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The enolase superfamily (ENS) has served as a paradigm for understanding how enzymes that share a conserved structure, as well as a common partial reaction (i.e., metal-assisted, Brønsted base-catalyzed enol(ate) formation), evolved from a common progenitor to catalyze mechanistically diverse reactions. Enzymes of the mandelate racemase (MR)-subgroup of the ENS share interdigitating loops between adjacent, 2-fold symmetry-related protomers of the tightly associated homodimers that comprise their quaternary structures. For the MR-subgroup members MR and d-tartrate dehydratase (TarD), the tip of the loop contributes a binding determinant to the adjacent active site (i.e., Leu 93 and Lys 102, respectively). To assess the role of Leu 93 of MR in substrate specificity and catalysis, we constructed L93 variants bearing hydrophobic (L93A, L93F, and L93W), polar neutral (L93N), acidic (L93D), or basic (L93K and L93R) residues at position 93. Gel filtration-HPLC revealed that wild-type MR and all L93 MR variants, apart from L93R MR (dimeric), were tetrameric in solution. The catalytic efficiency (k

Background Comparisons between frailty assessment tools for waitlist candidates are a recognized priority area for kidney transplantation. We compared the prevalence of frailty using three established tools in a cohort of waitlist candidates. Methods Waitlist candidates were prospectively enrolled from 2016 to 2020 across five centers. Frailty was measured using the Frailty Phenotype (FP), a 37-variable frailty index (FI), and the Clinical Frailty Scale (CFS). The FI and CFS were dichotomized using established cutoffs. Agreement was compared using κ coefficients. Area under the receiver operating characteristic (ROC) curves were generated to compare the FI and CFS (treated as continuous measures) with the FP. Unadjusted associations between each frailty measure and time to death or waitlist withdrawal were determined using an unadjusted Cox proportional hazards model. Results Of 542 enrolled patients, 64% were male, 80% were White, and the mean age was 54±14 years. The prevalence of frailty by the FP was 16%. The mean FI score was 0.23±0.14, and the prevalence of frailty was 38% (score of ≥0.25). The median CFS score was three (IQR, 2–3), and the prevalence was 15% (score of ≥4). The κ values comparing the FP with the FI (0.44) and CFS (0.27) showed fair to moderate agreement. The area under the ROC curves for the FP and FI/CFS were 0.86 (good) and 0.69 (poor), respectively. Frailty by the CFS (HR, 2.10; 95% CI, 1.04 to 4.24) and FI (HR, 1.79; 95% CI, 1.00 to 3.21) was associated with death or permanent withdrawal. The association between frailty by the FP and death/withdrawal was not statistically significant (HR, 1.78; 95% CI, 0.79 to 3.71). Conclusion Frailty prevalence varies by the measurement tool used, and agreement between these measurements is fair to moderate. This has implications for determining the optimal frailty screening tool for use in those being evaluated for kidney transplant.

Neutrophils are important innate immune cells involved in microbial clearance at sites of infection. However, the roles of neutrophils in cancer development are not clear. We hypothesized that neutrophils might mediate antitumor effects at the initial tumor formation stage. To test this hypothesis, we injected 2x106 mouse lung Lewis cancer cells into the flanks of C57BL/6 and Cxcr2-/- mice, the latter lack the receptor for ELR+ CXC neutrophil chemokines. We observed increased tumor growth in Cxcr2-/- mice as compared with controls within 3 weeks of injection. FACS analysis indicated fewer neutrophils and more IL-17-producing cells in the tumors of Cxcr2-/- mice than those of C57BL/6 mice. We further tested the roles of neutrophils using anti-Ly6G antibody depletion at the time of tumor cell injection. We demonstrated that tumors grew faster in the neutrophil depleted group, while the percentages of IL-17-producing cells in the tumors and the levels of G-CSF in the plasma were also increased in the absence of neutrophils. To test whether neutrophils have a direct cytotoxic effect on LLC cells, we performed in vitro cell killing assays. We demonstrated neutrophils isolated from both circulation and bone marrow exerted cytotoxic effects. In conclusion, by using Cxcr2-/- and neutrophil antibody-depleted C57BL/6 mice, we have demonstrated that initial tumor growth of LLC cells is enhanced in association with inaccessibility of neutrophils and activation of the IL-17/G-CSF axis.

Ndfip1 is an adaptor that promotes polyubiquitylation by the E3 ubiquitin ligase Itch. Mice that lack either Ndfip1 or Itch develop a severe TH2-mediated inflammatory disease at sites of environmental antigen exposure, including the lung. Here we demonstrate that lungs from Ndfip1-/- mice showed increased numbers of neutrophils and TH17 cells. This was not because Ndfip1-/- T cells are intrinsically biased towards TH17 differentiation. In fact, fewer Ndfip1-/- T cells differentiated into TH17 cells in vitro due to the overproduction of IL-4 production. Rather, TH17 differentiation in vivo was increased in Ndfip1-/- mice due to elevated numbers of IL-6 producing eosinophils. Interestingly, mice lacking both Ndfip1 and IL-4 had levels of IL-6 comparable to WT controls and these Ndfip1-/- IL-4-/- mice had fewer TH17 cells in their lungs compared to Ndfip1-/- IL-4+/+ mice. These results indicate that TH2 inflammation, such as that observed in Ndfip1-/- mice, can increase TH17 differentiation by recruiting IL-6 producing eosinophils into secondary lymphoid organs and tissues. This may explain why TH17 cells develop within an ongoing TH2 inflammatory response, such as asthma.