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Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that recognize microbial vitamin B metabolites and have emerging roles in infectious disease, autoimmunity, and cancer. Although MAIT cells are identified by a semi-invariant TCR, their phenotypic and functional heterogeneity is not well understood. Here we present an integrated single cell transcriptomic analysis of over 76,000 human MAIT cells during early and prolonged Ag-specific activation with the MR1 ligand 5-OP-RU and nonspecific TCR stimulation. We show that MAIT cells span a broad range of homeostatic, effector, helper, tissue-infiltrating, regulatory, and exhausted phenotypes, with distinct gene expression programs associated with CD4+ or CD8+ coexpression. During early activation, MAIT cells rapidly adopt a cytotoxic phenotype characterized by high expression of GZMB, IFNG and TNF. In contrast, prolonged stimulation induces heterogeneous states defined by proliferation, cytotoxicity, immune modulation, and exhaustion. We further demonstrate a FOXP3 expressing MAIT cell subset that phenotypically resembles conventional regulatory T cells. Moreover, scRNAseq-defined MAIT cell subpopulations were also detected in individuals recently exposed to Mycobacterium tuberculosis, confirming their presence during human infection. To our knowledge, our study provides the first comprehensive atlas of human MAIT cells in activation conditions and defines substantial functional heterogeneity, suggesting complex roles in health and disease.

A dual stimuli-responsive nanoplatform was rationally designed for controlled drug delivery. The nanosheets of graphene oxide (GO) were first modified with aminated mesoporous silica (NH2-mSiO2), and then methotrexate (MTX) was loaded into the mesopores of mSiO2. Alginate (Alg) acted as the "gatekeeper" was then anchored to the MTX-loaded GO/NH2-mSiO2 by amidation reaction, achieving the encapsulation of MTX in the core-shell structured GO/mSiO2@Alg. Due to the high pH sensitivity of amide bond and the excellent photothermal conversion ability of GO, the constructed nanoplatform could be used for pH and near-infrared (NIR) controlled delivery of MTX. The results of cell viability test demonstrate the high inhibitory rate of the dual stimuli-responsive nanoplatform toward hepatoma (HepG2) cells.

Fluoxazolevir is an aryloxazole-based entry inhibitor of hepatitis C virus (HCV). We show that fluoxazolevir inhibits fusion of HCV with hepatic cells by binding HCV envelope protein 1 to prevent fusion. Nine of ten fluoxazolevir resistance-associated substitutions are in envelope protein 1, and four are in a putative fusion peptide. Pharmacokinetic studies in mice, rats and dogs revealed that fluoxazolevir localizes to the liver. A 4-week intraperitoneal regimen of fluoxazolevir in humanized chimeric mice infected with HCV genotypes 1b, 2a or 3 resulted in a 2-log reduction in viraemia, without evidence of drug resistance. In comparison, daclatasvir, an approved HCV drug, suppressed more than 3 log of viraemia but is associated with the emergence of resistance-associated substitutions in mice. Combination therapy using fluoxazolevir and daclatasvir cleared HCV genotypes 1b and 3 in mice. Fluoxazolevir combined with glecaprevir and pibrentasvir was also effective in clearing multidrug-resistant HCV replication in mice. Fluoxazolevir may be promising as the next generation of combination drug cocktails for HCV treatment. Preclinical studies show that fluoxazolevir, which inhibits the fusion of hepatitis C virus (HCV) with hepatic cells by binding viral envelope protein 1, could be useful in drug cocktails to treat HCV.

Despite recent advances in therapy, allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative option for a range of high-risk hematologic malignancies. However, acute graft-versus-host disease (aGVHD) continues to limit the long-term success of HSCT, and new therapies are still needed. We previously demonstrated that aGVHD depends on the ability of donor conventional T cells (Tcons) to express the lymph node trafficking receptor, CC-Chemokine Receptor 7 (CCR7). Consequently, we examined the ability of cosalane, a recently identified CCR7 small-molecule antagonist, to attenuate aGVHD in mouse HSCT model systems. Here we show that the systemic administration of cosalane to transplant recipients after allogeneic HSCT did not prevent aGVHD. However, we were able to significantly reduce aGVHD by briefly incubating donor Tcons with cosalane ex vivo before transplantation. Cosalane did not result in Tcon toxicity and did not affect their activation or expansion. Instead, cosalane prevented donor Tcon trafficking into host secondary lymphoid tissues very early after transplantation and limited their subsequent accumulation within the liver and colon. Cosalane did not appear to impair the intrinsic ability of donor Tcons to produce inflammatory cytokines. Furthermore, cosalane-treated Tcons retained their graft-versus-leukemia (GVL) potential and rejected a murine P815 inoculum after transplantation. Collectively, our data indicate that a brief application of cosalane to donor Tcons before HSCT significantly reduces aGVHD in relevant preclinical models while generally sparing beneficial GVL effects, and that cosalane might represent a viable new approach for aGVHD prophylaxis.

