Binding of Per- and Polyfluoroalkyl Substances to ß-Lactoglobulin from Bovine Milk.

Per- and polyfluoroalkyl substances (PFAS) are known for their high environmental persistence and potential toxicity. The presence of PFAS has been reported in many dairy products. However, the mechanisms underlying the accumulation of PFAS in these products remain unclear. Here, we used native mass spectrometry and molecular dynamics simulations to probe the interactions between 19 PFAS of environmental concern and two isoforms of the major bovine whey protein ß-lactoglobulin (ß-LG). We observed that six of these PFAS bound to both protein isoforms with low- to mid-micromolar dissociation constants. Based on quantitative, competitive binding experiments with endogenous ligands, PFAS can bind orthosterically and preferentially to ß-LG's hydrophobic ligand-binding calyx. ß-Cyclodextrin can also suppress binding of PFAS to ß-LG owing to the ability of ß-cyclodextrin to directly sequester PFAS from solution. This research sheds light on PFAS-ß-LG binding, suggesting that such interactions could impact lipid-fatty acid transport in bovine mammary glands at high PFAS concentrations. Furthermore, our results highlight the potential use of ß-cyclodextrin in mitigating PFAS binding, providing insights toward the development of strategies to reduce PFAS accumulation in dairy products and other biological systems.

Inhibition of pyruvate dehydrogenase accelerates anaerobic glycolysis under postmortem simulating conditions.

This research aimed to explore the potential influence of mitochondria on the rate of anaerobic glycolysis. We hypothesized that mitochondria could reduce the rate of anaerobic glycolysis and pH decline by metabolizing a portion of glycolytic pyruvate. We utilized an in vitro model and incorporated CPI-613 and Avidin to inhibit pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), respectively. Four treatments were tested: 400 µM CPI-613, 1.5 U/ml Avidin, 400 µM CPI-613 + 1.5 U/ml Avidin, or control. Glycolytic metabolites and pH of the in vitro model were evaluated throughout a 1440-min incubation period. CPI-613-containing treatments, with or without Avidin, decreased pH levels and increased glycogen degradation and lactate accumulation compared to the control and Avidin treatments (P < 0.05), indicating increased glycolytic flux. In a different experiment, two treatments, 400 µM CPI-613 or control, were employed to track the fates of pyruvate using [13C6]glucose. CPI-613 reduced the contribution of glucose carbon to tricarboxylic acid cycle intermediates compared to control (P < 0.05). To test whether the acceleration of acidification in reactions containing CPI-613 was due to an increase in the activity of key enzymes of glycogenolysis and glycolysis, we evaluated the activities of glycogen phosphorylase, phosphofructokinase, and pyruvate kinase in the presence or absence of 400 µM CPI-613. The CPI-613 treatment did not elicit an alteration in the activity of these three enzymes. These findings indicate that inhibiting PDH increases the rate of anaerobic glycolysis and pH decline, suggesting that mitochondria are potential regulators of postmortem metabolism.

Molecular Geometries and Vibrational Contributions to Reaction Thermochemistry Are Surprisingly Insensitive to the Choice of Basis Sets.

Calculation of molecular geometries and harmonic vibrational frequencies are pre-requisites for thermochemistry calculations. Contrary to conventional wisdom, this paper demonstrates that quantum chemical predictions of the thermochemistry of many gas and solution phase chemical reactions appear to be very insensitive to the choice of basis sets. For a large test set of 80 diverse organic and transition-metal-containing reactions, variations in reaction free energy based on geometries and frequencies calculated using a variety of double and triple-zeta basis sets from the Pople, Jensen, Ahlrichs, and Dunning families are typically less than 4 kJ mol-1, especially when the quasiharmonic oscillator correction is applied to mitigate the effects of low-frequency modes. Our analysis indicates that for many organic molecules and their transition states, high-level revDSD-PBEP86-D4 and DLPNO-CCSD(T)/(aug-)cc-pVTZ single-point energies usually vary by less than 2 kJ mol-1 on density functional theory geometries optimized using basis sets ranging from 6-31+G(d) to aug-pcseg-2 and aug-cc-pVTZ. In cases where these single-point energies vary significantly, indicating sensitivity of molecular geometries to the choice of basis set, there is often substantial cancellation of errors when the reaction energy or barrier is calculated. The study concludes that the choice of basis set for molecular geometry and frequencies, particularly those considered in this study, is not critical for the accuracy of thermochemistry calculations in the gas or solution phase.

