The genes coding for the enzymes of oxidative degradation of nicotinic acid have recently been identified in several species of aerobic bacteria, namely, Pseudomonas putida KT2440, Bordetella bronchiseptica RB50, and Bacillus niacini. One of the enzymes involved in an early step of this pathway is a flavin-dependent monooxygenase (6-hydroxynicotinic acid 3-monooxygenase; NicC) that catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP), with concomitant oxidation of NADH to NAD+. The nicC genes from B. bronchiseptica RB50 and P. putida have been cloned, and the purified enzymes have been characterized functionally and structurally. Global fits of the steady-state kinetic data show that both enzymes are efficient catalysts, with an apparent k cat/K M 6‑HNA of 5.0 × 104 M–1 s–1 for B. bronchiseptica NicC. The pH dependence of V max/[E] t fits a double-bell model showing an optimum around pH 8 with apparent pK as of 7.24 ± 0.08 and 8.64 ± 0.08, whereas the apparent catalytic efficiency (k cat/K M 6‑HNA) is maximal around pH 7 and decreases at high pH with an apparent pK a of 7.60 ± 0.06. The enzyme’s relative affinity for 6-hydroxynicotinaldehyde, a neutral analogue that shows competitive inhibition with respect to 6-HNA, is weak (K i = 3000 ± 400 μM) in comparison to the apparent binding of 6-HNA (K M = 85 ± 13 μM). The crystal structure for P. putida NicC has been solved to 2.1 Å using SAD phasing, and the 6-HNA substrate has been modeled into the active site. Together these data provide insight into potential reaction mechanisms for this novel decarboxylative hydroxylation reaction.

Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (α-globin, β-globin, ALAS2) were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR) and transcription factors (GATA1, GATA2, FOG1) were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-γ mRNA is highly increased by L. donovani infection. Expression of the IFN-γ inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL), was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by cytokine-mediated apoptosis of erythroblasts.

No abstract

OBJECTIVES: Overutilization of laboratory services is now recognized as harmful to patients and wasteful. In fact, the American Board of Internal Medicine’s Choosing Wisely campaign recommends against ordering routine testing that does not answer a clinical question. Per peer benchmarking, our institution as a whole occupied an extreme outlier position at the 100th percentile for laboratory utilization. We sought to address this problem starting in our medical ICUs with a quality improvement project. DESIGN: Quality improvement project using the design, measure, analyze, improve, and control process. The primary endpoint was a sustained reduction in laboratory utilization. Counterbalance metrics were also followed, and these included mortality, renal replacement therapy initiation rates, stat laboratory orders, and central catheter–associated blood stream infections. SETTING: The medical ICU at the Ohio State University Medical Center. PATIENTS: All patients admitted to the medical ICU from March 2019 to March 2020. INTERVENTIONS: Root causes were identified and addressed with the implementation of a wide range of interventions involving a multidisciplinary team led by trainee physicians. MEASUREMENTS AND MAIN RESULTS: There was a sustained 20% reduction in the number of tests performed per patient day, with no change in the counterbalance metrics. CONCLUSIONS: Trainees can affect positive change in the culture and processes at their institutions to safely reduce laboratory utilization.

No abstract