The three-dimensional structure of an unusually active hydrolytic antibody with a phosphonate transition state analog (hapten) bound to the active site has been solved to 2.5 A resolution. The antibody (17E8) catalyzes the hydrolysis of norleucine and methionine phenyl esters and is selective for amino acid esters that have the natural alpha-carbon L configuration. A plot of the pH-dependence of the antibody-catalyzed reaction is bell-shaped with an activity maximum at pH 9.5; experiments on mechanism lend support to the formation of a covalent acyl-antibody intermediate. The structural and kinetic data are complementary and support a hydrolytic mechanism for the antibody that is remarkably similar to that of the serine proteases. The antibody active site contains a Ser-His dyad structure proximal to the phosphorous atom of the bound hapten that resembles two of the three components of the Ser-His-Asp catalytic triad of serine proteases. The antibody active site also contains a Lys residue to stabilize oxyanion formation, and a hydrophobic binding pocket for specific substrate recognition of norleucine and methionine side chains. The structure identifies active site residues that mediate catalysis and suggests specific mutations that may improve the catalytic efficiency of the antibody. This high resolution structure of a catalytic antibody-hapten complex shows that antibodies can converge on active site structures that have arisen through natural enzyme evolution.

BackgroundSevere acute kidney injury (AKI) after cardiac surgery is associated with poor clinical outcomes. This study evaluated the potential use of miR-21 as a risk marker for postoperative AKI progression and other poor outcomes.Methodology/Principal FindingsThe study included 120 adult patients undergoing cardiac surgery: 40 non-AKI controls, 39 patients with progressive AKI, and 41 with non-progressive AKI. Urine and plasma levels of miR-21 were assessed by quantitative real-time PCR (RT-qPCR). Associations between miR-21 levels and AKI progression were determined by estimating areas under receiver operating characteristic curves (AUC). We demonstrated that up-regulated urine and plasma levels of miR-21 in patients with AKI were both associated with AKI progression. The AUCs for urine and plasma levels of miR-21 associated with established AKI were 0.68 (95%CI: 0.59–0.78) and 0.80 (95%CI: 0.73–0.88), respectively. Multiple logistic regression analysis, adjusting for clinical variables, indicated that the prognostic predictive power of urine and plasma miR-21 levels for AKI progression were represented by AUCs of 0.81 (95%CI: 0.72–0.91) and 0.83 (95%CI: 0.74–0.92), respectively. Urinary and plasma miR-21 levels also predicted the need for postoperative renal replacement therapy (RRT), development of Acute Kidney Injury Network (AKIN) stage 3 AKI, 30-day in-hospital mortality and prolonged stay in hospital or ICU. Urine miR-21 was a better outcome predictor than plasma miR-21, being associated with higher (1.4- to 2.6-fold) unadjusted odds ratio for progression of AKI and other poor outcomes.ConclusionsUrinary and plasma miR-21 are associated with severe AKI and other poor postoperative outcomes of cardiac surgery, indicating their potential use as prognostic markers.

Sentinel nodes (SNs), the nodes nearest a primary tumor on the direct lymphatic drainage path, are the site of earliest metastases, and in melanoma show striking immune modulation. We evaluated SNs from breast cancer patients for evidence of similar immune perturbation. The purpose of this study was to evaluate whether SNs from patients with breast cancer show the alterations in the histology and cytology of the paracortical areas seen in SNs from patients with melanoma. Formalin-fixed and paraffin-embedded sections from 32 SNs and 32 nonsentinel nodes (NSNs) from patients with breast cancer were evaluated. Sections were stained with hematoxylin and eosin and with antibodies to S-100 protein and HLA-DR, DQ, and DP to identify interdigitating dendritic cells (IDCs), and by an antibody to CD43RA to delineate T lymphocytes. By computerized image analysis we evaluated the distribution, frequency, immunophenotype, and activation status of IDCs and associated T lymphocytes in SNs and NSNs. Average areas occupied by S-100-positive dendritic cells (DCs) in SNs and NSNs were 0.13% and 19.98%, respectively, of total nodal area (p < 0.0001). The average density of S-100-positive IDCs in SNs was 11.00/mm2 and in NSNs was 257.88/mm2 (p < 0.0001). In SNs 43.55% of DCs (4.93/mm2) were nondendritic, 51.92% (5.69/mm2) had short dendrites, and 5.2% were mature with long dendrites (0.62/mm2). In SNs the ratio of immature to mature IDCs was 7.95:1. In NSNs, 8.09% of DCs (8.5/mm2) were nondendritic, 28.22% (67.46/mm2) had short dendrites, and 63.07% (145.96/mm2) were mature DCs with long dendrites. The ratio of immature to mature DCs in NSNs was 1:6.66. The average areas occupied by HLA class II-positive DCs in SNs and NSNs were 4.21% and 31.82%, respectively, of total nodal area. The frequency of coexpression of S-100 and HLA class II by immature IDCs without dendrites was 11.27% in SNs and 15.00% in NSNs. In both SNs and NSNs (p < 0.001) all mature S-100-positive IDCs with long dendrites expressed HLA class II. CD43RA-positive T lymphocytes occupied 20.06% of total nodal area in SNs and 63.57% in NSNs (p < 0.0001). The SNs from breast cancer patients are profoundly immune modulated with, by comparison to NSNs, markedly reduced paracortical areas, densities of paracortical DCs, frequency of S-100-positive IDCs coexpressing HLA class II, and a predominance of immature nondendritic and poorly dendritic DCs.

Nonalcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease worldwide. Due to the growing economic burden of NAFLD on public health, it has become an emergent target for clinical intervention. DUSP12 is a member of the dual specificity phosphatase (DUSP) family, which plays important roles in brown adipocyte differentiation, microbial infection, and cardiac hypertrophy. However, the role of DUSP12 in NAFLD has yet to be clarified. Here, we reveal that DUSP12 protects against hepatic steatosis and inflammation in L02 cells after palmitic acid/oleic acid treatment. We demonstrate that hepatocyte specific DUSP12-deficient mice exhibit high-fat diet (HFD)-induced and high-fat high-cholesterol diet-induced hyperinsulinemia and liver steatosis and decreased insulin sensitivity. Consistently, DUSP12 overexpression in hepatocyte could reduce HFDinduced hepatic steatosis, insulin resistance, and inflammation. At the molecular level, steatosis in the absence of DUSP12 was characterized by elevated apoptosis signal-regulating kinase 1 (ASK1), which mediates the mitogenactivated protein kinase (MAPK) pathway and hepatic metabolism. DUSP12 physically binds to ASK1, promotes its dephosphorylation, and inhibits its action on ASK1-related proteins, JUN N-terminal kinase, and p38 MAPK in order to inhibit lipogenesis under high-fat conditions. Conclusion: DUSP12 acts as a positive regulator in hepatic steatosis and offers potential therapeutic opportunities for NAFLD. (Hepatology 2019;70:1099-1118). SEE EDITORIAL ON PAGE 1091N onalcoholic fatty liver disease (NAFLD) is the most common diffuse liver disease and is characterized by lipid accumulation in hepatocytes in the absence of excessive alcohol consumption. (1) The prevalence of NAFLD increases worryingly with obesity and other diseases of metabolic syndrome (MS) and is expected to be the leading cause of liver failure in the future. (2) Accumulating studies show that NAFLD not only is linked to an increased risk of liver-related mortality or morbidity

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