MM/GBSA prediction of relative binding affinities of carbonic anhydrase inhibitors: effect of atomic charges and comparison with Autodock4Zn.

Carbonic anhydrase is an attractive drug target for the treatment of many diseases. This paper examines the ability of end-state MM/GBSA methods to rank inhibitors of carbonic anhydrase in terms of their binding affinities. The MM/GBSA binding energies were evaluated using different atomic charge schemes (Mulliken, ESP and NPA) at different levels of theories, including Hartree-Fock, B3LYP-D3(BJ), and M06-2X with the 6-31G(d,p) basis set. For a large test set of 32 diverse inhibitors, the use of B3LYP-D3(BJ) ESP atomic charges yielded the strongest correlation with experiment (R2 = 0.77). The use of the recently enhanced Autodock Vina and zinc optimised AD4Zn force field also predicted ligand binding affinities with moderately strong correlation (R2 = 0.64) at significantly lower computational cost. However, the docked poses deviate significantly from crystal structures. Overall, this study demonstrates the applicability of docking to estimate ligand binding affinities for a diverse range of CA inhibitors, and indicates that more theoretically robust MM/GBSA simulations show promise for improving the accuracy of predicted binding affinities, as long as a validated set of parameters is used.

Predicting Solvent Effects on SN2 Reaction Rates: Comparison of QM/MM, Implicit, and MM Explicit Solvent Models.

Solvents are one of the key variables in the optimization of a synthesis yield or properties of a synthesis product. In this paper, contemporary solvent models are applied to predict the rates of SN2 reactions in a range of aqueous and non-aqueous solvents. High-level CCSD(T)/CBS//M06-2X/6-31+G(d) gas phase energies were combined with solvation free energies from SMD, SM12, and ADF-COSMO-RS continuum solvent models, as well as molecular mechanics (MM) explicit solvent models with different atomic charge schemes to predict the rate constants of three SN2 reactions in eight protic and aprotic solvents. It is revealed that the prediction of rate constants in organic solvents is not necessarily less challenging than in water and popular solvent models struggle to predict their rate constants to within 3 log units of experimental values. Among the continuum solvent models, the ADF-COSMO-RS model performed the best in predicting absolute rate contants while the SM12 model was best at predicting relative rate constants with an average accuracy of about 1.5 and 0.8 log units, respectively. The use of computationally more demanding MM explicit solvent models did not translate to improvements in absolute rate constants but was quite effective at predicting relative rate constants due to systematic error cancellation. Free energy barriers obtained from umbrella sampling with explicit solvent QM/MM simulations led to excellent agreement with experimental values, provided that a validated level of theory is used to treat the QM region.

Spectro 1-A Potential Spectrophotometer for Measuring Color and Myoglobin Forms in Beef.

