Host ANP32A mediates the assembly of the influenza virus replicase.

Aquatic birds represent a vast reservoir from which new pandemic influenza A viruses can emerge1. Influenza viruses contain a negative-sense segmented RNA genome that is transcribed and replicated by the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein complexes2,3. RNA polymerases of avian influenza A viruses (FluPolA) replicate viral RNA inefficiently in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity4. Host-adaptive mutations, particularly a glutamic-acid-to-lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit, enable avian FluPolA to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here we report cryo-electron microscopy structures of influenza C virus polymerase (FluPolC) in complex with human and chicken ANP32A. In both structures, two FluPolC molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain of ANP32A. The C-terminal low-complexity acidic region of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, suggesting a mechanism for how the adaptive PB2(E627K) mutation enables the replication of viral RNA in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a viral ribonucleoprotein complex.

Structures of influenza A virus RNA polymerase offer insight into viral genome replication.

Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans1,2. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPolA) composed of PB1, PB2 and PA subunits3-5. Although the high-resolution crystal structure of FluPolA of bat influenza A virus has previously been reported6, there are no complete structures available for human and avian FluPolA. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication-which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase7-10-remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPolA from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0-4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPolA forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPolA bound to the cRNA template reveals a binding site for the 3' cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPolA dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPolA dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPolA, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPolA that could be targeted in the development of antiviral drugs.

Van Gogh-like 2 is essential for the architectural patterning of the mammalian biliary tree.

BACKGROUND & AIMS: In the developing liver, bipotent epithelial progenitor cells undergo lineage segregation to form hepatocytes, which constitute the bulk of the liver parenchyma, and biliary epithelial cells (cholangiocytes), which comprise the bile duct (a complex tubular network that is critical for normal liver function). Notch and TGFß signalling promote the formation of a sheet of biliary epithelial cells, the ductal plate, that organises into discontinuous tubular structures. How these structures elongate and connect to form a continuous duct remains undefined. We aimed to define the mechanisms by which the ductal plate transitions from a simple sheet of epithelial cells into a complex and connected bile duct. METHODS: By combining single-cell RNA sequencing of embryonic mouse livers with genetic tools and organoid models we functionally dissected the role of planar cell polarity in duct patterning. RESULTS: We show that the planar cell polarity protein VANGL2 is expressed late in intrahepatic bile duct development and patterns the formation of cell-cell contacts between biliary cells. The patterning of these cell contacts regulates the normal polarisation of the actin cytoskeleton within biliary cells and loss of Vangl2 function results in the abnormal distribution of cortical actin remodelling, leading to the failure of bile duct formation. CONCLUSIONS: Planar cell polarity is a critical step in the post-specification sculpture of the bile duct and is essential for establishing normal tissue architecture. IMPACT AND IMPLICATIONS: Like other branched tissues, such as the lung and kidney, the bile ducts use planar cell polarity signalling to coordinate cell movements; however, how these biochemical signals are linked to ductular patterning remains unclear. Here we show that the core planar cell polarity protein VANGL2 patterns how cell-cell contacts form in the mammalian bile duct and how ductular cells transmit confluent mechanical changes along the length of a duct. This work sheds light on how biological tubes are patterned across mammalian tissues (including within the liver) and will be important in how we promote ductular growth in patients where the duct is mis-patterned or poorly formed.

Semiparametric Allocation of Subjects to Cohort Strata.

BACKGROUND: We illustrate a method for stratum assignment in small cohort studies that avoids modeling assumptions. METHODS: Off-the-shelf software ( rgenoud ) made stratum assignments to minimize a loss function built on within-stratum and population-adjusted Euclidean distances. RESULTS: In 100 trials using simulated data of 300 records with a binary treatment and four dissimilar covariate treatment predictors, minimizing a loss based on Euclidean distance reduced covariate imbalance by a median of 99%. Stratification by propensity score and weighting records by the inverse of their probability of treatment reduced imbalance by 76%-89% and 83%-94%, respectively. Loss minimization applied to a cohort of 361 children undergoing immunotherapy achieved nearly complete elimination of covariate differences for important treatment predictors. CONCLUSION: With the availability of semiparametric stratum-assignment algorithms, analysts can tailor loss functions to meet design goals. Here, a loss function that emphasized covariate balance performed well under limited testing.

