Does knowledge and attitude of healthcare professionals working in critical care areas affect their willingness to offer the option of organ donation? results of a tertiary hospital survey.

INTRODUCTION: Organ donation (OD) rates in Malaysia have remained suboptimal for decades. Healthcare professionals (HCPs) working in critical care areas are responsible for diagnosing brain death (BD) and initiating the OD process. Impact of their knowledge and attitudes on willingness to offer the option of OD to families of potential donors is unknown. METHODS: Knowledge and attitudes about BD, OD, and organ transplantation (OT) of critical care HCPs in a Malaysian transplant centre were studied using a validated questionnaire. Responses were analysed using multivariable analysis with willingness to offer the option of OD to families of potential donors as dependent variable. RESULTS: Age (p = 0.04), profession (doctors > nurses, p < 0.001), religion (Buddhists > others, p = 0.013) [but not ethnicity], higher knowledge scores for Brain Death Test, Brain Death Knowledge, Organ Donation and Transplantation, and overall knowledge score (p < 0.001) were associated with greater odds of offering OD to families. Belief in the reliable diagnosis of BD, confidence in explaining BD, and belief that OD will not affect religious services were significantly associated with willingness to offer OD, while HCPs who were willing to personally donate organs had greatest odds (p < 0.001). Other factors that significantly influenced HCPs' willingness to offer included their perception about families' willingness to donate, body disfigurement, and confidence in OT. CONCLUSIONS: Overall, HCPs had highly positive attitudes. However, potential barriers in offering OD to families were identified. Proven interventions from international experience could help address these issues and likely improve OD rates in Malaysia.

Demographics of healthcare professionals' knowledge and attitude toward deceased organ donation: Survey of critical care areas in a tertiary hospital.

INTRODUCTION: Organ donation rate in Malaysia is amongst the lowest in the World. Healthcare professionals (HCPs) working in critical care areas play an important role in the deceased organ donation (DOD) process. This study seeks to identify the demographics of HCPs working in the critical care areas and their knowledge and attitudes toward the DOD process. METHOD: A cross-sectional survey on the demographics, knowledge and attitudes of the doctors and nurses working in critical care areas was undertaken by the random sampling method, using a validated, structured questionnaire. HCP's knowledge and attitudes towards brain death (BD), DOD, organ transplantation (OT), and possession of organ donor card were compared against their demographics. RESULTS: Four hundred and twelve (72.9%) out of the total 565 HCPs in critical care areas responded of whom 163 (39.6%) were doctors and 249 (60.4%) were nurses. After adjusting for other factors, department of work and profession were highly correlated with the overall knowledge score (p<0.001 and p=0.003 respectively) and knowledge about BD (p<0.001 and p=0.013 respectively). HCPs from the neurosurgical intensive care unit (p<0.001) and doctors (p<0.001) had higher mean knowledge scores compared to their counterparts. Profession was most significantly correlated with having a positive attitude towards BD (p<0.001) and OT (p<0.001). CONCLUSION: Department, profession and ethnicity were the demographic characteristics that correlated with knowledge and attitudes of HCPs on organ donation. Efforts to improve DOD rates in Malaysia should include targeted interventions to address the knowledge and attitudes of HCPs working in critical care areas.

Generation and characterisation of monoclonal antibodies specific to avian influenza H7N9 haemagglutinin protein.

INTRODUCTION: Emerging virulent strains of influenza virus pose a serious public health threat with potential pandemic consequences. A novel avian influenza virus, H7N9, breached the species barrier from infected domestic poultry to humans in 2013 in China. Since then, it has caused numerous infections in humans with a close contact to poultry. MATERIALS AND METHODS: In this study, we describe the preliminary characterisation of five murine monoclonal antibodies (MAbs) developed against recombinant haemagglutinin (rHA) protein of avian H7N9 A/Anhui/1/2013 virus by their Western blot and enzyme-linked immunosorbent assay (ELISA) reactivity and binding affinity. RESULTS: Of the five MAbs, four were highly specific to H7N9 HA and did not show any cross-reactivity in ELISA with rHA protein from pandemic as well as seasonal H1N1, H2N2, H3N2, H5N1 and influenza virus B (B/Brisbane/60/2008). However, one of the MAbs, MA-24, in addition to HA protein of H7N9 also reacted strongly with HA protein of H3N2 and weakly with HA of pandemic and seasonal H1N1 and H2N2. All the five MAbs also reacted with H7N9 rHA in Western blot. The MAbs bound H7N9 rHA with an equilibrium dissociation constant (KD) ranging between 0.14 and 25.20 nM, indicating their high affinity to HA. CONCLUSIONS: These antibodies may be useful in developing diagnostic tools for the detection of influenza H7N9 virus infections.