The objective of this study was to compare the Color Muse Spectro 1 sensor to the HunterLab MiniScan XE Plus spectrophotometer for evaluating beef color. Color coordinates (lightness (L*), redness (a*), yellowness (b*), chroma (C*), and hue (h*)), myoglobin redox forms (metmyoglobin (MMb), deoxymyoglobin (DMb), and oxymyoglobin (OMb)), and metmyoglobin reducing ability (MRA) were measured on beef steaks over a 5-days storage period. The results indicated that L*, b*, C*, MMb%, OMb%, and MRA% values obtained with Spectro 1 were comparable to those of MiniScan. However, Spectro 1 values for a* were overestimated compared to MiniScan (p < 0.05), whereas those for h* and DMb% were underestimated (p < 0.05). Regardless, Spectro 1 had the capability to detect changes in color coordinates, myoglobin forms, and MRA throughout the storage period. Bland−Altman plots demonstrated that L*, b*, and C* are interchangeable between the two instruments, but it was not the case for a*, h*, myoglobin forms, and MRA. Color coordinates measured by Spectro 1 exhibited excellent stability over time, evidenced by the low total color difference (ΔE*ab) values. Collectively, these findings indicate that Spectro 1 is a potential alternative spectrophotometer for studying meat color and myoglobin redox forms.

Cattle breed type and anabolic implants impact calpastatin expression and abundance of mRNA associated with protein turnover in the longissimus thoracis of feedlot steers.

Two methods that the beef cattle industry can use to improve efficiency, sustainability, and economic viability are growth promotants and crossbreeding cattle of different breed types. In the United States, over 90% of cattle receive an anabolic implant at some point during production resulting in an overall increase in skeletal muscle growth. Recent research suggests that the two main cattle breed types, Bos indicus and Bos taurus, respond differently to anabolic implants. The objective of this study was to characterize changes that occur in skeletal muscle following implanting in Bos indicus influenced steers or Bos taurus steers. Twenty steers were stratified by initial weight in a 2 × 2 factorial design examining two different breeds: Angus (AN; n = 10) or Santa Gertrudis influenced (SG; n = 10), and two implant strategies: no implant (CON; n = 10) or a combined implant containing 120 mg TBA and 24 mg E2 (IMP; n = 10; Revalor-S, Merck Animal Health). Skeletal muscle biopsies were taken from the longissimus thoracis (LT) 2 and 10 d post-implantation. The mRNA abundance of 24 genes associated with skeletal muscle growth were examined, as well as the protein expression of µ-calpain and calpastatin. Succinate dehydrogenase mRNA abundance was impacted (P = 0.05) by a breed × treatment interaction 2 d post-implanting, with SG-CON having a greater increased abundance than all other steers. A tendency for a breed × treatment interaction was observed for calpain-6 mRNA (P = 0.07), with SG-CON having greater abundance than AN-CON and SG-IMP. Additionally, calpastatin protein expression was altered (P = 0.01) by a breed × treatment interaction, with SG-CON and SG-IMP steers having increased expression (P = 0.01) compared with AN-CON steers. At 2 d post-implanting, a breed × treatment interaction was observed with SG-CON steers having greater (P = 0.05) mRNA abundance of mitogen-activated protein kinase compared with AN-CON steers. Furthermore, breed affected (P = 0.05) calpastatin abundance with AN steers having increased (P = 0.05) abundance 2 d post-implanting compared with SG steers. Meanwhile, implants tended to affect (P = 0.09) muscle RING finger protein-1 mRNA abundance, with CON steers having increased (P = 0.09) abundance compared with that of IMP steers. These findings suggest that cattle breed type and anabolic implants impact calpastatin expression and mRNA abundance associated with protein turnover in the LT of feedlot steers 2 and 10 d post-implantation.

Two methods that the beef cattle industry can use to potentially improve efficiency, sustainability, and economic viability are growth promotants and crossbreeding cattle of different breed types. In the United States, over 90% of cattle receive at least one anabolic implant during the production cycle resulting in improvements in production and overall economic and environmental sustainability. Research suggests that the two main cattle breed types, Bos indicus and Bos taurus, respond differently to different anabolic implant strategies. The objective of this study was to characterize changes that occur in the skeletal muscle following implanting in Bos indicus influenced animals and Bos taurus animals. This research measured mRNA abundance of 24 genes associated with skeletal muscle growth, and protein expression of calpain-1 and calpastatin. The findings of this research suggest that anabolic implants and cattle breed type interact to cause changes in mRNA abundance in the longissimus thoracis that are related to protein turnover of skeletal muscle. Furthermore, calpastatin protein abundance was also altered by this breed × treatment interaction. This research demonstrates that anabolic implants cause molecular changes in skeletal muscle of feedlot steers, with some of these changes being breed dependent.

