Perturbations in protein dynamics associated with ligand binding to the blood coagulation enzymes thrombin and Factor XIII.

In blood coagulation, the serine protease thrombin is a multifaceted enzyme that interacts with multiple proteins. Thrombin utilizes two anion binding exosites (ABE-I and II) to supplement binding to fibrinogen and to the platelet receptor GpIba, two proteins instrumental in clot formation. Approximately 7% to 15% of the fibrinogen ? chain exists as the highly anionic ?\u27 variant ( 410 PEHPAETEY S DSLY S PEDDL 427) , which has been shown to bind ABE-II. GpIba possesses a similar anionic stretch of residues ( 269 DEGDTDLY S DY S Y S PEEDTEG 286 ); however, the exact destination of thrombin binding is more ambiguous. 1D and 2D solution NMR have been employed to characterize the structural features of the bound ?\u27 and GpIba peptides. The results indicate that the ?\u27 residues A 414 -L 427 make significant contact with the enzyme, a turn is present between residues Y S 422 -D 425 , and there is a hydrophobic cluster involving Y S 418 -Y S 422 . For the GpIba peptide, NMR results suggest the peptide exists in an extended conformation with residues D 274 -E 285 contributing to binding. Hydrogen deuterium exchange (HDX) coupled with MALDI-TOF MS indicate that at a 20:1 peptide-enzyme ratio both peptides are interacting with residues within ABE-II fragments. Both peptides affect the dynamics of HDX for regions not associated with ABE-II including ABE-I, the autolysis loop, and the A-chain. Curiously, at 40:1 GpIba peptide to thrombin, the GpIba peptide appears to interact with ABE-I, with unique allosteric consequences. Thrombin also activates FXIII, and the resultant transglutaminase covalently crosslinks fibrin through the formation of isopeptide bonds. Studies were performed to examine the effects of activation and inhibition on the conformational dynamics of FXIII using MALDI-TOF MS. A peptide-based FXIIIa inhibitor has been developed with the glutamine isostere 6-diazo-5-oxo-norleucine (DON). The K9 DON peptide blocks chemical modification of C 409 within the catalytic core and promotes significant HDX protection for the ß barrel 1 fragment 526-546. These results suggest that inhibition of FXIIIa leads to local and long-range effects on protein dynamics. Finally, thrombin activation of FXIII appears to influence regions near the activation peptide and Ca 2+ -activated FXIII presents more evidence for the existence of additional Ca 2+ binding sites. Ultimately, these studies further detail the dynamic nature of thrombin and FXIII

Relationship Between Stages of Change and HPV Vaccine Attitudes and Beliefs in Baccalaureate Nursing Students

The purpose of this study was to: (a) determine if there is a relationship between attitudes/beliefs about Human papillomavirus (HPV) vaccination and stages of change and (b) investigate gender differences in attitudes/beliefs and stages of change in undergraduate baccalaureate nursing students. The study employs a cross-sectional and descriptive correlational design and it was guided by the Trans-theoretical Model of Change (TMC). The convenience sample was comprised of 131 participants at a large urban public university in Midwest United States. Data were collected with online surveys distributed via university email. A positive, moderate relationship was found between HPV vaccination attitudes/beliefs and stage of change (r=.36, p\u3c.001). Attitudes/beliefs and readiness to change were measured using multiple regression. An independent t-test was used to determine difference in male and female mean belief and attitude scores. Results show that those with more favorable attitudes/beliefs about HPV vaccination, as well as those who felt supported by others regarding obtaining the vaccine, were more likely to either be in the process of getting vaccinated or had already been vaccinated. Also, although there is no statistical difference in attitudes/beliefs about the HPV vaccine between males and females, males were less likely to have made efforts to be vaccinated compared with females (t= -2.99, p=.003). Therefore, it is possible that there is a gender disparity in knowledge about the vaccine or how to obtain it. The findings warrant further investigation as to what prevents males from being vaccinated

Cerebral Vasoreactivity Is Impaired Beyond Symptom Resolution Following Concussion in Collegiate Athletes

