Cutinase is described as playing various roles in fungal-plant pathogen interactions, such as eliciting host-derived signals, fungal spore attachment and carbon acquisition during saprophytic growth. However, the characteristics of the cutinase genes, their expression in compatible interactions and their roles in pathogenesis have not been reported in Curvularia lunata, an important leaf spot pathogen of maize in China. Therefore, a cutinase gene family analysis could have profound significance. In this study, we identified 13 cutinase genes (ClCUT1 to ClCUT13) in the C. lunata genome. Multiple sequence alignment showed that most fungal cutinase proteins had one highly conserved GYSQG motif and a similar DxVCxG[ST]-[LIVMF](3)-x(3)H motif. Gene structure analyses of the cutinases revealed a complex intron-exon pattern with differences in the position and number of introns and exons. Based on phylogenetic relationship analysis, C. lunata cutinases and 78 known cutinase proteins from other fungi were classified into four groups with subgroups, but the C. lunata cutinases clustered in only three of the four groups. Motif analyses showed that each group of cutinases from C. lunata had a common motif. Real-time PCR indicated that transcript levels of the cutinase genes in a compatible interaction between pathogen and host had varied expression patterns. Interestingly, the transcript levels of ClCUT7 gradually increased during early pathogenesis with the most significant up-regulation at 3 h post-inoculation. When ClCUT7 was deleted, pathogenicity of the mutant decreased on unwounded maize (Zea mays) leaves. On wounded maize leaves, however, the mutant caused symptoms similar to the wild-type strain. Moreover, the ClCUT7 mutant had an approximately 10 % reduction in growth rate when cutin was the sole carbon source. In conclusion, we identified and characterized the cutinase family genes of C. lunata, analyzed their expression patterns in a compatible host-pathogen interaction, and explored the role of ClCUT7 in pathogenicity. This work will increase our understanding of cutinase genes in other fungal-plant pathogens.

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Background Heart failure ( HF ) and atrial fibrillation ( AF ) are rising in prevalence and pose a substantial public health burden. Methods and Results We evaluated temporal trends specific to age, sex, race, and geographic region in rates of HF ‐ and AF ‐related morbidity, mortality, and years of potential life lost at age 75 years between 1991 and 2015 in the United States. For trends in hospitalization with a primary diagnosis of HF versus AF , we used data for patients aged ≥30 years from 1993 to 2014 from the Nationwide Inpatient Sample. For trends in death due to HF versus AF , we used data from 1991 to 2015 from the National Center for Health Statistics. Over the past 25 years, the age‐adjusted rates of hospitalization declined for HF (−1.72% per year) but increased for AF (+1.61% per year). HF mortality rates remained unchanged, whereas those for AF increased (+11.2% per year). Years of potential life lost increased for both HF (+0.4% per year) and AF (+9.8% per year). Trends in HF and AF morbidity rates varied moderately by age group, whereas mortality rates varied by age and race. HF and AF hospitalization and mortality rates rose for individuals aged <50 years. HF hospitalization rates declined in all 4 US census regions, whereas AF rates increased. Conclusions We observed divergent trends of decreasing hospitalization and mortality rates for HF versus increasing rates for AF . Variations in disease burden by race and geography warrant specific targeting of “at risk” groups in selected US regions. Additional studies are warranted to evaluate the rising burden of both conditions in younger adults.

Histatin 1 is a histidine-rich phosphoprotein present in human parotid saliva that possesses candidacidal activity and functions in mineralization by adsorbing to hydroxyapatite. The objective of the present study was to develop a system for recombinant production of histatin 1 and to examine the role of phosphorylation in the functional activities of this molecule. Native histatin 1 (containing a phosphoserine at residue 2) was purified from parotid saliva, whereas a bacterial expression system was used to produce a recombinant form of histatin 1 (re-Hst1) that lacked phosphorylated serine. Histatin 1 cDNA was inserted into the vector pGEX-3X, which expresses foreign genes as soluble fusion proteins attached to the carboxyl-terminus of glutathione S-transferase (GST). The GST/re-Hst1 fusion protein was isolated from cell lysates by affinity chromatography on glutathione (GSH)-Sepharose and digested with cyanogen bromide to separate re-Hst1 from the GST fusion partner. The digest was subjected to reversed-phase high-performance liquid chromatography on a C18 column, and re-Hst1 was eluted as a well-defined peak. The yield of re-Hst1 was 4 mg/L of bacterial culture. Amino-terminal sequencing and amino acid analysis confirmed the final product as re-Hst1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that native histatin 1 and re-Hst1 had the same apparent molecular weights, while cationic PAGE showed that re-Hst1 was more basic. Phosphate analysis indicated 1 mol phosphate/mol of native histatin 1, while re-Hst1 lacked any detectable phosphate. Re-Hst1 demonstrated candidacidal activity comparable to that of native histatin 1, but displayed substantially lower binding to hydroxyapatite. These results show that phosphorylation of histatin 1 at residue 2 contributes significantly to its ability to bind to hydroxyapatite.