Stratified analysis in comparative effectiveness studies that emulate randomized trials.

PURPOSE: For observational cohort studies that employ matching by propensity scores (PS), preliminary stratification by consequential predictors of outcome better emulates stratified randomization and potentially reduces variance and bias through relaxed dependence on modeling assumptions. We assessed the impact of pre-stratification in two real-life examples. For both, prior evidence from placebo-controlled randomized clinical trials (RCTs) suggested small or no risk reduction, but observational analysis suggested protection, presumably the result of confounding bias. STUDY DESIGN AND SETTING: The study populations consisted of Medicare beneficiaries (2014-18) with type 2 diabetes initiating either (i) empagliflozin versus dipeptidyl peptidase-4 inhibitors (DPP-4i) or (ii) empagliflozin versus glucagon-like peptide-1 receptor agonists (GLP-1RA). The outcome was myocardial infarction or stroke. We estimated hazard ratios (HR) and rate differences (RD) after controlling for 143 pre-exposure covariates via 1:1 PS matching after (1) PS estimation in the total cohort (total-cohort PS-matching) and (2) PS estimation separately by baseline cardiovascular disease (stratified PS matching). RESULTS: Stratified PS matching resulted in HRs that exceeded those from total-cohort PS-matching by 13% and 9%, respectively, for the comparisons of empagliflozin to DPP-4i and GLP-1RA. Against both comparators, HRs and RDs after stratified PS matching were closer to the null, with slightly higher variances (2%-3%) than those after total-cohort PS matching. CONCLUSION: Stratified PS matching produced effect estimates closer to the expected trial findings than total-cohort PS matching. The price paid in increased variance was minimal.

Characterization of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification.

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.

Management of Lobular Neoplasia Diagnosed by Core Biopsy.

Lobular neoplasia (LN) involves proliferative changes within the breast lobules. LN is divided into lobular carcinoma in situ (LCIS) and atypical lobular hyperplasia (ALH). LCIS can be further subdivided into three subtypes: classic LCIS, pleomorphic LCIS, and LCIS with necrosis (florid type). Because classic LCIS is now considered as a benign etiology, current guidelines recommend close follow-up with imaging versus surgical excision. The goal of our study was to determine if the diagnosis of classic LN on core needle biopsy (CNB) merits surgical excision. This is a retrospective, observational study conducted at Mount Auburn Hospital, Cambridge, MA, from May 17, 2017, through June 30, 2020. We reviewed the data of breast biopsies conducted at our hospital over this period and included patients who were diagnosed with classic LN (LCIS and/or ALH) and excluded patients having any other atypical lesions on CNB. All known cancer patients were excluded. Of the 2707 CNBs performed during the study period, we identified 68 women who were diagnosed with ALH or LCIS on CNB. CNB was performed for an abnormal mammogram in the majority of patients (60; 88%) while 7(10.3%) had an abnormal breast magnetic resonance imaging study (MRI), and 1 had an abnormal ultrasound (US). A total of 58 patients (85%) underwent excisional biopsy, of which 3 (5.2%) showed malignancy, including 2 cases of DCIS and 1 invasive carcinoma. In addition, there was 1 case (1.7%) with pleomorphic LCIS and 11 cases with ADH (15.5%). The management of LN found on core biopsy is evolving, with some advocating surgical excision and others recommending observation. Our data show a change in diagnosis with excisional biopsy in 13 (22.4%) of patients with 2 cases of DCIS, 1 invasive carcinoma, 1 pleomorphic LCIS, and 9 cases of ADH, diagnosed on excisional biopsy. While ALH and classic LCIS are considered benign, the choice of ongoing surveillance versus excisional biopsy should be made with shared decision making with the patient, with consideration of personal and family history, as well as patient preferences.

The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme.

SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5' capping of viral RNAs. The formation of the 5' 7-methylguanosine (m7G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5' triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5' end of viral RNA via a 5' to 5' triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.

Trends in pulmonary tuberculosis mortality between 1985 and 2018: an observational analysis.