Compromised cerebral blood flow (CBF) regulation is linked to impaired functional outcome following concussion. Cerebral vasoreactivity (CVR), an important mechanism in CBF regulation, is the ability of cerebral blood vessels to alter blood flow during dynamic changes in arterial carbon-dioxide (CO₂). PURPOSE: The purpose of this study was to examine CVR in an ongoing prospective cohort of collegiate athletes during acute (day-3) and sub-acute (day-21) phases following concussion and compare them with non-injured athletes. METHODS: Sixteen male and female collegiate athletes (21±1 years) with sports-related concussion and 16 sports matched non-injured controls (21±1 years) were enrolled in the study. For injured athletes, data was collected during the acute and sub-acute phase following concussion and for the controls data was collected at one time point. Symptom severity and cognition were assessed using the Sports Concussion Assessment Tool-3rd Edition. Continuous middle cerebral artery blood flow velocity (MCAV) was obtained with transcranial Doppler ultrasonography (TCD) while subjects were seated in an upright position. End-tidal CO₂ (PetCo₂) was measured with an infrared CO₂ analyzer attached to a nasal cannula. MCAV was evaluated in response to changes in PetCo₂ for 2-minutes each during normal breathing (normocapnia), inspiring a gas mixture containing 8% CO₂, 21% oxygen (hypercapnia) and, hyperventilating (hypocapnia). CVR was analyzed as the slope of the linear relationship between PetCo₂ and MCAV, which was expressed as the percent change in CBF velocity per mmHg change in PetCo₂. Independent and paired t-tests were used to compare symptom severity, and CVR between acute and sub-acute phase following concussion with the controls. RESULTS: As anticipated, concussed athletes exhibited higher symptom severity (26.3±0.5 versus 5±7 P= 0.0007) and lower cognition (26.5±1.6 versus 28.3±2.4 P=0.03) during acute phase compared to the controls. Although symptoms and cognition were resolved during the sub-acute phase, CVR was lower in the acute phase compared to the non-injured controls (1.7±0.5U versus 2.3±0.3U, P=0.0006) and it continued to be blunted in the sub-acute phase following concussion (1.9±0.5U P=0.04). CONCLUSION: Despite improvements in symptom and cognition, cerebral vasoreactivity appears to be impaired in the sub-acute phase following concussion. Cerebral vasoreactivity utilizing TCD may be a useful vascular biomarker for physiological recovery and aid in accurate return-to play decision-making

Untangling the Links Among Athletic Involvement, Gender, Race, and Adolescent Academic Outcomes

Although previous research has established that high school sports participation may be associated with positive academic outcomes, the parameters of the relationship remain unclear. Using a longitudinal sample of nearly 600 Western New York adolescents, this study examined gender- and race-specific differences in the impact of two dimensions of adolescent athletic involvement (“jock” identity and athlete status) on changes in school grades and school misconduct over a two-year interval. Female and black adolescents who identified themselves as “jocks” reported lower grades than those who did not, whereas female athletes reported higher grades than female nonathletes. Jocks also reported significantly more misconduct (including skipping school, cutting classes, having someone from home called to the school for disciplinary purposes, and being sent to the principal’s office) than nonjocks. Gender moderated the relationship between athlete status and school misconduct; athletic participation had a less salutary effect on misconduct for girls than for boys

The role of chromatin accessibility in directing the widespread, overlapping patterns of Drosophila transcription factor binding

Abstract Background In Drosophila embryos, many biochemically and functionally unrelated transcription factors bind quantitatively to highly overlapping sets of genomic regions, with much of the lowest levels of binding being incidental, non-functional interactions on DNA. The primary biochemical mechanisms that drive these genome-wide occupancy patterns have yet to be established. Results Here we use data resulting from the DNaseI digestion of isolated embryo nuclei to provide a biophysical measure of the degree to which proteins can access different regions of the genome. We show that the in vivo binding patterns of 21 developmental regulators are quantitatively correlated with DNA accessibility in chromatin. Furthermore, we find that levels of factor occupancy in vivo correlate much more with the degree of chromatin accessibility than with occupancy predicted from in vitro affinity measurements using purified protein and naked DNA. Within accessible regions, however, the intrinsic affinity of the factor for DNA does play a role in determining net occupancy, with even weak affinity recognition sites contributing. Finally, we show that programmed changes in chromatin accessibility between different developmental stages correlate with quantitative alterations in factor binding. Conclusions Based on these and other results, we propose a general mechanism to explain the widespread, overlapping DNA binding by animal transcription factors. In this view, transcription factors are expressed at sufficiently high concentrations in cells such that they can occupy their recognition sequences in highly accessible chromatin without the aid of physical cooperative interactions with other proteins, leading to highly overlapping, graded binding of unrelated factors

Stem Cell Transplantation As A Dynamical System: Are Clinical Outcomes Deterministic?

Outcomes in stem cell transplantation (SCT) are modeled using probability theory. However the clinical course following SCT appears to demonstrate many characteristics of dynamical systems, especially when outcomes are considered in the context of immune reconstitution. Dynamical systems tend to evolve over time according to mathematically determined rules. Characteristically, the future states of the system are predicated on the states preceding them, and there is sensitivity to initial conditions. In SCT, the interaction between donor T cells and the recipient may be considered as such a system in which, graft source, conditioning and early immunosuppression profoundly influence immune reconstitution over time. This eventually determines clinical outcomes, either the emergence of tolerance or the development of graft versus host disease. In this paper parallels between SCT and dynamical systems are explored and a conceptual framework for developing mathematical models to understand disparate transplant outcomes is proposed.Comment: 23 pages, 4 figures. Updated version with additional data, 2 new figures and editorial revisions. New authors adde

Clinical Assessment of Potential Drug Interactions of Faldaprevir, a Hepatitis C Virus Protease Inhibitor, With Darunavir/Ritonavir, Efavirenz, and Tenofovir

Faldaprevir is a potent hepatitis C virus NS3/4A protease inhibitor. The findings from 3 phase 1 studies reported here suggest that faldaprevir can be safely coadministered with commonly used antiretroviral