A community partnership to evaluate the feasibility of addressing food insecurity among adult patients in an urban healthcare system.

BACKGROUND: Food insecurity (FI) is a significant public health problem. Possible sequelae of prolonged food insecurity include kidney disease, obesity, and diabetes. Our objective was to assess the feasibility of a partnership between Henry Ford Health System (HFHS) and Gleaners Community Foodbank of Southeastern Michigan to implement and evaluate a food supplementation intervention initiated in a hospital outpatient clinic setting. METHODS: We established a protocol for using the Hunger Vital Signs to screen HFHS internal medicine patients for food insecurity and established the data sharing infrastructure and agreements necessary for an HFHS-Gleaners partnership that would allow home delivery of food to consenting patients. We evaluated the food supplementation program using a quasi-experimental design and constructing a historical comparison group using the electronic medical record. Patients identified as food insecure through screening were enrolled in the program and received food supplementation twice per month for a total of 12 months, mostly by home delivery. The feasibility outcomes included successful clinic-based screening and enrollment and successful food delivery to consenting patients. Our evaluation compared healthcare utilization between the intervention and historical comparison group during a 12-month observation period using a difference-in-differences (DID) analysis. RESULTS: Of 1691 patients screened, 353 patients (20.9%) met the criteria for FI, of which 340/353 (96.3%) consented, and 256/340 (75.3%) were matched and had data sufficient for analysis. Food deliveries were successfully made to 89.9% of participant households. At follow-up, the intervention group showed greater reductions in emergency department visits than the comparison group, -41.5% and -25.3% reduction, respectively. Similar results were observed for hospitalizations, -55.9% and -17.6% reduction for intervention and control groups, respectively. DID regression analysis also showed lower trends in ED visits and hospitalizations for the intervention group compared to the comparison group. CONCLUSIONS: Results suggest that community-health system partnerships to address patient-reported food insecurity are feasible and potentially could reduce healthcare utilization in these patients. A larger, randomized trial may be the next step in fully evaluating this intervention, perhaps with more outcomes (e.g., medication adherence), and additional covariates (e.g., housing insecurity and financial strain).

Food insecurity in Detroit: exploring the relationship between patient-reported food insecurity and proximity to healthful grocery stores.

OBJECTIVE: The objective of the current study was to determine if patients of a large health care system in Detroit who self-identify as food insecure live further away from healthy grocery stores compared with food secure patients. Second, we explored whether food insecurity and distance to healthy grocery stores are related to ecological measures of vehicle availability in the area of residence. DESIGN: A secondary data analysis that uses baseline data from a pilot intervention/feasibility study. SETTING: Detroit, Michigan, USA. PARTICIPANTS: Patients of Henry Ford Health System were screened for food insecurity to determine eligibility for a pilot intervention/feasibility study (i.e. Henry's Groceries for Health), conducted through a collaboration with Gleaners Community Foodbank of Southeastern Michigan. Only patients residing in Detroit city limits (including Highland Park and Hamtramck) were included in the secondary analysis. Of the 1,100 patients included in the analysis, 336 (31 %) were food insecure. RESULTS: After accounting for socio-demographic factors associated with food insecurity, we did not find evidence that food insecure patients lived further away from healthier grocery stores, nor was this modified by ecological measures of vehicle access. However, some neighbourhoods were identified as having a significantly higher risk of food insecurity. CONCLUSIONS: Food insecure patients in Detroit are perhaps limited by social and political determinants and not their immediate neighbourhood geography or physical access to healthy grocery stores. Future research should explore the complexity in linkages between household socio-economic factors, socio-cultural dynamics and the neighbourhood food environment.

