Behavioural interactions, kin and disease susceptibility in the bumblebee Bombus terrestris

Behavioural interactions are often analysed in terms of their costs and benefits to the actors [Hamilton, (1964) J. Theor. Biol.7 1-16; Gadagkar, (1993) Trends Ecol. Evol.8 232-234; Foster et al., (2001) Ann. Zool. Fenn.38 229-238]. Using the bumblebee Bombus terrestris, we wish to distinguish between two possible determinants of interaction behaviour between conspecifics, namely kin-directed behaviour that reflects genetic distance between individuals, or, alternatively, interactions guided by a functional distance between individuals, specifically, with respect to disease susceptibility. We find no relationship between contact rate of individuals and the genetic distance of their respective colonies. Interestingly, we do find a significant negative correlation between contact rate and the distance between the two colonies in susceptibility to a spectrum of parasite strains. This cannot be explained by either of the a priori alternatives so we propose two further testable hypotheses to explain our results.     

Solution structure of the Ca2+-Binding EGF3-4 pair from vitamin K-dependent protein S: identification of an unusual fold in EGF3

Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the psi angle of Glu186 from 160 degrees +/- 40 degrees , as seen in ten other EGF domains, to approximately 0 degrees +/- 15 degrees . NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S.     

Human ras-converting enzyme (hRCE1) endoproteolytic activity on K-ras-derived peptides

A human gene responsible for one of the steps in Ras post-translational modification and membrane localization, hRCE1, encodes a 35-kDa membrane-associated endoprotease. We examined hRCE1 activity using farnesylated 9 aa peptides with the core sequence, KSKTKC(farnesyl)VIM [(farnesyl) = (f)], from the C-terminus of K-Ras. We first demonstrated hRCE1 specificity in cleavage location and endoproteolysis. We then describe a direct fluorescent microtiter plate assay. We demonstrated that hRCE1 protease cleaved KSKTKC(f)VIM peptides between the C(f) and V positions, generating KSKTKC(f) and the corresponding tripeptides as products. We found that the sequence KSKTKC(f)VI was a better substrate for hRCE1 than KSKTKC(f)VIM. We also found that hRCE1 cleaved modified versions of KSKTKC(f)VIM that incorporated either MCA or ABZ fluorescent chromophores at the N-terminus, and quenching-group-containing amino acids at the V or M, but not the I, amino acid positions of VIM. The quenching-group-containing amino acids used were either Q(S) (dinitrophenyldiaminopropionic acid) or Q(L) (lysine epsilon-dinitrophenyl). Cleavage of KSKTKC(f)VIM and modified versions of this peptide by hRCE1 was initially evaluated by HPLC product resolution and quantitation. The hRCE1 cleavage of quenched peptides enabled us to directly monitor proteolytic activity in a 96-well microtiter fluorescent plate assay. The microtiter format assay was validated by its sensitivity to RPI, an inhibitor of prenyl protein protease. A direct fluorescent assay provides an effective tool for further characterization of this enzyme and also for detection of novel inhibitors.     

Discovery of Novel Plasmodium falciparum Pre-Erythrocytic Antigens for Vaccine Development

Background

Nearly 100% protection against malaria infection can be achieved in humans by immunization with P. falciparum radiation-attenuated sporozoites (RAS). Although it is thought that protection is mediated by T cell and antibody responses, only a few of the many pre-erythrocytic (sporozoite and liver stage) antigens that are targeted by these responses have been identified.

Methodology

Twenty seven P. falciparum pre-erythrocytic antigens were selected using bioinformatics analysis and expression databases and were expressed in a wheat germ cell-free protein expression system. Recombinant proteins were recognized by plasma from RAS-immunized subjects, and 21 induced detectable antibody responses in mice and rabbit and sera from these immunized animals were used to characterize these antigens. All 21 proteins localized to the sporozoite: five localized to the surface, seven localized to the micronemes, cytoplasm, endoplasmic reticulum or nucleus, two localized to the surface and cytoplasm, and seven remain undetermined. PBMC from RAS-immunized volunteers elicited positive ex vivo or cultured ELISpot responses against peptides from 20 of the 21 antigens.