BACKGROUND: Pulmonary tuberculosis (TB) is a major source of global morbidity and mortality. Latent infection has enabled it to spread to a quarter of the world's population. The late 1980s and early 1990s saw an increase in the number of TB cases related to the HIV epidemic, and the spread of multidrug-resistant TB. Few studies have reported pulmonary TB mortality trends. Our study reports and compares trends in pulmonary TB mortality. METHODS: We utilized the World Health Organization (WHO) mortality database from 1985 through 2018 to analyze TB mortality using the International Classification of Diseases-10 codes. Based on the availability and quality of data, we investigated 33 countries including two countries from the Americas; 28 countries from Europe; and 3 countries from the Western Pacific region. Mortality rates were dichotomized by sex. We computed age-standardized death rates per 100,000 population using the world standard population. Time trends were investigated using joinpoint regression analysis. RESULTS: We observed a uniform decrease in mortality in all countries across the study period except the Republic of Moldova, which showed an increase in female mortality (+ 0.12 per 100,000 population). Among all countries, Lithuania had the greatest reduction in male mortality (-12) between 1993-2018, and Hungary had the greatest reduction in female mortality (-1.57) between 1985-2017. For males, Slovenia had the most rapid recent declining trend with an estimated annual percentage change (EAPC) of -47% (2003-2016), whereas Croatia showed the fastest increase (EAPC, + 25.0% [2015-2017]). For females, New Zealand had the most rapid declining trend (EAPC, -47.2% [1985-2015]), whereas Croatia showed a rapid increase (EAPC, + 24.9% [2014-2017]). CONCLUSIONS: Pulmonary TB mortality is disproportionately higher among Central and Eastern European countries. This communicable disease cannot be eliminated from any one region without a global approach. Priority action areas include ensuring early diagnosis and successful treatment to the most vulnerable groups such as people of foreign origin from countries with a high burden of TB and incarcerated population. Incomplete reporting of TB-related epidemiological data to WHO excluded high-burden countries and limited our study to 33 countries only. Improvement in reporting is crucial to accurately identify changes in epidemiology, the effect of new treatments, and management approaches.

Using Icon Arrays to Communicate Gambling Information Reduces the Appeal of Scratch Card Games.

Past work has demonstrated that presenting statistical information in a foreground-background icon array can improve risk understanding, reduce decision-making biases, and decrease the salience of low-probability risks. In the present study, we assess whether presenting readily available gambling information within a foreground-background icon array influences individuals' gambling-related judgments (e.g., their perceived likelihood of winning a prize). Across two experiments (N = 1151), we find that using icon arrays to present gambling information reduces the appeal of scratch card games. That is, participants presented with gambling information in a foreground-background icon array, as opposed to a non-graphical numerical format, reported feeling less likely to win a prize, less excitement to play, and less urge to gamble on a scratch card game presented in a hypothetical gambling task. Overall, we conclude that presenting gambling information in an icon array format represents a simple yet promising tool for correcting gamblers' often overly-optimistic perceptions and reducing the appeal of negative expected value scratch card games.

Push Outcomes Bias Perceptions of Scratch Card Games.

In the domain of scratch card gambling, "pushes" refer to outcomes in which a prize is won that is equal to the cost of a scratch card game. Despite resulting in no net monetary gain, these outcomes are categorized as wins by lottery operators, effectively inflating published scratch card information (e.g., posted odds of winning). Additionally, the experience of obtaining a push shares similarities (e.g., the revealing of matching symbols) with the experience of obtaining a win and thus may be experienced similarly to wins by gamblers. Across four studies (N = 1502), we examined the impact of push outcomes on participants' perceptions of scratch card games. In Studies 1 and 2, participants reported feeling more likely to win, more excitement to play, and a stronger urge to gamble when presented with a scratch card that categorized push outcomes as wins compared to when presented a scratch card that did not categorize these outcomes as wins. In Study 3, participants experiencing a push outcome prior to a loss reported feeling more likely to win compared to those not experiencing a push outcome yet experiencing the same net monetary loss. In Study 4, push outcomes were found to elicit more excitement and a stronger urge to gamble compared to losses but less excitement and a weaker urge to gamble compared to wins. Overall, the present investigation suggests that push outcomes, a prevalent feature of scratch card games, can bias gambling-related judgments and increase the appeal of scratch card games.

The acute effects of azithromycin use on cardiovascular mortality as compared with amoxicillin-clavulanate in US Veterans.

