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Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats

Maopu buckwheat finished Baijiu (MBFB), a unique Chinese Baijiu, is mainly composed of base Baijiu and tartary buckwheat extract (TBE). However, there is a lack of studies reporting its flavor. In this study, the key odorants in MBFB were characterized for the first time using a sensomics approach. Forty-nine odorants were identified using gas chromatography-mass spectrometry/olfactory analysis, coupled with aroma extraction dilution analysis. They were quantified using multiple methods and their odor activity values were calculated. Ethyl hexanoate, ethyl butanoate, butanoic acid, hexanoic acid, dimethyl trisulfide, p-cresol, β-damascenone, and 3-methylbutanal were confirmed as the key odorants in MBFB using recombination and omission experiments. Furthermore, the interaction between TBE and odorants were revealed that TBE enhanced the sweet notes, while it inhibited the alcoholic, acidic, and fruity notes, which might be because TBE promoted the volatility of most esters, but inhibited the volatilities of butanoic acid and hexanoic acid.

SummaryDuring mitosis of human cells, the kinase Bub1 orchestrates chromosome segregation through phosphorylating histone H2A and the anaphase-promoting complex/cyclosome activator Cdc20. Bub1-mediated H2A-T120 phosphorylation (H2A-pT120) at kinetochores promotes centromeric sister-chromatid cohesion, whereas Cdc20 phosphorylation by Bub1 contributes to spindle checkpoint signaling. Here, we show that phosphorylation at the P+1 substrate-binding loop of human Bub1 enhances its activity toward H2A but has no effect on its activity toward Cdc20. We determine the crystal structure of phosphorylated Bub1. A comparison between structures of phosphorylated and unphosphorylated Bub1 reveals phosphorylation-triggered reorganization of the P+1 loop. This activating phosphorylation of Bub1 is constitutive during the cell cycle. Enrichment of H2A-pT120 at mitotic kinetochores requires kinetochore targeting of Bub1. The P+1 loop phosphorylation of Bub1 appears to occur through intramolecular autophosphorylation. Our study provides structural and functional insights into substrate-specific regulation of a key mitotic kinase and expands the repertoire of kinase activation mechanisms.

A given protein generally has only one native tertiary fold, which is the conformation with the lowest Gibbs free energy. Mad2, a protein involved in the spindle checkpoint, however, has two natively folded states with similar Gibbs free energies. Through binding to its target Cdc20, Mad2 inhibits the multisubunit ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C), and delays the onset of anaphase until all sister chromatids achieve bipolar attachment to the mitotic spindle. Without ligand binding or covalent modifications, Mad2 adopts two topologically and functionally distinct native folds in equilibrium under physiological conditions. The transition between the two Mad2 states is regulated by multiple mechanisms and is central to the activation and inactivation of the spindle checkpoint. This review summarizes recent structural and biochemical studies on the two-state behavior of Mad2 and discusses the generality and implications of structural malleability of proteins.

Summary The status of spindle checkpoint signaling depends on the balance of two opposing dynamic processes that regulate the highly unusual two-state behavior of Mad2. In mitosis, a Mad1-Mad2 core complex recruits cytosolic Mad2 to kinetochores through Mad2 dimerization and converts Mad2 to a conformer amenable to Cdc20 binding, thereby facilitating checkpoint activation. p31 comet inactivates the checkpoint through binding to Mad1- or Cdc20-bound Mad2, thereby preventing Mad2 activation and promoting the dissociation of the Mad2-Cdc20 complex. Here, we report the crystal structure of the Mad2-p31 comet complex. The C-terminal region of Mad2 that undergoes rearrangement in different Mad2 conformers is a major structural determinant for p31 comet binding, explaining the specificity of p31 comet toward Mad1- or Cdc20-bound Mad2. p31 comet adopts a fold strikingly similar to that of Mad2 and binds at the dimerization interface of Mad2. Thus, p31 comet exploits the two-state behavior of Mad2 to block its activation by acting as an "anti-Mad2."

SummaryThe spindle checkpoint forms an intricate signaling circuit to sense unattached kinetochores, to inhibit the anaphase-promoting complex/cyclosome (APC/C), and to delay anaphase onset. Using clever genetic experiments in the budding yeast, Lau and Murray define the endpoint of checkpoint signaling and provide key mechanistic insights into checkpoint inhibition of APC/C.

HCN, a new 3D NMR technique for stepwise coherence transfer from 1H to 13C to 15N and reverse through direct spin couplings 1JCH and 1JCN, is presented as a method for detection and assignment of histidine and tryptophan side-chain 1H, 13C, and 15N resonances in uniformly 13C/15N-labeled proteins. Product-operator calculations of cross-peak volumes vs adjustable delay tau 3 were employed for determination of optimal tau 3. For the phosphatidylinositol 3-kinase (PI3K SH3 domain, MW = 9.6 kD) at pH 6, H(C)N, the 1H/15N projection, produced observable cross peaks within 20 min. and was completely selective for the single tryptophan and single histidine. The 3D HCN experiment yielded well-defined cross peaks in 20 h for the 13C/15N-labeled origin-specific DNA binding domain from simian virus 40 T-antigen (T-ag-OBD131-259, MW = 15.4 kD) at pH 5.5. Resonances from all six histidines in T-ag-OBD were observed, and 11 of the 12 1H and 13C chemical shifts and 10 of the 12 15N chemical shifts were determined. The 13C dimension proved essential in assignment of the multiply overlapping 1H and 15N resonances. From the spectra recorded at a single pH, three of the imidazoles were essentially neutral and the other three were partially protonated (22-37%). HCN yielded strong cross peaks after 18 h on a 2.0 mM sample of phenylmethanesulfonyl fluoride (PMSF)-inhibited alpha-lytic protease (MW = 19.8 kD) at pH 4.4. No spectra have been obtained, however, of native or boronic acid-inhibited alpha-lytic protease after 18 h at various temperatures ranging from 5 to 55 degrees C, probably due to efficient relaxation of active-site imidazole 1H and/or 15N nuclei.