Conclusions

These T cell and antibody responses support our approach of using reagents from RAS-immunized subjects to screen potential vaccine antigens, and have led to the identification of a panel of novel P. falciparum antigens. These results provide evidence to further evaluate these antigens as vaccine candidates.

Trial Registration

ClinicalTrials.gov NCT00870987 ClinicalTrials.gov NCT00392015     

Risk of Post-Discharge Venous Thromboembolism and Associated Mortality in General Surgery: A Population-Based Cohort Study Using Linked Hospital and Primary Care Data in England

Background

Trends towards day case surgery and enhanced recovery mean that postoperative venous thromboembolism (VTE) may increasingly arise after hospital discharge. However, hospital data alone are unable to capture adverse events that occur outside of the hospital setting. The National Institute for Health and Care Excellence has suggested the use of primary care data to quantify hospital care-related VTE. Data in surgical patients using these resources is lacking. The aim of this study was to measure VTE risk and associated mortality in general surgery using linked primary care and hospital databases, to improve our understanding of harm from VTE that arises beyond hospital stay.

Methods

This was a longitudinal cohort study using nationally linked primary care (Clinical Practice Research Datalink, CPRD), hospital administrative (Hospital Episodes Statistics, HES), population statistics (Office of National Statistics, ONS) and National Cancer Intelligence Network databases. Routinely collected information was used to quantify 90-day in-hospital VTE, 90-day post-discharge VTE and 90-day mortality in adults undergoing one of twelve general surgical procedures between 1st April 1997 and 31st March 2012. The earliest postoperative recording of deep vein thrombosis or pulmonary embolism in CPRD, HES and ONS was counted in each patient. Covariates from multiple datasets were combined to derive detailed prediction models for VTE and mortality. Limitation included the capture of VTE presenting to healthcare only and the lack of information on adherence to pharmacological thromboprophylaxis as there was no data linkage to hospital pharmacy records.

Results

There were 981 VTE events captured within 90 days of surgery in 168005 procedures (23.7/1000 patient-years). Overall, primary care data increased the detection of postoperative VTE by a factor of 1.38 (981/710) when compared with using HES and ONS only. Total VTE rates ranged between 3.2/1000 patient-years in haemorrhoidectomy to 118.3/1000 patient-years in esophagogastric resection. Predictors of VTE included emergency surgery (OR = 1.91 95%CI 1.60–2.28, p<0.001), age (OR = 1.02 95%CI 1.02–1.03, p<0.001), body mass index (OR = 1.03 95%CI 1.01–1.04, p<0.001), previous VTE (OR = 8.07 95%CI 6.61–9.83, p<0.001), length of stay (OR = 1.00 95%CI 1.00–1.00, p = 0.007) and cancer stages II (OR = 1.38 95%CI 1.03–1.87, p = 0.033), III (OR = 1.50 95%CI 1.11–2.01, p = 0.008) and IV (OR = 1.63 95%CI 1.03–2.59, p = 0.038). Major organ resections had the greatest odds of VTE when adjusted for other risk factors including length of hospital stay. Post-discharge VTE accounted for 64.8% (636/981) of all recorded VTE. In-hospital VTE (165.4/1000 patient-years) was recorded more frequently than post-discharge VTE (16.2/1000 patient-years). Both in-hospital (OR = 2.07 95%CI 1.51–2.85, p<0.001) and post-discharge (OR = 4.03 95%CI 2.95–5.51, p<0.001) VTE independently predicted 90-day mortality. In patients who died and VTE was recorded on HES or CPRD (n = 56), VTE was one of the causes of death in 37.5% (21/56) of cases.

Conclusions

A large proportion of postoperative VTE was detected in primary care. Evaluation of linked databases was a useful way of measuring postoperative VTE at population level. These resources identified a significant association between post-discharge VTE and mortality in general surgery.