Dental management of hemophiliac child under general anesthesia.

Hemophilia is the most common inherited bleeding disorder. Hemophilic patients should be cosidered as special patients. There is no contraindication to general dental treatment for hemophiliacs, as they generally do not involve bleeding. But caution must be used with any surgical procedures that involve the local and general anesthesia. Such patients should always be managed in the setting of specialized units with appropriate clinical expertise and laboratory support. Recent advances in the management of hemophilia have enabled many hemophiliac patients to receive surgical dental procedures in an outpatient dental care on a routine basis. The purpose of this case report is to provide a few management strategies when providing full mouth rehabilitation under anesthesia and replacement therapies that are available. In addition, overviews of possible complication that may be encountered when providing such treatment are discussed here.

Static fracture resistance of ultra high molecular weight polyethylene using the single specimen normalization method.

Fracture of Ultra High Molecular Weight Polyethylene (UHMWPE) components used in total joint replacements is a clinical concern. UHMWPE materials exhibits stable crack growth under static loading, therefore, their fracture resistance is generally characterized using the J-R curve. The multiple specimen method recommended by ASTM for evaluation of the J-R curve for polymers is time and material intensive. In this study, the applicability of a single specimen method based on load normalization to predict J-R curves of UHMWPE materials is evaluated. The normalization method involves determination of a deformation function. In this study, the J-R curves obtained using a power law based deformation function and the LMN curve based deformation function were compared. The results support the use of the power law based deformation function when using the single specimen approach to predict J-R curves for UHMWPE materials.

Evaluation of J-initiation fracture toughness of ultra high molecular weight polyethylene used in total joint replacements.

Fracture of ultra high molecular weight polyethylene (UHMWPE) total joint replacement components is a clinical concern. Thus, it is important to characterize the fracture resistance of UHMWPE. To determine J-initiation fracture toughness (J(Q)) for metals and metallic alloys, ASTM E1820 recommends a procedure based on an empirical crack blunting line. This approach has been found to overestimate the initiation toughness of tough polymers like UHMWPE. Therefore, in this study, a novel experimental approach based on crack tip opening displacement (CTOD) was utilized to evaluate J(Q) of UHMWPE materials. J-initiation fracture toughness was experimentally measured in ambient air and a physiologically-relevant 37°C PBS environment for three different formulations of UHMWPE and compared to the blunting line approach. The CTOD method was found to provide J(Q) values comparable to the blunting line approach for the UHMWPE materials and environments examined in this study. The CTOD method used in this study is based on experimental observation and, thus, does not rely on an empirical relationship or fracture surface measurements. Therefore, determining J(Q) using the experimentally based CTOD method proposed in this study may be a more reliable approach for UHMWPE and other tough polymers than the blunting line approach.

The prevalence of disorders of haemostasis in adolescents with menorrhagia referred to a haemophilia treatment centre.

Menorrhagia at the time of menarche is relatively common and historically attributed primarily to immaturity of the pituitary-ovarian-uterine axis. Intuitively, a proportion of these patients should have an underlying disorder of haemostasis, given the 5-20% prevalence of von Willebrand's disease and the > or =20% prevalence of platelet dysfunction in light of recent epidemiological studies in menorrhagia, although the average age of the patients in those studies has been approximately 35 years. However, there are a few comprehensive studies in the adolescent population determining whether widespread haemostasis evaluation should be carried out in adolescents presenting with menorrhagia. A retrospective chart review study of disorders of haemostasis was carried out in 61 consecutive adolescent patients, ages 11-19 at the time of evaluation referred to the Hemophilia Treatment Center (HTC)/Hematology unit. The mean and median ages were 15 +/- 2.2 and 14 years (11, 19), respectively. Standard evaluation included complete blood count, prothrombin time, partial thromboplastin time, von Willebrand factor (VWF) levels and platelet aggregation. The proportion of patients with VWF deficiency was 22/61 (36%) [95% confidence interval (CI), 24-49%]; the proportion of patients with platelet aggregation abnormalities was 4/61 (7%) (95% CI, 2-16%). There was no difference in the frequency of additional muco-cutaneous bleeding symptoms. A relatively high proportion of adolescents are identified with an underlying disorder of haemostasis when referred to an HTC for evaluation of menorrhagia. This involves in part a selective referral bias, but underscores the role of the HTC in evaluating adolescents referred with menorrhagia for an underlying bleeding disorder, given the relatively high yield of haemostatic disorders detected in this setting.