Asian Americans represent an understudied racial category in health disparity research. Using data from the National Asian American Survey, we examined self-rated health (SRH) disparities in eight Asian subgroups compared to Whites, explored the moderating effect of nativity status, and investigated the mediating effect of socioeconomic status. None of the Asian subgroups fared better than Whites. Across Asian subgroups, South Asians, Japanese, and Filipinos had the best SRH, with Cambodians being the most disadvantaged. Nativity was a significant moderator in that SRH disadvantages were only manifested among immigrants for Chinese, Korean, Hmong, and Vietnamese and only among natives for Filipinos. For most groups showing SRH disadvantages, SES played partial mediating roles. Education showed a higher explanatory power than income for inter-ethnic SRH disparities. Contrary to popular perception, Asian Americans are not the model minority in terms of SRH. Cultural influences on SRH reporting biases were discussed.

CC-chemokine receptor 7 (CCR7) is a G protein-coupled receptor expressed on a variety of immune cells. CCR7 plays a critical role in the migration of lymphocytes into secondary lymphoid tissues. CCR7 expression, however, has been linked to numerous disease states. Due to its therapeutic relevance and absence of available CCR7 inhibitors, we undertook a high-throughput screen (HTS) to identify small-molecule antagonists of the receptor. Here, we describe a robust HTS approach using a commercially available β-galactosidase enzyme fragment complementation system and confirmatory transwell chemotaxis assays. This work resulted in the identification of several compounds with activity against CCR7. The most potent of these was subsequently determined to be cosalane, a cholesterol derivative previously designed as a therapeutic for human immunodeficiency virus. Cosalane inhibited both human and murine CCR7 in response to both CCL19 and CCL21 agonists at physiologic concentrations. Furthermore, cosalane produced durable inhibition of the receptor following a cellular incubation period with subsequent washout. Overall, our work describes the development of an HTS-compatible assay, completion of a large HTS campaign, and demonstration for the first time that cosalane is a validated CCR7 antagonist. These efforts could pave the way for new approaches to address CCR7-associated disease processes.

Reliance on hepatitis C virus (HCV) replicon systems and protein-based screening assays has led to treatments that target HCV viral replication proteins. The model does not encompass other viral replication cycle steps such as entry, processing, assembly and secretion, or viral host factors. We previously applied a phenotypic high-throughput screening platform based on an infectious HCV system and discovered an aryloxazole-based anti-HCV hit. Structure– activity relationship studies revealed several compounds exhibiting EC50 values below 100 nM. Lead compounds showed inhibition of the HCV pseudoparticle entry, suggesting a different mode of action from existing HCV drugs. Hit 7a and lead 7ii both showed synergistic effects in combination with existing HCV drugs. In vivo pharmacokinetics studies of 7ii showed high liver distribution and long half-life without obvious hepatotoxicity. The lead compounds are promising as preclinical candidates for the treatment of HCV infection and as molecular probes to study HCV pathogenesis.

In this study, we examined the association between participation in the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) and breastfeeding outcomes before and after the 2009 revisions. Four-thousand-three-hundred-and-eight WIC-eligible children younger than 60 months were included from the 2005&ndash, 2014 National Health and Nutrition Examination Survey (NHANES). We compared two birth cohorts with regard to their associations between WIC participation and being ever-breastfed and breastfed at 6 months. We estimated the average effect of the treatment for the treated to assess the causal effect of WIC participation on breastfeeding based on propensity score matching. The results showed that WIC-eligible participating children born between 2000 and 2008 were significantly less likely than WIC-eligible nonparticipating children to ever receive breastfeeding (p &lt, 0.05) or to be breastfed at 6 months (p &lt, 0.05). Among children born between 2009 and 2014, WIC-eligible participating children were no longer less likely to ever receive breastfeeding compared to WIC-eligible nonparticipating children, the gap remained in breastfeeding at 6-months (p &lt, 0.05). The disparities in prevalence of ever breastfed between WIC-eligible participants and nonparticipants have been eliminated since the 2009 WIC revision. More efforts are needed to improve breastfeeding persistence among WIC-participating mother&ndash, infant dyads.