PURPOSE: Azithromycin is a common first-line antibiotic for respiratory infection; however, there is conflicting evidence regarding risk of cardiovascular death. We assessed cardiovascular and noncardiovascular mortality associated with azithromycin versus amoxicillin-clavulanate among US Veterans treated for nonear-nose-throat respiratory infection ("respiratory") or ear-nose-throat infection indication. METHODS: Electronic health record data from the US Veterans Health Administration database were used to identify Veterans (30-74 years) with outpatient dispensings of oral azithromycin versus amoxicillin-clavulanate for respiratory or ear-nose-throat infection (January 01, 2000-December 31, 2014). Outcomes assessed were risk of cardiovascular death and noncardiovascular death within 1-5 and 6-10 days postdispensing. Inverse probability of treatment-weighted proportional hazards models and binomial regression models were used to estimate hazard ratios (HRs) and compute risk differences (RD) per million courses of therapy. Cardiac death (subset of cardiovascular death) was assessed in sensitivity analyses. RESULTS: There were 629 345 azithromycin and 168 429 amoxicillin-clavulanate dispensings for respiratory indications, 143 783 azithromycin, and 203 142 amoxicillin-clavulanate dispensings for ear-nose-throat indications. For respiratory indications, azithromycin was not associated with a significantly different risk of cardiovascular death versus amoxicillin-clavulanate within 1-5 days postdispensing (HR [95% confidence interval (CI)]: 1.12 [0.63, 2.00]; RD [95% CI]: 11 [-43, 64] deaths/million courses of therapy). No elevated risk for azithromycin was found for ear-nose-throat indications. Pooled results for both indications via meta-analysis showed no association between antibiotics and cardiovascular mortality. There was no significant difference in risk of noncardiovascular or cardiac death between antibiotics postdispensing. CONCLUSION: Azithromycin was not associated with elevated risk of cardiovascular or noncardiovascular death versus amoxicillin-clavulanate among US Veterans.

Mutation of an Influenza Virus Polymerase 3' RNA Promoter Binding Site Inhibits Transcription Elongation.

Influenza viruses encode a viral RNA-dependent RNA polymerase (FluPol), which is responsible for transcribing and replicating the negative-sense viral RNA (vRNA) genome. FluPol transcribes vRNA using a host-capped mRNA primer and replicates it by synthesizing a positive-sense cRNA intermediate, which is copied back into vRNA. To carry out these functions, FluPol interacts with vRNA and cRNA using conserved promoter elements at the 5' and 3' termini. Recent structural studies have identified a new surface binding site for the 3' vRNA and cRNA promoters on FluPol, referred to as the mode B site. However, the role of this binding site in FluPol function is unknown. In this study, we used a combination of cell-based and biochemical assays to show that the mode B site is important for both viral genome transcription and replication in influenza A virus. Furthermore, we show that the mode B site is not needed for initiating transcription in vitro but is required to synthesize a full-length product. This is consistent with a model in which the 3' terminus of the vRNA template binds in the mode B site during elongation. Our data provide the first functional insights into the role of the mode B site on FluPol, which advances our understanding of FluPol function and influenza virus replication.IMPORTANCE Influenza viruses are responsible for up to 650,000 deaths per year through seasonal epidemics, and pandemics have caused tens of millions of deaths in the past. Most current therapeutics suffer from widespread resistance, creating a need for new drug targets against influenza virus. The virus encodes an RNA-dependent RNA polymerase, which replicates and transcribes the vRNA genome. The polymerase interacts with vRNA and the complementary replicative intermediate cRNA using several specific binding sites; however, the functions associated with these binding sites remain unknown. Here, we functionally characterize a binding site for the 3' vRNA and cRNA promoters. Our data offer insight into the mechanism of viral genome transcription by the influenza virus polymerase and may be applicable to other related viruses.

Long-term risk of hepatocellular carcinoma mortality in 23220 hospitalized patients treated with micafungin or other parenteral antifungals.

