Chemical Synthesis of Staphyloferrin B Affords Insight into the Molecular Structure, Iron Chelation, and Biological Activity of a Polycarboxylate Siderophore Deployed by the Human Pathogen

Staphyloferrin B (SB) is a citrate-based polycarboxylate siderophore produced and utilized by the human pathogen Staphylococcus aureus for acquiring iron when colonizing the vertebrate host. The first chemical synthesis of SB is reported, which enables further molecular and biological characterization and provides access to structural analogues of the siderophore. Under conditions of iron limitation, addition of synthetic SB to bacterial growth medium recovered the growth of the antibiotic resistant community isolate S. aureus USA300 JE2. Two structural analogues of SB, epiSB and SBimide, were also synthesized and employed to investigate how epimerization of the citric acid moiety or imide formation influence its function as a siderophore. Epimerization of the citric acid stereocenter perturbed the iron-binding properties and siderophore function of SB as evidenced by experimental and computational modeling studies. Although epiSB provided growth recovery to S. aureus USA300 JE2 cultured in iron-deficient medium, the effect was attenuated relative to that of SB. Moreover, SB more effectively sequestered the Fe(III) bound to human holo-transferrin, an iron source of S. aureus, than epiSB. SBimide is an imide analogous to the imide forms of other citric acid siderophores that are often observed when these molecules are isolated from natural sources. Here, SBimide is shown to be unstable, converting to native SB at physiological pH. SB is considered to be a virulence factor of S. aureus, a pathogen that poses a particular threat to public health because of the number of drug-resistant strains emerging in hospital and community settings. Iron acquisition by S. aureus is important for its ability to colonize the human host and cause disease, and new chemical insights into the structure and function of SB will inform the search for new therapeutic strategies for combating S. aureus infections.Alfred Benzon Foundation (Postdoctoral fellowship)Pacific Southwest Regional Center of ExcellenceAlfred P. Sloan Foundatio

The impact of shock wave therapy at varied energy and dose levels on functional and structural changes in erectile tissue

OBJECTIVES: Only minimal literature exists on consequences of shock wave therapy (SWT) on erectile function in treatment of Peyronie's disease (PD). This study was undertaken to define SWT impact at varied energy/dose levels at different time points on functional and structural changes in erectile tissue. METHODS: In 45 rats 2000 shock waves (sw) at 2 BAR were applied to the penis weekly sorted by one, two, and three sessions (high-dose/energy level, HD-1, HD-2, HD-3). Each group was followed for 1, 7, or 28 d before measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP). Fifteen control animals (C1, C7, C28) underwent anesthesia alone. Another 15 animals were exposed to three SWT sessions applying 1000 sw at 1 BAR and analyzed identically (low-dose/energy level, LD-3-1, -7, -28). Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling assay was used to define the apoptotic index (AI) and Masson's trichrome (MT) staining was prepared to evaluate smooth muscle-to-collagen ratios. RESULTS: ICP/MAP ratios for all C groups displayed a mean of 64%. All SWT groups demonstrated significantly reduced ICP/MAP ratios compared to their corresponding C groups (p0.05). AIs for the LD-3 groups were significantly lower compared to the HD-3 groups (p<0.001), but all AIs were significantly increased compared to C groups (p<0.01). CONCLUSIONS: Overall, at both energy/dose levels, SWT resulted in a time- and treatment-dependent reduction of ICP/MAP ratios, which might be mediated partly through apoptosis and collagenization of corporal smooth muscle

The functional and structural consequences of cavernous nerve injury are ameliorated by sildenafil citrate

