Efficient overexpression and purification of severe acute respiratory syndrome coronavirus 2 nucleocapsid proteins in Escherichia coli.

The fundamental biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.

Mapping the Initial Stages of a Protective Pathway that Enhances Catalytic Turnover by a Lytic Polysaccharide Monooxygenase.

Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII-(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII-tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues "on the fly" to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.

Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant O6-Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an O6-Alkylguanine Adductome.

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, O6-methylguanine O6-methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA O6-alkylguanines (O6-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple O6-alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different O6-alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known O6-MethylG (O6-MeG) levels. It was digested with trypsin, and ASPs were detected and quantified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. ASPs containing S-methyl, S-ethyl, S-propyl, S-hydroxyethyl, S-carboxymethyl, S-benzyl, and S-pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding O6-alkGs. The LOQ of ASPs containing S-methylcysteine detected after MGMT incubation with Me-CT-DNA was <0.05 pmol O6-MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing S-methylcysteine at levels that correlated with those of O6-MeG determined previously by HPLC-radioimmunoassay (r2 = 0.74; p = 0.014). O6-CMG, a putative O6-hydroxyethylG adduct, and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of O6-alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterized. The methodology establishes a platform for characterizing the human DNA O6-alkG adductome and, given the mutagenic potential of O6-alkGs, can provide mechanistic information about cancer pathogenesis.

Community-based case management and health care use in older adults: outcomes of a collaborative multiagency approach.

Case management (CM) establishes valuable connections between clients and needed supports. There are, however, mixed results on its prediction of health care use. This quasi-experimental record review (N = 96) of a collaborative CM consortium examined predictors of health care use among older adults (aged older than 60 years) who had received CM for at least 6 months. Descriptive, bivariate, and multivariate models were used to predict differences in outcomes (hospitalization and emergency room [ER] visits) between participants who received CM and those who did not. Hospitalization rates were significantly lower among CM recipients. Living alone, identifying as White/non-Hispanic, and higher numbers of medications were significant copredictors of hospital use. CM was not a significant predictor of ER use. Race/ethnicity, however, remained significant in that White/non-Hispanic participants were 4 times likely to have at least 1 ER visit than persons of color. Implications for future research are discussed to promote better understanding of the effectiveness of CM, particularly within ethnically and racially diverse communities. Further inquiry is needed around the experiences of older adults of color, those who live alone, or who take multiple medications as needed. Further testing of a consortium model is necessary to determine value added through a multiagency approach.

Bile salts enhance the susceptibility of the peach allergenic lipid transfer protein, Pru p 3, to in vitro gastrointestinal proteolysis.

Sensitisation to the lipid transfer protein Pru p 3 is associated with severe allergic reactions to peach, the proteins stability being thought to play a role in its allergenicity. Lipid binding increases susceptibility of Pru p 3 to digestion and so the impact of bile salts on the in vitro gastrointestinal digestibility of Pru p 3 was investigated and digestion products mapped by SDS-PAGE and mass spectrometry. Bile salts enhanced the digestibility of Pru p 3 resulting in an ensemble of around 100 peptides spanning the protein's sequence which were linked by disulphide bonds into structures of ~ 5-6 kDa. IgE binding studies with a serum panel from peach allergic subjects showed digestion reduced, but did not abolish, the IgE reactivity of Pru p 3. These data show the importance of including bile salts in vitro digestion systems and emphasise the need to profile of digestion in a manner that allows identification of immunologically relevant disulphide-linked peptide aggregates.

Preschool Fantasy-Reality Discrimination: Influences of Trait and Primed Fearfulness.

Distinguishing between fantasy and reality is an important developmental milestone, achieved in the preschool years, that has been linked to children's cognitive capabilities and emotional knowledge. The authors examined the influence of both trait and prime fearfulness on preschoolers' ability to differentiate between fantastic and real situations. Forty 3-5-year-old preschoolers were administered a fearfulness assessment, a standard theory-of-mind task, and a fantasy-reality discrimination task (with or without a fear prime). When primed to experience fear, participants who were high in trait fearfulness made more mistakes distinguishing between fantastic and real events than did participants who were low in trait fearfulness. In the absence of a fear prime there was no difference in performance between these two groups. These findings were independent of participants' age, gender, and theory of mind abilities and suggest that preschoolers who are high in trait fearfulness are more prone to difficulty in discriminating between fantasy and reality when actively experiencing fear. This study illuminates important factors that contribute to the errors in fantasy-reality judgments frequently seen in preschoolers.

Muslim American adolescents' explanations of changing religious practices: Cultural tools in cultural contexts.

To examine the culturally embedded nature of religious practices, we conducted a mixed-methods study in which Muslim American adolescents described how and why their religious practices had changed in recent years (see Etengoff & Daiute, 2013, J. Adolesc. Res., 28, 690). Participants included 201 Muslim adolescents (ages 13-19) from predominantly immigrant families; all were contestants in a Muslim Inter-Scholastic Tournament regional competition. Participants completed surveys including an item regarding whether their religious practices had changed, and for those who answered affirmatively, open-ended questions about the change. Additional measures assessed ethnic identity and perceived discrimination. As hypothesized, the 60% of participants who reported a change in religious practices described this shift as a response to new contexts, people, and religious knowledge. Those who reported a change also reported higher levels of ethnic identity exploration and perceived discrimination. Overall, Muslim American adolescents' descriptions portrayed religious practices as developing through reciprocal interactions with culture. More generally, participants' descriptions point to the viability of a model in which religious practices change and in turn are changed by cultural contexts. Statement of contribution What is already known on this subject? Religious development is viewed as taking place in relational systems with reciprocity between individuals and surrounding contexts. Variations in contexts predict variations in religious development, but mechanisms of development are not well understood. Muslim Americans, including adolescents, show high levels of religious involvement and experience unique cultural and religious contexts. Muslim American emerging adults describe their religious practices as responsive to sociocultural contexts. What does the study add? This study focuses on Muslim American adolescents, a group that has received little research attention, especially in regard to religious development. Participants reported a wide array of changes in religious practices, and they described these changes as responses to social and cultural influences. Participants' descriptions of changing practices can be understood through a Vygotskian framework in which religious practices are cultural tools that both respond to and shape surrounding cultural contexts.