The historical view of β-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis (Mtb) in the presence of a β-lactamase inhibitor. However, most antimycobacterial β-lactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 β-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3’ that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a β-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A β-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent β-lactam. Thus, the antimycobacterial activity of pyrithione-containing cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states.

Whether minority concentration in a neighborhood exposes residents to, or protects them from, health risks has generated burgeoning scholarly interests; yet endogeneity as a result of neighborhood selection largely remains unclear in the literature. This study addresses such endogeneity and simultaneously investigates the roles of co-ethnic density and immigrant enclaves in influencing high blood pressure and high cholesterol level among Latinos, the largest minority group in the United States. Pooled cross-sectional data that included both native and foreign-born Latinos of Puerto Rican, Mexican, and other origins (N = 1563) from the 2006 and 2008 Southeastern Pennsylvania Household Health Survey were linked to census-tract profiles from the 2005-2009 American Community Survey. Results from both multilevel regression and propensity score matching analysis confirmed the deleterious effect of residential co-ethnic density on Latino adults' health risks over and above individual risk factors. We also found selection bias associated with the observed protective effect of immigrant concentration, which is likely a result of residential preference.

Commensals rule the MAITrix Mucosal-associated invariant T (MAIT) cells play an important role in mucosal homeostasis. MAIT cells recognize microbial small molecules presented by the major histocompatibility complex class Ib molecule MR1. MAIT cells are absent in germ-free mice, and the mechanisms by which microbiota control MAIT cell development are unknown (see the Perspective by Oh and Unutmaz). Legoux et al. show that, in mice, development of MAIT cells within the thymus is governed by the bacterial product 5-(2-oxopropylideneamino)-6- d -ribitylaminouracil, which rapidly traffics from the mucosa to the thymus, where it is captured by MR1 and presented to developing MAIT cells. Constantinides et al. report that MAIT cell induction only occurs during a limited, early-life window and requires exposure to defined microbes that produce riboflavin derivatives. Continual interactions between MAIT cells and commensals in the skin modulates tissue repair functions. Together, these papers highlight how the microbiota can direct immune cell development and subsequent function at mucosal sites by secreting compounds that act like self-antigens. Science , this issue p. 494 , p. eaax6624 ; see also p. 419

Innate immune responses that control early Mtb infection are poorly understood, but understanding these responses may inform vaccination and immunotherapy strategies. Innate T cells that respond to conserved bacterial ligands such as mucosal-associated invariant T (MAIT) and γδ T cells are prime candidates to mediate these early innate responses but have not been examined in subjects who have been recently exposed to Mtb. We recruited a cohort living in the same household with an active tuberculosis (TB) case and examined the abundance and functional phenotypes of 3 innate T cell populations reactive to M. tuberculosis: γδ T, invariant NK T (iNKT), and MAIT cells. Both MAIT and γδ T cells from subjects with Mtb exposure display ex vivo phenotypes consistent with recent activation. However, both MAIT and γδ T cell subsets have distinct response profiles, with CD4+ MAIT and γδ T cells accumulating after infection. Examination of exposed but uninfected contacts demonstrates that resistance to initial infection is accompanied by robust MAIT cell CD25 expression and granzyme B production coupled with a depressed CD69 and IFNγ response. Finally, we demonstrate that MAIT cell abundance and function correlate with the abundance of specific gut microbes, suggesting that responses to initial infection may be modulated by the intestinal microbiome.

