Engineering and Directed Evolution of a Ca2+ Binding Site A-Deficient AprE Mutant Reveal an Essential Contribution of the Loop Leu75–Leu82 to Enzyme Activity

An aprE mutant from B. subtilis 168 lacking the connecting loop Leu75–Leu82 which is predicted to encode a Ca2+ binding site was constructed. Expression of the mutant gene (aprEΔLeu75–Leu82) produced B. subtilis colonies lacking protease activity. Intrinsic fluorescence analysis revealed spectral differences between wild-type AprE and AprEΔL75–L82. An AprEΔL75–L82 variant with reestablished enzyme activity was selected by directed evolution. The novel mutations Thr66Met/Gly102Asp located in positions which are predicted to be important for catalytic activity were identified in this variant. Although these mutations restored hydrolysis, they had no effect with respect to thermal inactivation of AprEΔL75–L82 T66M G102D. These results support the proposal that in addition to function as a calcium binding site, the loop that connects β-sheet e3 with α-helix c plays a structural role on enzyme activity of AprE from B. subtilis 168

Meaning-making from wordless (or nearly wordless) picturebooks: what educational research expects and what readers have to say

Wordless (or nearly wordless) picturebooks are intriguing in terms of how readers make meaning from them. This article offers a conceptualization of existing studies in the field of education that use wordless picturebooks with young readers. While some of these studies contribute to understanding meaning-making, the pragmatic use of wordless picturebooks often does not take account of their particular nature and of the heightened role of the reader, leading to a mismatch between what the picturebook expects from the implied reader and the researchers’ expectations of what ‘real’ readers must do with these books. By highlighting observations from children’s literature scholarship and reader-response studies, this article aims to encourage a more interdisciplinary understanding of meaning-making. It also seeks to persuade educational researchers and mediators to consider investigative approaches that are not based on verbalization but are more in tune with the invitations that wordless picturebooks extend to young readers

Comparison of clinical characteristics of Zika and dengue symptomatic infections and other acute illnesses of unidentified origin in Mexico.

BackgroundOur purpose was to provide a detailed clinical description, of symptoms and laboratory abnormalities, and temporality in patients with confirmed Zika and dengue infections, and other acute illnesses of unidentified origin (AIUO).Methods/ principal findingsThis was a two-year, multicenter, observational, prospective, cohort study. We collected data from patients meeting the Pan American Health Organization's modified case-definition criteria for probable Zika infection. We identified Zika, dengue chikungunya by RT-PCR in serum and urine. We compared characteristics between patients with confirmed Zika and dengue infections, Zika and AIUO, and Dengue and AIUO at baseline, Days 3,7,28 and 180 of follow-up. Most episodes (67%) consistent with the PAHO definition of probable Zika could not be confirmed as due to any flavivirus and classified as Acute Illnesses of Unidentified Origin (AIUO). Infections by Zika and dengue accounted for 8.4% and 16% of episodes. Dengue patients presented with fever, generalized non-macular rash, arthralgia, and petechiae more frequently than patients with Zika during the first 10 days of symptoms. Dengue patients presented with more laboratory abnormalities (lower neutrophils, lymphocytosis, thrombocytopenia and abnormal liver function tests), with thrombocytopenia lasting for 28 days. Zika patients had conjunctivitis, photophobia and localized macular rash more frequently than others. Few differences persisted longer than 10 days after symptoms initiation: conjunctivitis in Zika infections, and self-reported rash and petechia in dengue infections.ConclusionsOur study helps characterize the variety and duration of clinical features in patients with Zika, dengue and AIUO. The lack of diagnosis in most patients points to need for better diagnostics to assist clinicians in making specific etiologic diagnoses