INTRODUCTION: Radical prostatectomy (RP) is associated with erectile dysfunction (ED). A single, placebo-controlled, human study has assessed the effects of regular sildenafil use after RP and demonstrated an increased chance of preservation of preoperative erectile function. Aim. This study was undertaken to define the effects of such a regimen in an animal model. METHODS: Using the cavernous nerve (CN) crush injury model, animals were divided into a number of groups: no CN injury (sham), bilateral CN injury exposed to either no sildenafil (control) or sildenafil at two doses (10 and 20 mg/kg) subcutaneously daily for three different durations (3, 10, 28 days). MAIN OUTCOME MEASURES: At these time points, CN electrical stimulation was used to assess erectile function by mean intracavernosal pressure (ICP)/mean arterial pressure (MAP) ratio. For the structural analyses, whole rat penes were harvested. Staining for Masson's trichrome was utilized to calculate the smooth muscle-collagen ratio. Immunohistochemical antibody staining was performed for endothelial (CD31 and eNOS) and neural (GAP43, NGF, and nNOS) factors and immunoblotting was performed to analyze the AKT/eNOS pathway. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay was used for the assessment of apoptotic indices and the CN architecture was evaluated by transmission electron microscopy (TEM). RESULTS: Erectile function was improved with sildenafil in a time- and dose-dependent fashion with maximization of erectile function recovery occurring with daily 20 mg/kg at the 28-day time point. Sildenafil use resulted in smooth muscle-collagen ratio protection and CD31 and eNOS expression preservation. Sildenafil reduced apoptotic indices significantly compared with control. Animals exposed to sildenafil had increased phosphorylation of akt and eNOS. Tem demonstrated distinct differences in architecture between control and sildenafil groups toward an increased amount of myelinized nerve fibers. CONCLUSIONS: Sildenafil use in the CN crush injury model preserves erectile function that appears to be mediated predominantly through preservation of smooth muscle content and endothelial function as well as through reduction in apoptosis

Autophagy-Independent Lysosomal Targeting Regulated by ULK1/2-FIP200 and ATG9

Iron is vital for many homeostatic processes, and its liberation from ferritin nanocages occurs in the lysosome. Studies indicate that ferritin and its binding partner nuclear receptor coactivator-4 (NCOA4) are targeted to lysosomes by a form of selective autophagy. By using genome-scale functional screening, we identify an alternative lysosomal transport pathway for ferritin that requires FIP200, ATG9A, VPS34, and TAX1BP1 but lacks involvement of the ATG8 lipidation machinery that constitutes classical macroautophagy. TAX1BP1 binds directly to NCOA4 and is required for lysosomal trafficking of ferritin under basal and iron-depleted conditions. Under basal conditions ULK1/2-FIP200 controls ferritin turnover, but its deletion leads to TAX1BP1-dependent activation of TBK1 that regulates redistribution of ATG9A to the Golgi enabling continued trafficking of ferritin. Cells expressing an amyotrophic lateral sclerosis (ALS)-associated TBK1 allele are incapable of degrading ferritin suggesting a molecular mechanism that explains the presence of iron deposits in patient brain biopsies

Possible axonal regrowth in late recovery from the minimally conscious state

We used diffusion tensor imaging (DTI) to study 2 patients with traumatic brain injury. The first patient recovered reliable expressive language after 19 years in a minimally conscious state (MCS); the second had remained in MCS for 6 years. Comparison of white matter integrity in the patients and 20 normal subjects using histograms of apparent diffusion constants and diffusion anisotropy identified widespread altered diffusivity and decreased anisotropy in the damaged white matter. These findings remained unchanged over an 18-month interval between 2 studies in the first patient. In addition, in this patient, we identified large, bilateral regions of posterior white matter with significantly increased anisotropy that reduced over 18 months. In contrast, notable increases in anisotropy within the midline cerebellar white matter in the second study correlated with marked clinical improvements in motor functions. This finding was further correlated with an increase in resting metabolism measured by PET in this subregion. Aberrant white matter structures were evident in the second patient’s DTI images but were not clinically correlated. We propose that axonal regrowth may underlie these findings and provide a biological mechanism for late recovery. Our results are discussed in the context of recent experimental studies that support this inference