This study investigated the independent association between neighborhood racial/ethnic diversity and metabolic syndrome among US adults, and focused on how this association differed across individual and neighborhood characteristics (i.e., race/ethnicity, sex, age, urbanity, neighborhood poverty). Objectively-measured biomarker data from 2003 to 2008 National Health and Nutrition Examination Survey were linked to census-tract profiles from 2000 decennial census (N = 10,122). Multilevel random intercept logistic regression models were estimated to examine the contextual effects of tract-level racial/ethnic diversity on individual risks of metabolic syndrome. Overall, more than 20% of the study population were identified as having metabolic syndrome, although the prevalence also varied across demographic subgroups and specific biomarkers. Multilevel analyses showed that increased racial/ethnic diversity within a census tract was associated with decreased likelihood of having metabolic syndrome (OR 0.71, 95% CI 0.52–0.96), particularly among female (OR 0.64; 95% CI 0.43–0.96), young adults (OR 0.60; 95% CI 0.39–0.93), and residents living in urban (OR 0.67; 95% CI 0.48–0.93) or poverty neighborhoods (OR 0.54; 95% CI 0.31–0.95). The findings point to the potential benefits of neighborhood racial/ethnic diversity on individual health risks.

SummaryThis study examines the associations between parental and sibling rural-to-urban migration and blood pressure (BP) of rural left-behind children (LBC) in rural China. Analysis was based on the 2000, 2004, 2006 and 2009 waves of longitudinal data from the China Health and Nutrition Survey, which is an ongoing prospective survey covering nine provinces with an individual-level response rate of 88%. Blood pressure levels were measured by trained examiners at three consecutive times on the same visit and the means of three measurements were used as the final BP values. An ordinal BP measure was then created using a recently validated age–sex-specified distribution for Chinese children and adolescents, distinguishing normal BP, pre-hypertension and hypertension. Random effect modelling was performed. Different migration circumstances play different roles in LBC's BP with mother-only and both-parent migration being particularly detrimental and father-only and sibling-only migration either having no association or a negative association with LBC's BP levels or odds of high BP. In conclusion, the link between family migration and left-behind children's blood pressure is complex, and depends on who is the person out-migrating.

Inhibition of androgen biosynthesis is clinically effective for treating androgen-responsive prostate cancer. Abiraterone is a clinical first-in-class inhibitor of cytochrome P450 17A1 (CYP17A1) required for androgen biosynthesis. However, abiraterone also causes hypertension, hypokalemia, and edema, likely due in part to off-target inhibition of another steroidogenic cytochrome P450, CYP21A2. Abiraterone analogs were designed based on structural evidence that B-ring substituents may favorably interact with polar residues in binding CYP17A1 and sterically clash with residues in the CYP21A2 active site. The best analogs increased selectivity of CYP17A1 inhibition up to 84-fold compared with 6.6-fold for abiraterone. Cocrystallization with CYP17A1 validated the intended new contacts with CYP17A1 active site residues. Docking these analogs into CYP21A2 identified steric clashes that likely underlie decreased binding and CYP21A2 inhibition. Overall, these analogs may offer a clinical advantage in the form of reduced side effects.

Hepatocellular carcinoma (HCC) continues to be a leading cause of cancer related death in the world. Conventional chemotherapeutic agents such as cisplatin (CDDP) have an unsatisfactory efficacy on HCC due to the poor response, severe toxicity and drug resistance. Curcumin (CUR) could improve the chemosensitivity of HCC to chemotherapy drugs by regulating a variety of signaling pathways. Herein, we describe a combination strategy using co-loaded liposomes to effectively deliver and release CDDP and curcumin (CUR) to HCC for overcoming the unsatisfactory clinical outcome of CDDP monotherapy. In the study, CDDP and CUR co-loaded liposomes (CDDP/CUR-Lip) were prepared by a reverse microemulsion and film dispersion method and their average particle size 294.6 ± 14.8 nm with uniform size distribution. In vitro study showed that the nano sized CDDP/CUR-Lip could synchronously release both CDDP and CUR to achieve the synergistic effect against HCC cells based on the optimal ratio (1:8) of both drugs. Compared with free drug or encapsulated mono-drug therapy, CDDP/CUR-Lip demonstrated the higher anti-tumor activity in vitro against HepG2 cells with the IC50 of 0.62 μM. In addition, CDDP/CUR-Lip also increased intracellular ROS level during the HCC cells treatment. Furthermore, compared with single drug formulation, CDDP/CUR-Lip showed the elongated retention time (t1/2 = 2.38 h) and improved antitumor effect in both mouse hepatoma H22 and human HCC HepG2 xenograft models with reduced side effects. In conclusion, CDDP/CUR-Lip provide an attractive and potential strategy to attain synergistic effect of CDDP and CUR for the treatment of HCC.