Specialty dentistry for the hemophiliac: is there a protocol in place?

Restorative dental care for the hemophiliac patient is of paramount importance for the fact that advanced dental conditions and subsequent treatments prove to be more complicated and risky. Quite often, dental health is neglected by hemophiliacs for fear of bleeding during procedures. Surprisingly, even dental specialists avoid these candidates and contribute to the conversion ofa simple dental patient to an oral surgical patient. The complexities involved in diagnosing a bleeding disorder and the rarity of a standardized protocol to handle such patients contribute to this problem. This article prescribes a simple protocol to diagnose bleeding disorders and a modified scheme for endodontic and periodontal therapy in a hemophiliac patient.

Effect of signal peptide on the stability and folding kinetics of maltose binding protein.

While the role of the signal sequence in targeting proteins to specific subcellular compartments is well characterized, there are fewer studies that characterize its effects on the stability and folding kinetics of the protein. We report a detailed characterization of the folding kinetics and thermodynamic stabilities of maltose binding protein (MBP) and its precursor form, preMBP. Isothermal GdmCl and urea denaturation as a function of temperature and thermal denaturation studies have been carried out to compare stabilities of the two proteins. preMBP was found to be destabilized by about 2-6 kcal/mol (20-40%) with respect to MBP. Rapid cleavage of the signal peptide by various proteases shows that the signal peptide is accessible in the native form of preMBP. The observed rate constant of the major slow phase in folding was decreased 5-fold in preMBP relative to MBP. The rate constants of unfolding were similar at 25 degrees C, but preMBP also exhibited a large burst phase change in unfolding that was absent in MBP. At 10 degrees C, preMBP exhibited a higher unfolding rate than MBP as well as a large burst phase. The appreciable destabilization of MBP by signal peptide is functionally relevant, because it enhances the likelihood of finding the protein in an unfolded translocation-competent form and may influence the interactions of the protein with the translocation machinery. Destabilization is likely to result from favorable interactions between the hydrophobic signal peptide and other hydrophobic regions that are exposed in the unfolded state.

Structural consequences of replacement of an alpha-helical Pro residue in Escherichia coli thioredoxin.

While it is well known that introduction of Pro residues into the interior of protein alpha-helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein alpha-helix. We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1-1.5 kcal/mol in the temperature range 280-330 K. This paper describes the structure of the P40S mutant at a resolution of 1.8 A. In wild-type thioredoxin, P40 is located in the interior of helix two, a long alpha-helix that extends from residues 32 to 49 with a kink at residue 40. Structural differences between the wild-type and P40S are largely localized to the above helix. In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36. The helix remains kinked. In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39. However, these are absent in P40S. Instead, these main chain atoms are hydrogen bonded to water molecules. The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin.

Spontaneous hepatic rupture: a report of five cases.

BACKGROUND: Spontaneous hepatic rupture is a well recognised but rare condition. Because of the difficulty in diagnosis, it is often associated with a high mortality rate. Pregnant women with HELLP syndrome are more prone to hepatic rupture, but it can also occur with other liver pathology. Different modalities of treatment, including liver resection, packing, hepatic artery ligation and even liver transplantation have been described for this condition. PATIENTS AND RESULTS: We report a series of five cases, three of which were associated with pregnancy and two with no identifiable pathology. Pre-operative diagnosis was not made in any of these cases.Two of the five patients had hepatic resection, two had peri-hepatic packing and one was treated with laparoscopic drainage. DISCUSSION: From our experience we conclude that no single form of treatment is applicable to all cases of hepatic rupture.The treatment should be individualised,depending on the extent of hepatic rupture and the expertise available, to obtain best outcome.