Ultrasonication of beef improves calpain-1 autolysis and caspase-3 activity by elevating cytosolic calcium and inducing mitochondrial dysfunction.

The objective of this study was to investigate if ultrasonication of bovine longissimus thoracis et lumborum (LTL) steaks increases calpain-1 and caspase-3 activities, and if so, to explore the underlying mechanisms that trigger their activation. Post-rigor bovine LTL steaks were subjected to ultrasonication at 40 kHz and 12 W/cm2 for 40 min and subsequently aged for 14 d at 4 °C. Ultrasonication improved beef tenderness (P < 0.05) without negatively impacting pH, color, or cook loss (P > 0.05). Improved tenderness in the ultrasonicated steaks was associated with greater degradation of titin, desmin, troponin-T, and calpastatin and increased calpain-1 autolysis and caspase-3 activity (P < 0.05). In addition, ultrasonicated steaks had greater levels of cytosolic calcium and reactive oxygen species and lower mitochondrial oxygen consumption rate (P < 0.05). These data indicate that improved beef tenderness following ultrasonication is, in part, a function of increased calpain-1 and caspase-3 activities, potentially by elevating cytosolic calcium and inducing mitochondrial dysfunction, respectively.

Relationship between behavioral inhibition and approach motivation systems (BIS/BAS) and intrinsic brain network connectivity in adult cannabis users.

Dampened behavioral inhibition and overactive behavioral approach motivation systems (i.e. BIS/BAS) are associated with cannabis use disorder (CUD), although the underlying neural mechanisms of these alterations have not yet been examined. The brain's executive control network (ECN) plays a role in decision-making and is associated with BIS/BAS. In this study, we tested the hypothesis that altered ECN resting-state functional connectivity (rsFC) underlies dysfunctional behavioral inhibition and approach motivation in cannabis users. To that end, we collected resting-state functional magnetic resonance imaging scans in 86 cannabis using adults and 59 non-using adults to examine group differences in the relationship between ECN rsFC and BIS/BAS. Our results showed that BIS was positively correlated with left ECN rsFC in cannabis users, while it was positively correlated with right ECN rsFC in non-users. There was a trend-level moderation effect of group on the association between BIS/BAS and ECN rsFC, showing a weaker association in BIS/BAS and ECN rsFC in cannabis users compared to non-users. An exploratory mediation analysis found that the severity of CUD mediated the relationship between users' BIS scores and left ECN rsFC. These findings suggest that cannabis use may lead to dysregulation in typical ECN functional organization related to BIS/BAS.

Residual Effects of Cannabis Use on Effort-Based Decision-Making.

OBJECTIVE: Acute Δ9-tetrahydrocannabinol (THC) administration in humans (Lawn etal., 2016) and rats (Silveira, Adams, Morena, Hill, & Winstanley, 2016) has been associated with decreased effort allocation that may explain amotivation during acute cannabis intoxication. To date, however, whether residual effects of cannabis use on effort-based decision-making are present and observable in humans have not yet been determined. The goal of this study was to test whether prolonged cannabis use has residual effects on effort-based decision-making in 24-hr abstinent cannabis using adults. METHOD: We evaluated performance on the Effort Expenditure for Reward Task (EEfRT) in 41 adult cannabis users (mean age = 24.63 years, 21 males) and 45 nonusers (mean age = 23.90 years, 19 males). A mixed 2x3x3 ANOVA with age as a covariate was performed to examine the effect of group, probability of winning, and reward amount on EEfRT performance. EEfRT performance was operationalized as % of trials for which the hard (vs. easy) condition was chosen. Pearson's correlations were conducted to test the relationship between EEfRT performance and measures of cannabis use, anhedonia and motivation. RESULTS: We found that cannabis users selected hard trials significantly more than nonusers regardless of win probability or reward level. Frequency of cannabis use was positively correlated with amount of % hard trials chosen. There were no significant correlations between % hard trials chosen, self-reported anhedonia, or motivation. CONCLUSIONS: These results suggest that unlike acute effects, residual effects of cannabis following 24 hrs of abstinence are associated with greater effort allocation during effort-based decision-making.