This study investigates the association between neighborhood racial composition and adult obesity risks by race and gender, and explores whether neighborhood social and built environment mediates the observed protective or detrimental effects of racial composition on obesity risks. Cross-sectional data from the 2006 and 2008 Southeastern Pennsylvania Household Health Survey are merged with census-tract profiles from 2005–2009 American Community Survey and Geographic Information System-based built-environment data. The analytical sample includes 12,730 whites and 4,290 blacks residing in 953 census tracts. Results from multilevel analysis suggest that black concentration is associated with higher obesity risks only for white women, and this association is mediated by lower neighborhood social cohesion and socioeconomic status (SES) in black-concentrated neighborhoods. After controlling for neighborhood SES, black concentration and street connectivity are associated with lower obesity risks for white men. No association between black concentration and obesity is found for blacks. The findings point to the intersections of race and gender in neighborhood effects on obesity risks, and highlight the importance of various aspects of neighborhood social and built environment and their complex roles in obesity prevention by socio-demographic groups.

A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM.

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N 2 ,N 4 - d i be nzyl q uinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.

We previously found that the p97 cofactor, p47, significantly decreased the potency of some ATP-competitive p97 inhibitors such as ML240 [2-(2-amino-1H-benzo[d]imidazol-1-yl)-N-benzyl-8-methoxyquinazolin-4-amine] and ML241 [2-(2H-benzo[b][1,4]oxazin-4(3H)-yl)-N-benzyl-5,6,7,8 tetrahydroquinazolin-4-amine]. In this study, we aimed to evaluate inhibitor potencies against two additional p97 cofactor complexes, p97-p37 and p97-Npl4-Ufd1. We focused on these two cofactor complexes, because the protein sequence of p37 is 50 % identical to that of p47, and the Npl4-Ufd1 heterodimer (NU) is the most-studied p97 cofactor complex. We screened 200 p97 inhibitor analogues for their ability to inhibit the ATPase activity of p97 alone and of p97-p37 and p97-NU complexes. In contrast to the effect of p47, p37 and NU did not significantly change the potencies of most of the compounds. These results highlight differences among p97 cofactors in influencing p97 conformation and effects of inhibitors on p97 complexes, as compared to p97 alone. Continued efforts are needed to advance the development of complex-specific p97 inhibitors.

The formation of drug-protein adducts via metabolic activation and covalent binding may stimulate an immune response or may result in direct cell toxicity. Protein covalent binding is a potentially pivotal step in the development of idiosyncratic adverse drug reactions (IADRs). Trimethoprim (TMP)-sulfamethoxazole (SMX) is a combination antibiotic that commonly causes IADRs. Recent data suggest that the contribution of the TMP component of TMP-SMX to IADRs may be underappreciated. We previously demonstrated that TMP is bioactivated to chemically reactive intermediates that can be trapped in vitro by N-acetyl cysteine (NAC), and we have detected TMP-NAC adducts (i.e., mercapturic acids) in the urine of patients taking TMP-SMX. However, the occurrence and extent of TMP covalent binding to proteins was unknown. To determine the ability of TMP to form protein adducts, we incubated [(14)C]TMP with human liver microsomes in the presence and absence of NADPH. We observed protein covalent binding that was NADPH dependent and increased with incubation time and concentration of both protein and TMP. The estimated covalent binding was 0.8 nmol Eq TMP/mg protein, which is comparable to the level of covalent binding for several other drugs that have been associated with covalent binding-induced toxicity and/or IADRs. NAC and selective inhibitors of CYP2B6 and CYP3A4 significantly reduced TMP covalent binding. These results demonstrate for the first time that TMP bioactivation can lead directly to protein adduct formation, suggesting that TMP has been overlooked as a potential contributor of TMP-SMX IADRs.

Mucosal-associated invariant T (MAIT) cells are an abundant class of innate T cells restricted by the MHC I-related molecule MR1. MAIT cells can recognize bacterially-derived metabolic intermediates from the riboflavin pathway presented by MR1 and are postulated to play a role in innate antibacterial immunity through production of cytokines and direct bacterial killing. MR1 tetramers, typically stabilized by the adduct of 5-amino-6-D-ribitylaminouracil (5-A-RU) and methylglyoxal (MeG), are important tools for the study of MAIT cells. A long-standing problem with 5-A-RU is that it is unstable upon storage. Herein we report an efficient synthetic approach to the HCl salt of this ligand, which has improved stability during storage. We also show that synthetic 5-A-RU•HCl produced by this method may be used in protocols for the stimulation of human MAIT cells and production of both human and mouse MR1 tetramers for MAIT cell identification.

