activation of autophagy results in the amelioration of muscle pathology. Seven adult patients affected by COL6 myopathies underwent a controlled low-protein diet for 12 mo and we evaluated the presence of autophagosomes and the mRNA and protein levels for BECN1/Beclin 1 and MAP1LC3B/LC3B in muscle biopsies and blood leukocytes. Safety measures were assessed, including muscle strength, motor and respiratory function, and metabolic parameters. After one y of low-protein diet, autophagic markers were increased in skeletal muscle and blood leukocytes of patients. The treatment was safe as shown by preservation of lean:fat percentage of body composition, muscle strength and function. Moreover, the decreased incidence of myofiber apoptosis indicated benefits in muscle homeostasis, and the metabolic changes pointed at improved mitochondrial function. These data provide evidence that a low-protein diet is able to activate autophagy and is safe and tolerable in patients with COL6 myopathies, pointing at autophagy activation as a potential target for therapeutic applications. In addition, our findings indicate that blood leukocytes are a promising noninvasive tool for monitoring autophagy activation in patients.

The non-essential amino acid neurotransmitter glycine (Gly) may serve as a biomarker for brain tumors. Using 36 biopsies from patients with brain tumors [12 glioblastoma multiforme (GBM); 10 low-grade (LG), including 7 schwannoma and 3 pylocytic astrocytoma; 7 meningioma (MN); 7 brain metastases (MT), including 3 adenocarcinoma and 4 breast cancer] and 9 control biopsies from patients undergoing surgery for epilepsy, we tested the hypothesis that the presence of glycine may distinguish among these brain tumor types. Using high-resolution magic angle spinning (HRMAS) 1H magnetic resonance spectroscopy (MRS), we determined a theoretically optimum echo time (TE) of 50 ms for distinguishing Gly signals from overlapping myo-inositol (Myo) signals and tested our methodology in phantom and biopsy specimens. Quantitative analysis revealed higher levels of Gly in tumor biopsies (all combined) relative to controls; Gly levels were significantly elevated in LG, MT and GBM biopsies (P≤0.05). Residual Myo levels were elevated in LG and MT and reduced in MN and GBM (P<0.05 vs. control levels). We observed higher levels of Gly in GBM as compared to LG tumors (P=0.05). Meanwhile, although Gly levels in GBM and MT did not differ significantly from each other, the Gly:Myo ratio did distinguish GBM from MT (P<0.003) and from all other groups, a distinction that has not been adequately made previously. We conclude from these findings that Gly can serve as a biomarker for brain tumors and that the Gly:Myo ratio may be a useful index for brain tumor classification.