Carbonic anhydrases are mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO 2 /HCO 3 − . Previously, the X-ray crystal structures of CO 2 -bound holo (zinc-bound) and apo (zinc-free) human carbonic anhydrase IIs (hCA IIs) were captured at high resolution. Here, we present sequential timeframe structures of holo-[T = 0 s (CO 2 -bound), 50 s, 3 min, 10 min, 25 min, and 1 h] and apo-hCA IIs [T = 0 s, 50 s, 3 min, and 10 min] during the "slow" release of CO 2 . Two active site waters, W DW (deep water) and W DW ′ (this study), replace the vacated space created on CO 2 release, and another water, W I (intermediate water), is seen to translocate to the proton wire position W1. In addition, on the rim of the active site pocket, a water W2′ (this study), in close proximity to residue His64 and W2, gradually exits the active site, whereas His64 concurrently rotates from pointing away ("out") to pointing toward ("in") active site rotameric conformation. This study provides for the first time, to our knowledge, structural "snapshots" of hCA II intermediate states during the formation of the His64-mediated proton wire that is induced as CO 2 is released. Comparison of the holo-and apo-hCA II structures shows that the solvent network rearrangements require the presence of the zinc ion. − (general reviews are in refs. 1-5). In the hydration direction, the first step of catalysis is the conversion of CO 2 into bicarbonate through the nucleophilic attack of the reactive Znbound hydroxide, and the resultant bicarbonate is subsequently displaced from the zinc by a water molecule (expression 1) (6). The second step of catalysis is the transfer of a proton from the Zn-bound water to bulk solvent for the regeneration of the Znbound hydroxide. The general base for proton transfer (PT), B, can be either a proton acceptor in solution (water) or a residue (His64) in the enzyme (expression 2):

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We report the results of a 2-year longitudinal comparison of grade 3 and grade 4 English-as-a-second-language learners in an experimental, comprehension-based program and those in a regular (i.e., more typical) language learning program. The goal was to examine the extent to which sustained, long-term comprehension practice in both listening and reading—in the virtual absence of any speaking—can help develop learners’ second language (L2) pronunciation. We analyzed learners’ sentences from an elicited imitation task using several accuracy and fluency measures as well as listener ratings of accentedness, comprehensibility, and fluency. We found no differences between the two programs at the end of year 1. However, at the end of year 2, there were some differences—namely, in the listener ratings of fluency and comprehensibility—that favored learners in the regular program. These findings highlight the beneficial effects of comprehension practice for the development of L2 pronunciation but also point to some potential limits of this practice.

We report a novel transcriptomic analysis workflow called LiEB (Life cycle of Epstein-Barr virus) to characterize distributions of oncogenic virus, Epstein-Barr virus (EBV) infection in human tumors. We analyzed 851 The Cancer Genome Atlas whole-transcriptome sequencing (WTS) data to investigate EBV infection by life cycle information using three-step LiEB workflow: 1) characterize virus infection generally; 2) align transcriptome sequences against a hybrid human-EBV genome, and 3) quantify EBV gene expression. Our results agreed with EBV infection status of public cell line data. Analysis in stomach adenocarcinoma identified EBV-positive cases involving PIK3CA mutations and/or CDKN2A silencing with biologically more determination, compared to previous reports. In this study, we found that a small number of colorectal adenocarcinoma cases involved with EBV lytic gene expression. Expression of EBV lytic genes was also observed in 3% of external colon cancer cohort upon WTS analysis. Gene set enrichment analysis showed elevated expression of genes related to E2F targeting and interferon-gamma responses in EBV-associated tumors. Finally, we suggest that interpretation of EBV life cycle is essential when analyzing its infection in tumors, and LiEB provides high capability of detecting EBV-positive tumors. Observation of EBV lytic gene expression in a subset of colon cancers warrants further research.

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