We previously found that p97 ATPase inhibitors 2-(2-amino-1H-benzo[d]imidazol-1-yl)-N-benzyl-8-methoxyquinazolin-4-amine (ML240) and 2-(2H-benzo[b][1,4]oxazin-4(3H)-yl)-N-benzyl-5,6,7,8-tetrahydroquinazolin-4-amine (ML241) specifically target the D2 domain of wild-type p97. In addition, one of the major p97 cofactors, p47, decreases their potencies by ∼50-fold. In contrast, N(2) ,N(4) -dibenzylquinazoline-2,4-diamine (DBeQ) targets both the D1 and D2 domains and shows only a four- to sixfold decrease in potency against the p97-p47 complex. To elucidate structure-activity relationships for the inhibitors, we screened 200 p97 inhibitor analogues for their ability to inhibit the ATPase activity of either or both of the D1 or D2 domains, as well for their effects on p47 potency. The selectivity of 29 of these compounds was further examined by eight-dose titrations. Four compounds showed modest selectivity for inhibiting the ATPase activity of D1. Eleven compounds inhibited D2 with greater potencies, and four showed similar potencies against D1 and D2. p47 decreased the potencies of the majority of the compounds and increased the potencies of five compounds. These results highlight the possibility of developing domain-selective and complex-specific p97 inhibitors in order to further elucidate the physiological roles of p97 and its cofactors.

The p97 AAA (ATPase associated with diverse cellular activities), also called VCP (valosin-containing protein), is an important therapeutic target for cancer and neurodegenerative diseases. p97 forms a hexamer composed of two AAA domains (D1 and D2) that form two stacked rings and an N-terminal domain that binds numerous cofactor proteins. The interplay between the three domains in p97 is complex, and a deeper biochemical understanding is needed in order to design selective p97 inhibitors as therapeutic agents. It is clear that the D2 ATPase domain hydrolyzes ATP in vitro, but whether D1 contributes to ATPase activity is controversial. Here, we use Walker A and B mutants to demonstrate that D1 is capable of hydrolyzing ATP and show for the first time that nucleotide binding in the D2 domain increases the catalytic efficiency (k cat/K m) of D1 ATP hydrolysis 280-fold, by increasing k cat 7-fold and decreasing K m about 40-fold. We further show that an ND1 construct lacking D2 but including the linker between D1 and D2 is catalytically active, resolving a conflict in the literature. Applying enzymatic observations to small-molecule inhibitors, we show that four p97 inhibitors (DBeQ, ML240, ML241, and NMS-873) have differential responses to Walker A and B mutations, to disease-causing IBMPFD mutations, and to the presence of the N domain binding cofactor protein p47. These differential effects provide the first evidence that p97 cofactors and disease mutations can alter p97 inhibitor potency and suggest the possibility of developing context-dependent inhibitors of p97. © 2014 Elsevier Ltd.

In this study we scrutinize prevalence of current smoking and binge drinking among adult US immigrants, and examine whether age at migration predicts these two behaviors and moderates the effect of length of residence. Immigrant groups include those from Latin America/Caribbean, East and South Asia, Sub-Saharan Africa, Europe/Central Asia, and Middle East/North Africa. Multivariate logistic regressions are estimated using cross-sectional data from the New Immigrant Survey (N = 7,397). Results show that patterns of smoking and binge drinking vary by gender and by region of origins. In addition, arriving at age 0–9 are directly associated with higher odds of binge drinking among adult women. Among adult men, age at migration moderates the association between length of residence and substance use. Specifically, length of residence has more detrimental effects for adolescent immigrants (arriving at age 10–18) on smoking, while its detrimental effects are more pronounced for childhood immigrants (arriving at age 0–9) on binge drinking. We interpret our findings within the critical period model in epidemiological research, concluding that adolescence and childhood are critical life stages that are associated with differential effects of length of residence when looking at smoking and binge drinking among immigrant men.

The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (J. Med. Chem. 2012, 55:3319-3330) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye primuline. This study further characterizes a subset of these primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset were potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with Kd’s that reflect their potency in assays. The fluorescent properties of the primuline derivatives both in vitro and in cells are also described. The primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC50 = 5 ± 2 µM). In cells, the most effective primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.