Accuracy of DLPNO-CCSD(T): Effect of Basis Set and System Size.

The DLPNO-CCSD(T) method is designed to study large molecular systems at significantly reduced cost relative to its canonical counterpart. However, the error in this approach is also size-extensive and relies on cancellation of errors for the calculation of relative energies. This work provides a direct comparison of canonical CCSD(T) and TightPNO DLPNO-CCSD(T) calculations of reaction energies and barriers of a broad range of chemical reactions. The dataset includes acidities, anion binding affinities, enolization, Diels-Alder, nucleophilic substitution, and atom transfer reactions and complements existing theoretical datasets in terms of system size as well as new reaction types (e.g., anion binding affinities and chlorine atom transfer reactions). The performance of DLPNO-CCSD(T) was further examined with respect to systematic variation of basis set and system size and amounts of nonbonded interaction present in the system. The errors in the DLPNO-CCSD(T) were found to be relatively insensitive to the choice of basis set for small systems but increase monotonically with system size. Additionally, calculations of barriers appear to be more challenging than reaction energies with errors exceeding 5 kJ mol-1 for many Diels-Alder reactions. Further tests on three realistic organic reactions reveal the impact of the DLPNO approximation in calculating absolute and relative barriers that are important for predictions such as stereoselectivity.

Reproductive ecology and postpollination development in the hydrophilous monocot Ruppia maritima.

PREMISE: Water-pollination (hydrophily) is a rare but important pollination mechanism that has allowed angiosperms to colonize marine and aquatic habitats. Hydrophilous plants face unique reproductive challenges, and many have evolved characteristic pollen traits and pollination strategies that may have downstream consequences for pollen performance. However, little is known about reproductive development in the life history stage between pollination and fertilization (the progamic phase) in hydrophilous plants. The purpose of this study was to characterize reproductive ecology and postpollination development in water-pollinated Ruppia maritima L. METHODS: Naturally pollinated inflorescences of R. maritima were collected from the field. Experimental pollinations using both putatively outcross and self pollen were conducted in the greenhouse and inflorescences were collected at appropriate intervals after pollination. Pollen reception, pollen germination, pollen tube growth, and carpel morphology were characterized. RESULTS: Ruppia maritima exhibits incomplete protogyny, allowing for delayed selfing. Pollen germinated within 15 min after pollination. The average shortest possible pollen tube pathway was 425 µm and pollen tubes first reached the ovule at 45 min after pollination. The mean adjusted pollen tube growth rate was 551 µm/h. CONCLUSIONS: Ruppia pollen is adapted for rapid pollen germination, which is likely advantageous in an aquatic habitat. Small effective pollen loads suggest that pollen competition intensity is low. Selection for traits such as a long period of stigma receptivity, fast pollen germination, and carpel morphology likely played a larger role in shaping postpollination reproductive development in Ruppia than evolution in pollen tube growth rates.

A Multiple Correspondence Analysis of Patterns of CBD Use in Hemp and Marijuana Users.