BACKGROUND: Liver tumours observed in rats exposed to micafungin led to a black box warning upon approval in Europe in 2008. Micafungin's risk for liver carcinogenicity in humans has not been investigated. We sought to describe the risk of fatal hepatocellular carcinoma (HCC) among persons who received micafungin and other parenteral antifungals (PAFs) with up to 12 years of follow-up. METHODS: We assembled a US multicentre cohort of hospitalized patients who received micafungin or other PAFs between 2005 and 2012. We used propensity score (PS) matching on patient characteristics from electronic medical records to compare rates of HCC mortality identified through the National Death Index though to the end of December 2016. We computed HRs and 95% CIs. RESULTS: A total of 40110 patients who received a PAF were identified; 6903 micafungin recipients (87% of those identified) were successfully matched to 16317 comparator PAF users. Ten incident HCC deaths, one in the micafungin-exposed group and nine among comparator PAF users, occurred in 71285 person-years of follow-up. The HCC mortality rate was 0.05 per 1000 person-years in micafungin patients and 0.17 per 1000 person-years in comparator PAF patients. The PS-matched HR for micafungin versus comparator PAF was 0.29 (95% CI 0.04-2.24). CONCLUSIONS: Both micafungin and comparator PAFs were associated with HCC mortality rates of <0.2 per 1000 person-years. Given the very low event rates, any potential risk for HCC should not play a role in clinical decisions regarding treatment with micafungin or other PAFs investigated in this study.

Configuration of the extra-renal venous system in relation to the left renal vein: A cadaveric study and new proposed classification.

The advent of laparoscopic live-donor nephrectomy for renal transplantation has prompted the need to define the precise anatomical relations of the left renal vein (LRV) and its tributaries. The left kidney is preferred as the greater length of the LRV facilitates implantation in the recipient. While previous studies have described variations in the LRV system, the connections between the left ascending lumbar vein (LALV) and LRV tributaries have been less well-defined. This study aims to further characterise the LALV and proposes a novel classification for its relation to other veins. Dissection of the LRV system, including the left suprarenal vein (LSV), left gonadal vein (LGV) and LALV, was performed in 38 cadavers. Their drainage points into the LRV were recorded, and measurements taken of the distances from these points to the junction of the LRV and inferior vena cava (IVC). The position of the LRV in relation to the aorta was anterior in 35 cases (92%), entirely posterior in 1 case (3%), and circumaortic in 2 cases (5%). Duplication of the LSV and LGV occurred in 6 (16%) and 10 (27%) cases respectively. A direct posterior connection between the LALV and LRV was identified in 32 (86%) cases. The drainage point of the LALV into the LRV lay between the IVC and LGV in 8 (25%) cases. In 20 cases (63%), the drainage points of the LALV and LGV were equidistant from the IVC; and in 5 cases (16%), those of the LALV and posterior branch of the LRV were equidistant from the IVC. In these two groups, the vessels shared a confluent trunk in 10 and 4 cases respectively. In 3 cases, connections were observed between all three vessels (LALV, LGV and posterior branch of LRV). No confluence trunk was shared by the LALV and LSV. These results confirm the high incidence of communicating LALVs, which represent a potentially troublesome source of operative bleeding if unrecognised. Confluent venous trunks may also present difficulties during vessel ligation prior to nephrectomy. It is suggested that a novel classification of the relation of the LALV based on these findings may assist in surgical planning and reduce complications.

Foreign Language does not Affect Gambling-Related Judgments.

Previous work has demonstrated that peoples' gambling-related judgments (e.g., perceived likelihood of winning) are often biased by non-diagnostic unclaimed prize information (i.e., the number of prizes still available to be won) resulting in non-optimal scratch card preferences. Another line of research suggests that people make less biased decisions (e.g., are less affected by the framing of a gamble) when using a foreign language. In the current study, we investigated whether using a foreign language (as opposed to one's native language) reduced the biasing effects of unclaimed prize information and consequently led to more optimal scratch card preferences. Across three experiments (N = 409), we found that people were equally biased by unclaimed prize information regardless of whether they completed our scratch card gambling task in their native (Polish) or foreign (English) language. In conclusion, it appears that using a foreign language does not help people be less biased in utilizing gambling-related information, and consequently does not lead to more optimal scratch card preferences.

Structural and Functional Characterization of Dynamic Oligomerization in Burkholderia cenocepacia HMG-CoA Reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), in most organisms, catalyzes the four-electron reduction of the thioester (S)-HMG-CoA to the primary alcohol (R)-mevalonate, utilizing NADPH as the hydride donor. In some organisms, including the opportunistic lung pathogen Burkholderia cenocepacia, it catalyzes the reverse reaction, utilizing NAD+ as a hydride acceptor in the oxidation of mevalonate. B. cenocepacia HMGR has been previously shown to exist as an ensemble of multiple non-additive oligomeric states, each with different levels of enzymatic activity, suggesting that the enzyme exhibits characteristics of the morpheein model of allostery. We have characterized a number of factors, including pH, substrate concentration, and enzyme concentration, that modulate the structural transitions that influence the interconversion among the multiple oligomers. We have also determined the crystal structure of B. cenocepacia HMGR in the hexameric state bound to coenzyme A and ADP. This hexameric assembly provides important clues about how the transition among oligomers might occur, and why B. cenocepacia HMGR, unique among characterized HMGRs, exhibits morpheein-like behavior.

