Cysteine Nucleophiles in Glycosidase Catalysis : Application of a Covalent β-L-Arabinofuranosidase Inhibitor

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)(3)(Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived beta-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This beta-l-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.Medical BiochemistryBio-organic Synthesi

Competing meanings of international experiences for early-career researchers: a collaborative autoethnographic approach

Although there is a pressing demand for international experience for early career researchers (ECRs), the meaning of these experiences arising from their day-to-day work responsibilities is still unclear. Accordingly, using our emic reflections for this autoethnographic study, we—five Japanese ECRs with years of international experiences—collaboratively explored how we made sense of our international experiences, that is, our distinct capital attained from international study and research experiences. We used Identity Trajectory as a conceptual tool to widely capture ECRs’ key experiences and sense-making. Our reflective conversations resulted in five major themes: (1) global personal network, (2) communicative competence, (3) scholarly community culture, (4) scholarly ambition and (5) pedagogical application. We consistently valued our attained capital, but simultaneously recognised dilemmas while engaging in our work. Lack of institutional support was critical, preventing us from using our international experiential capital and further developing as internationally active researchers. This study offers insights for those who may consider an academic career in Japan after returning from international sojourns and for policymakers promoting the internationalisation of Japanese higher education. Studies such as this one also contribute to the exploration of the value of international experiences for researchers in different contexts

Supplementary Material for: Colorectal Cancer with BRAF D594G Mutation Is Not Associated with Microsatellite Instability or Poor Prognosis

<i>Objective:</i><i>BRAF</i> D594G mutations in colorectal cancer patients are not clearly understood. We retrospectively investigated the clinicopathological features of colorectal cancers with <i>BRAF</i> D594G mutations. <i>Methods:</i> We selected 908 colorectal cancer patients who underwent surgical resection from January 2008 to January 2013, and assessed <i>BRAF</i>, <i>KRAS</i>, microsatellite instability, and CpG island methylator phenotype (CIMP). <i>Results:</i> We detected <i>BRAF</i> D594G in 7 patients and <i>BRAF</i> V600E in 45 patients. The clinicopathological features of cancers with <i>BRAF</i> D594G mutation were similar to those with <i>BRAF</i> wild-type, but differed from those with <i>BRAF</i> V600E mutations. Regarding microsatellite instability status, 44.4% of cases with <i>BRAF</i>V600E mutations exhibited high microsatellite instability, compared to 14.3% of those with <i>BRAF</i> D594G mutations and 4.4% of those with <i>BRAF</i> wild-type. There were no CIMP-positive tumors in cancers with <i>BRAF</i> D594G mutations, whereas 67.8% of tumors with <i>BRAF</i> V600E mutations were CIMP-positive. In stage IV cancers, the survival rates of patients at 2 years were 8.5, 50.0, and 68.2% in the <i>BRAF</i> V600E mutation, <i>BRAF</i> D594G mutation, and <i>BRAF</i> wild-type groups, respectively.<i>Conclusion:</i> Colorectal cancers with <i>BRAF</i> D594G mutations exhibit similar clinicopathological features, microsatellite instability status, and prognosis as those with <i>BRAF</i> wild-type