Enhanced Growth of Tetracycline-susceptible Soil Bacteria Treated with Iron and Oxytetracycline

Antibiotic resistance has become an important area of research because of the excessive use of antibiotics in clinical and agricultural settings that are driving the evolution of antibiotic resistant bacteria. However, drug tolerance is a naturally occurring phenomenon in soil communities, and is often linked to those soils that are exposed to heavy metals as well as antibiotics. Resistance to antibiotics maybe coupled with resistance to heavy metals in soil bacteria through efflux pumps that can be regulated by iron. Although considered s a heavy metal, iron is an essential component of life that regulates gene expression through the Ferric Uptake Regulator (Fur) protein. This master regulator protein is known to control siderophore production, and other biological pathways. As a suspected controller of biofilm formation, the role of Fur in environmental antibiotic resistance may be greater than is currently realized. In this study, we sought to explore a potential Fur-regulated drug tolerance pathway by understanding the response of soil bacteria when stressed with oxytetracycline and iron. Bacteria were collected from two locations in Miami Dade County. Isolates were first tested using Kirby-Bauer Disk Diffusion tests for antibiotic resistance/susceptibility and identified by 16S rDNA sequencing. A 96-well growth assay was developed to measure planktonic cell growth with 3 mM FeCl3, Oxytetracycline HCl, and the combination treatments. A Microtiter Dish Biofilm Formation Assay was employed and Fur diversity was evaluated. Tetracycline-susceptible bacterial isolates developed drug resistance with iron supplementation, but iron did not enhance biofilm formation. Development of a Fur-dependent drug resistance may be selected for, but further study is required to evaluate Fur evolution in the studied isolates. Gene expression analysis is also needed to further understand the ecological role of Fur and antibiotic resistance

Harnessing the Benefits of Endogenous Hydrogen Sulfide to Reduce Cardiovascular Disease

Cardiovascular disease is the leading cause of death in the U.S. While various studies have shown the beneficial impact of exogenous hydrogen sulfide (H2S)-releasing drugs, few have demonstrated the influence of endogenous H2S production. Modulating the predominant enzymatic sources of H2S—cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase—is an emerging and promising research area. This review frames the discussion of harnessing endogenous H2S within the context of a non-ischemic form of cardiomyopathy, termed diabetic cardiomyopathy, and heart failure. Also, we examine the current literature around therapeutic interventions, such as intermittent fasting and exercise, that stimulate H2S production

Adenosine A1 receptor activation increases myocardial protein <i>S</i>-nitrosothiols and elicits protection from ischemia-reperfusion injury in male and female hearts

<div><p>Nitric oxide (NO) plays an important role in cardioprotection, and recent work from our group and others has implicated protein <i>S</i>-nitrosylation (SNO) as a critical component of NO-mediated protection in different models, including ischemic pre- and post-conditioning and sex-dependent cardioprotection. However, studies have yet to examine whether protein SNO levels are similarly increased with pharmacologic preconditioning in male and female hearts, and whether an increase in protein SNO levels, which is protective in male hearts, is sufficient to increase baseline protection in female hearts. Therefore, we pharmacologically preconditioned male and female hearts with the adenosine A1 receptor agonist N6-cyclohexyl adenosine (CHA). CHA administration prior to ischemia significantly improved functional recovery in both male and female hearts compared to baseline in a Langendorff-perfused heart model of ischemia-reperfusion injury (% of preischemic function ± SE: male baseline: 37.5±3.4% vs. male CHA: 55.3±3.2%; female baseline: 61.4±5.7% vs. female CHA: 76.0±6.2%). In a separate set of hearts, we found that CHA increased p-Akt and p-eNOS levels. We also used SNO-resin-assisted capture with LC-MS/MS to identify SNO proteins in male and female hearts, and determined that CHA perfusion induced a modest increase in protein SNO levels in both male (11.4%) and female (12.3%) hearts compared to baseline. These findings support a potential role for protein SNO in a model of pharmacologic preconditioning, and provide evidence to suggest that a modest increase in protein SNO levels is sufficient to protect both male and female hearts from ischemic injury. In addition, a number of the SNO proteins identified with CHA treatment were also observed with other forms of cardioprotective stimuli in prior studies, further supporting a role for protein SNO in cardioprotection.</p></div

Label-free SNO protein levels for various targets.

<p>For common SNO proteins that were identified in male and female hearts, SNO levels were assessed via label-free peptide quantification for (A) aconitase, (B) electron transfer flavoprotein β, (C) lactate dehydrogenase, (D) voltage-dependent anion channel 2, (E) voltage-dependent anion channel 3, (F) isocitrate dehydrogenase α, (G) enoyl-CoA hydratase, and (H) sarcoplasmic reticulum Ca²⁺-ATPase 2a (male control: clear bar, male CHA: clear hashed bar, female control: black bar, female CHA: black hashed bar). Please note that for groups that do not contain a bar, the SNO peptide indicated was not detected.</p

Common SNO protein identifications resulting from different cardioprotective interventions.

<p>Common SNO protein identifications resulting from different cardioprotective interventions.</p

CHA increases GSNOR activity in male and female hearts.

<p>GSNOR activity was measured in control and CHA-perfused male and female hearts (male control: clear bar, male CHA: clear hashed bar, female control: black bar, female CHA: black hashed bar; n = 3 hearts/group; *p<0.05 vs. male CHA, **p<0.05 vs. male control, male CHA, female control).</p

Perfusion protocol for CHA-induced cardioprotection.

<p>Hearts were Langendorff-perfused during a 20 minute equilibration period with or without CHA, and then subjected to a 20 minute period of ischemia and 30 minutes of reperfusion.</p

CHA-induced changes in protein SNO in male and female hearts.

<p>(A) Venn diagram depicting common and unique SNO protein identifications in male control hearts (black circle) compared to CHA-perfused male hearts (grey circle). (B) Venn diagram depicting common and unique SNO protein identifications in female control hearts (black circle) compared to CHA-perfused female hearts (grey circle).</p

Post-ischemic ROS production is reduced in female hearts.

<p>Hydrogen peroxide production was assessed using Amplex Red in post-ischemic heart tissue and with purified α-ketoglutarate dehydrogenase. (A) Hydrogen peroxide production over time (male: open circles, female: closed circles) and (B) the rate of hydrogen peroxide production in post-ischemic male and female hearts (male: clear bar, female: black bar; n = 5 hearts/group; *p<0.05 vs. male). (C) Hydrogen peroxide production over time (control: open circles, GSNO: closed circles) and (D) the rate of hydrogen peroxide production with purified α-ketoglutarate dehydrogenase treated with and without GSNO (control: clear bar, GSNO: black bar; n = 6 replicates/group; *p<0.05 vs. control).</p