Klotho: An Elephant in Aging Research.

The year 2017 marked the 20th anniversary of the first publication describing Klotho. This single protein was and is remarkable in that its absence in mice conferred an accelerated aging, or progeroid, phenotype with a dramatically shortened life span. On the other hand, genetic overexpression extended both health span and life span by an impressive 30%. Not only has Klotho deficiency been linked to a number of debilitating age-related illnesses but many subsequent reports have lent credence to the idea that Klotho can compress the period of morbidity and extend the life span of both model organisms and humans. This suggests that Klotho functions as an integrator of organ systems, making it both a promising tool for advancing our understanding of the biology of aging and an intriguing target for interventional studies. In this review, we highlight advances in our understanding of Klotho as well as key challenges that have somewhat limited our view, and thus translational potential, of this potent protein.

Clinical and economic impact of meropenem resistance in Pseudomonas aeruginosa-infected patients.

BACKGROUND: The emergence of carbapenem resistance has had a significant impact on both clinical and economic outcomes. METHODS: A retrospective, observational cohort study was performed in a 433-bed tertiary care medical center. The cohort was established from all inpatients with Pseudomonas aeruginosa-positive cultures over a 3-year period. Two multivariate models were developed: a logistic regression model to evaluate the primary outcome of in-hospital mortality and a linear regression model to evaluate the secondary outcome of total hospital cost. RESULTS: The adjusted odds ratio for in-hospital mortality among patients with meropenem-resistant isolates was 2.89 (95% confidence interval [CI], 1.15-7.28). There were significantly more deaths in the meropenem-resistant group (28.1% vs 8.9%, P = .003). Patients with meropenem-resistant P aeruginosa experienced a 4-day increase in median length of stay versus those in the meropenem-susceptible group (14 vs 9 days, P = .004). Likewise, the percentage of patients who required intensive care unit (ICU) admission increased from 42% to 81.3% (P <.001). Meropenem resistance was also associated with a significant increase in total hospital cost by a factor of 1.42 among patients who were not admitted to the ICU (95% CI, 1.03-1.95). CONCLUSIONS: Our results demonstrate that meropenem resistance was a significant predictor of in-hospital mortality. Carbapenem resistance also resulted in a significant increase in hospital cost, but only among patients who were not admitted to the ICU.

A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay.

Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened ∼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.

Clinical and economic impact of a quality improvement initiative to enhance early recognition and treatment of sepsis.

BACKGROUND: Studies evaluating the clinical effectiveness of sepsis screening tools and methods to improve the time from diagnosis to antibiotic administration are needed to improve sepsis-related outcomes. OBJECTIVE: To evaluate the clinical and economic impact of a sepsis quality improvement initiative to improve early recognition and treatment of sepsis. METHODS: A retrospective observational study of adults with sepsis was performed in a 433-bed tertiary medical center. Baseline data were collected for 181 patients with sepsis diagnosis-related group (DRG) coding assignments from July through September 2013. The intervnetion group included 216 patients from October through December 2013. A First-Dose STAT Antibiotic policy was developed, and nurses were instructed to complete an electronic sepsis screening tool once per shift. Primary outcomes included in-hospital mortality and intensive care unit (ICU) length of stay (LOS). Secondary outcomes included overall LOS and cost per case. RESULTS: Nonsignificant decreases in overall LOS (7.43 ± 5.68 days vs 6.77 ± 5 days; P = 0.138) and in-hospital mortality (13.8% vs 8.8%; P = 0.113) were observed in patients with sepsis DRGs. Early recognition and treatment contributed to significant reductions in ICU LOS (5.85 ± 4.38 days vs 4.21 ± 3.64 days; P = 0.003) and total cost per case ($14 378 vs $12 311; P = 0.033). The percentage of highest disease-severity DRG coding assignments decreased from 7.9% to 0%. CONCLUSIONS: Strategies to improve early recognition and treatment of sepsis, including routine use of an electronic sepsis screening tool and implementation of a First-Dose STAT Antibiotic policy, contributed to significant reductions in ICU LOS and cost per case.

Identification of a potent sodium hydrogen exchanger isoform 1 (NHE1) inhibitor with a suitable profile for chronic dosing and demonstrated cardioprotective effects in a preclinical model of myocardial infarction in the rat.

Sodium-hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed transmembrane ion channel responsible for intracellular pH regulation. During myocardial ischemia, low pH activates NHE1 and causes increased intracellular calcium levels and aberrant cellular processes, leading to myocardial stunning, arrhythmias, and ultimately cell damage and death. The role of NHE1 in cardiac injury has prompted interest in the development of NHE1 inhibitors for the treatment of heart failure. This report outlines our efforts to identify a compound suitable for once daily, oral administration with low drug-drug interaction potential starting from NHE1 inhibitor sabiporide. Substitution of a piperidine for the piperazine of sabiporide followed by replacement of the pyrrole moiety and subsequent optimization to improve potency and eliminate off-target activities resulted in the identification of N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine (60). Pharmacological evaluation of 60 revealed a remarkable ability to prevent ischemic damage in an ex vivo model of ischemia reperfusion injury in isolated rat hearts.

Identification of pharmacological inhibitors of the MEK5/ERK5 pathway.

We have identified two novel MEK5 inhibitors, BIX02188 and BIX02189, which inhibited catalytic function of purified, MEK5 enzyme. The MEK5 inhibitors blocked phosphorylation of ERK5, without affecting phosphorylation of ERK1/2 in sorbitol-stimulated HeLa cells. The compounds also inhibited transcriptional activation of MEF2C, a downstream substrate of the MEK5/ERK5 signaling cascade, in a cellular trans-reporter assay system. These inhibitors offer novel pharmacological tools to better characterize the role of the MEK5/ERK5 pathway in various biological systems.

Glucose-regulated insulin production from genetically engineered human non-beta cells.

In this report we describe development and characterization of four human cell lines that are able to secrete insulin and C-peptide in response to higher concentrations of glucose. These cell lines have been developed by stably and constitutively expressing human proinsulin with a furin-cleavable site, whereas expression of furin is regulated by glucose concentration. These cell lines have been cloned and, therefore, the transgene in each cell is located in an identical location of the genome leading to a uniform expression. Cloning has also allowed us to identify cell lines with more desirable properties such as higher basal insulin secretion and/or better glucose responsiveness. We have further shown that the insulin produced by these cells is biologically active and induces normoglycemia when injected in diabetic animals. Our objective in initiating these studies was to identify a cell line that could serve as a surrogate beta cell line for therapeutic intervention in type I diabetic patients.