Background: With the passing of the 2018 Agriculture Improvement Act that legalized hemp-derived products, i.e., cannabidiol (CBD), the use of CBD has increased exponentially. To date, the few studies that have characterized individuals who use CBD suggest that co-use of CBD and tetrahydrocannabinol (THC)-dominant cannabis, i.e., marijuana, is highly prevalent. It is, therefore, important to investigate the relationship between CBD use and marijuana use to understand the antecedents and consequences of co-use of these two cannabis products. Methods: We conducted an online survey using structured questionnaires to determine differences in CBD users with (CBD+MJ) and without co-morbid marijuana use. Group comparisons were carried out using chi-square tests and ANOVA. Multiple correspondence analysis (MCA) with bootstrap ratio testing was performed to examine the relationship between the categorical data. Results: We received 182 survey responses from current CBD users. CBD+MJ had more types of CBD administration (F = 17.07, p < 0.001) and longer lifetime duration of CBD use (χ2 = 12.85, p < 0.05). Results from the MCA yielded two statistically significant dimensions that accounted for 77% of the total variance. Dimension 1 (representing 57% of the variance) associated CBD+MJ with indication of CBD use for medical ailments, use of CBD for more than once a day for longer than 2 years, applying CBD topically or consuming it via vaping or edibles, being female, and, having lower educational attainment. Dimension 2 (representing 20% of the variance) separated the groups primarily on smoking-related behaviors where CBD+MJ was associated with smoking CBD and nicotine. Conclusions: Identifying the factors that influence use of CBD and marijuana can inform future studies on the risks and benefits associated with each substance as well as the impacts of policies related to cannabis-based products.

Pollen structure and development in Nymphaeales: insights into character evolution in an ancient angiosperm lineage.

PREMISE OF THE STUDY: A knowledge of pollen characters in early-diverging angiosperm lineages is essential for understanding pollen evolution and the role of pollen in angiosperm diversification. In this paper, we report and synthesize data on mature pollen and pollen ontogeny from all genera of Nymphaeales within a comparative, phylogenetic context and consider pollen evolution in this early-diverging angiosperm lineage. We describe mature pollen characters for Euryale, Barclaya, and Nymphaea ondinea, taxa for which little to no structural data exist. METHODS: We studied mature pollen for all nymphaealean genera using light, scanning electron, and transmission electron microscopy. We reviewed published reports of nymphaealean pollen to provide a comprehensive discussion of pollen characters in water lilies. KEY RESULTS: Nymphaeales exhibit diversity in key pollen characters, including dispersal unit size, ornamentation, aperture morphology, and tapetum type. All Nymphaeales pollen are tectate-columellate, exhibiting one of two distinct patterns of infratectal ultrastructure-a thick infratectal space with robust columellae or a thin infratectal space with thin columellae. All genera have pollen with a lamellate endexine that becomes compressed in the proximal, but not distal wall. This endexine ultrastructure supports the operculate hypothesis for aperture origin. Nymphaeaceae pollen exhibit a membranous granular layer, which is a synapomorphy of the family. CONCLUSIONS: Variation in pollen characters indicates that significant potential for lability in pollen development was present in Nymphaeales at the time of its divergence from the rest of angiosperms. Structural and ontogenetic data are essential for interpreting pollen characters, such as infratectum and endexine ultrastructure in Nymphaeales.

Repeated evolution of tricellular (and bicellular) pollen.

PREMISE OF STUDY: Male gametophytes of seed plants are sexually immature at the time they are dispersed as pollen, but approximately 30% of flowering plants have tricellular pollen containing fully formed sperm at anthesis. The classic study of Brewbaker (1967: American Journal of Botany 54: 1069-1083) provided a powerful confirmation of the long-standing hypothesis that tricellular pollen had many parallel and irreversible origins within angiosperms. We readdressed the main questions of that study with modern comparative phylogenetic methods. METHODS: We used our own and more recent reports to greatly expand the Brewbaker data set. We modeled trait evolution for 2511 species on a time-calibrated angiosperm phylogeny using (1) Binary State Speciation and Extinction (BiSSE), which accounts for the effect of species diversification rates on character transition rates and, (2) the hidden rates model (HRM), which incorporates variation in transition rates across a phylogeny. KEY RESULTS: Seventy percent of species had bicellular pollen. BiSSE found a 1.9-fold higher bicellular to tricellular transition rate than in the reverse direction, and bicellular lineages had a 1.8-fold higher diversification rate than tricellular lineages. HRM found heterogeneity in evolutionary rates, with bidirectional transition rates in three of four rate classes. CONCLUSIONS: The tricellular condition is not irreversible. Pollen cell numbers are maintained at intermediate frequencies because lower net diversification rates of tricellular lineages are counterbalanced by slower state shifts to the bicellular condition. That tricellular lineages diversify slowly and give rise to bicellular lineages slowly reflects a linkage between the evolution of sporophyte lifestyles and the developmental lability of male gametophytes.