Graphical Depiction of Statistical Information Improves Gambling-Related Judgments.

The domain of gambling is rife with both diagnostic and non-diagnostic information. Previous studies examining scratch card gambling have demonstrated that people are often biased by intuitively appealing, yet non-diagnostic information (i.e., unclaimed prize information). The current study investigated how varying the presentation format of a diagnostic piece of information (i.e., payback percentage) could influence participants' use of this information when in conflict with unclaimed prize information. We hypothesized that when payback percentage information was presented in a graphical, as opposed to a numerical format, participants would be better at ignoring unclaimed prize information and correspondingly have their preferences become congruent with the true value of the presented scratch cards. In Experiment 1 (N = 201), with payback percentage presented in a numerical format, participants displayed a non-optimal preference for cards with greater numbers of unclaimed prizes and lower payback percentages. This preference was reversed in Experiment 2 (N = 201) when payback percentage was presented in a graphical format. In conclusion, the results of the current study demonstrate how judgments in a scratch card gambling domain can be improved by simply changing the presentation format of a single piece of information.

Structural and Biochemical Characterization of Cinnamoyl-CoA Reductases.

Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a strategic plant for bioenergy production, were deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Although SbCCR1 displayed higher affinity for caffeoyl-CoA or p-coumaroyl-CoA than for feruloyl-CoA, the enzyme showed significantly higher activity for the latter substrate. Through molecular docking and comparisons between the crystal structures of the Vitis vinifera dihydroflavonol reductase and SbCCR1, residues threonine-154 and tyrosine-310 were pinpointed as being involved in binding CoA-conjugated phenylpropanoids. Threonine-154 of SbCCR1 and other CCRs likely confers strong substrate specificity for feruloyl-CoA over other cinnamoyl-CoA thioesters, and the T154Y mutation in SbCCR1 led to broader substrate specificity and faster turnover. Through data mining using our structural and biochemical information, four additional putative CCR genes were discovered from sorghum genomic data. One of these, SbCCR2, displayed greater activity toward p-coumaroyl-CoA than did SbCCR1, which could imply a role in the synthesis of defense-related lignin. Taken together, these findings provide knowledge about critical residues and substrate preference among CCRs and provide, to our knowledge, the first three-dimensional structure information for a CCR from a monocot species.

The Enzyme Activity and Substrate Specificity of Two Major Cinnamyl Alcohol Dehydrogenases in Sorghum (Sorghum bicolor), SbCAD2 and SbCAD4.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis, reducing sinapaldehyde, coniferaldehyde, and p-coumaraldehyde to their corresponding alcohols in an NADPH-dependent manner. Because of its terminal location in monolignol biosynthesis, the variation in substrate specificity and activity of CAD can result in significant changes in overall composition and amount of lignin. Our in-depth characterization of two major CAD isoforms, SbCAD2 (Brown midrib 6 [bmr6]) and SbCAD4, in lignifying tissues of sorghum (Sorghum bicolor), a strategic plant for generating renewable chemicals and fuels, indicates their similarity in both structure and activity to Arabidopsis (Arabidopsis thaliana) CAD5 and Populus tremuloides sinapyl alcohol dehydrogenase, respectively. This first crystal structure of a monocot CAD combined with enzyme kinetic data and a catalytic model supported by site-directed mutagenesis allows full comparison with dicot CADs and elucidates the potential signature sequence for their substrate specificity and activity. The L119W/G301F-SbCAD4 double mutant displayed its substrate preference in the order coniferaldehyde > p-coumaraldehyde > sinapaldehyde, with higher catalytic efficiency than that of both wild-type SbCAD4 and SbCAD2. As SbCAD4 is the only major CAD isoform in bmr6 mutants, replacing SbCAD4 with L119W/G301F-SbCAD4 in bmr6 plants could produce a phenotype that is more amenable to biomass processing.