Evidence for the role of Epstein Barr Virus infections in the pathogenesis of acute coronary events.

BACKGROUND: The role of viral infections in the pathogenesis of atherosclerosis remains controversial largely due to inconsistent detection of the virus in atherosclerotic lesions. However, viral infections elicit a pro-inflammatory cascade known to be atherogenic and to precipitate acute ischemic events. We have published in vitro data that provide the foundation for a mechanism that reconciles these conflicting observations. To determine the relation between an early viral protein, deoxyuridine triphosphate nucleotidohydrolase (dUTPase), produced following reactivation of Epstein Barr Virus (EBV) to circulating pro-inflammatory cytokines, intercellular adhesion molecule-1 (ICAM-1) and acute coronary events. METHODOLOGY/PRINCIPAL FINDINGS: Blood samples were obtained from 299 patients undergoing percutaneous coronary intervention for stable angina (SA), unstable angina (UA), or acute myocardial infarction (AMI). Plasma concentrations of pro-inflammatory cytokines and neutralizing antibody against EBV-encoded dUTPase were compared in the three patient groups. AMI was associated with the highest measures of interleukin-6 (ANOVA p<0.05; 4.6 ± 2.6 pg/mL in patients with AMI vs. 3.2 ± 2.3 pg/mL in SA). ICAM-1 was significantly higher in patients with AMI (ANOVA p<0.05; 304 ± 116 pg/mL in AMI vs. 265 ± 86 pg/mL SA). The highest values of ICAM-1 were found in patients having an AMI and who were antibody positive for dUTPase (ANOVA p=0.008; 369 ± 183 pg/mL in AMI and positive for dUTPase vs. 249 ± 70 pg/mL in SA negative for dUTPase antibody). CONCLUSIONS/SIGNIFICANCE: These clinical data support a model, based on in vitro studies, by which EBV may precipitate AMI even under conditions of low viral load through the pro-inflammatory action of the early protein dUTPase that is produced even during incomplete viral replication. They further support the putative role of viral infections in the pathogenesis of atherosclerosis and coronary artery events.

Pollen tube development in two species of Trithuria (Hydatellaceae) with contrasting breeding systems.

Trithuria (Hydatellaceae; Nymphaeales) is unique among early-divergent angiosperms in that its species are extremely small and most have exceptionally short, annual life histories. Given the evolution of these extremes of size and development, we sought to understand whether post-pollination processes still varied predictably with breeding system in Trithuria. To address this question, we studied two Western Australian species, Trithuria austinensis (dioecious, obligately outcrossing) and Trithuria submersa (bisexual, highly selfing). To document developmental timing, carpels were hand-pollinated, collected at sequential time points, and examined with light and fluorescence microscopy. In both species, pollen tubes first entered ovules<1 h after pollination, but the pollen tube pathway of outcrossing T. austinensis was almost four times longer and its pollen tube growth rates were up to six times faster (≤2,166 vs. 321 µm/h) than those of T. submersa. T. austinensis also exhibited greater male investment, slower pollen germination, and greater pollen tube attrition. These differences in male gametophyte development are predicted for outcrossers versus selfers in phylogenetically derived angiosperms. These new data for Hydatellaceae reinforce the idea that an acceleration of pollen tube development occurred in the Nymphaeales stem lineage, before the origin of Hydatellaceae. We infer that a recent evolutionary transition to selfing in T. submersa has been accompanied by predictable modifications to reproductive development, which, because of the ancient relationship between Hydatellaceae and all other angiosperms, suggests that traits underlying the lability of flowering plant post-pollination biology were present early in their history.