Electron spin resonance and fluorescence studies of the bound-state conformation of a model protein substrate to the chaperone SecB.

SecB is a homotetrameric, cytosolic chaperone that forms part of the protein translocation machinery in Escherichia coli. We have investigated the bound-state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58-residue protein and contains three disulfide groups between residues 5 and 55, 14 and 38, as well as 30 and 51. Mutants of BPTI that contained only a single disulfide were reduced, and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide. The relative proximity of the labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy. The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB. Binding occurs at multiple sites on the substrate, and the binding site on each SecB monomer accommodates less than 21 substrate residues. In addition, we have labeled four solvent-accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The electron spin resonance data suggest that these cysteine residues are in close proximity (15 A) when no substrate protein is bound but move away to a distance of greater than 20 A when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein.

Reversible formation of on-pathway macroscopic aggregates during the folding of maltose binding protein.

Maltose binding protein (MBP) is widely used as a model for protein folding and export studies. We show here that macroscopic aggregates form transiently during the refolding of MBP at micromolar protein concentrations. Disaggregation occurs spontaneously without any aid, and the refolded material has structure and activity identical to those of the native, nondenatured protein. A considerable fraction of protein undergoing folding partitions into the aggregate phase and can be manually separated from the soluble phase by centrifugation. The separated MBP precipitate can be resolubilized and yields active, refolded protein. This demonstrates that both the soluble and aggregate phases contribute to the final yield of refolded protein. SecB, the cognate Escherichia coli cytosolic chaperone in vivo for MBP, reduces but does not entirely prevent aggregation, whereas GroEL and a variety of other control proteins have no effect. Kinetic studies using a variety of spectroscopic probes show that aggregation occurs through a collapsed intermediate with some secondary structure. The aggregate formed during refolding can convert directly to a near native state without going through the unfolded state. Further, optical and electron microscopic studies indicate that the MBP precipitate is not an amyloid.

Osmolytes stabilize ribonuclease S by stabilizing its fragments S protein and S peptide to compact folding-competent states.

Osmolytes stabilize proteins to thermal and chemical denaturation. We have studied the effects of the osmolytes sarcosine, betaine, trimethylamine-N-oxide, and taurine on the structure and stability of the protein.peptide complex RNase S using x-ray crystallography and titration calorimetry, respectively. The largest degree of stabilization is achieved with 6 m sarcosine, which increases the denaturation temperatures of RNase S and S pro by 24.6 and 17.4 degrees C, respectively, at pH 5 and protects both proteins against tryptic cleavage. Four crystal structures of RNase S in the presence of different osmolytes do not offer any evidence for osmolyte binding to the folded state of the protein or any perturbation in the water structure surrounding the protein. The degree of stabilization in 6 m sarcosine increases with temperature, ranging from -0.52 kcal mol(-1) at 20 degrees C to -5.4 kcal mol(-1) at 60 degrees C. The data support the thesis that osmolytes that stabilize proteins, do so by perturbing unfolded states, which change conformation to a compact, folding competent state in the presence of osmolyte. The increased stabilization thus results from a decrease in conformational entropy of the unfolded state.

Structural and thermodynamic consequences of introducing alpha-aminoisobutyric acid in the S peptide of ribonuclease S.

The S protein-S peptide interaction is a model system to study binding thermodynamics in proteins. We substituted alanine at position 4 in S peptide by alpha-aminoisobutyric acid (Aib) to investigate the effect of this substitution on the conformation of free S peptide and on its binding to S protein. The thermodynamic consequences of this replacement were studied using isothermal titration calorimetry. The structures of the free and complexed peptides were studied using circular dichroic spectroscopy and X-ray crystallography, respectively. The alanine4Aib replacement stabilizes the free S peptide helix and does not perturb the tertiary structure of RNase S. Surprisingly, and in contrast to the wild-type S peptide, the DeltaG degrees of binding of peptide to S pro, over the temperature range 5-30 degrees C, is virtually independent of temperature. At 25 degrees C, the DeltaDeltaG degrees, DeltaDeltaH degrees, DeltaDeltaS and DeltaDeltaCp of binding are 0.7 kcal/mol, 2.8 kcal/mol, 6 kcal/mol x K and -60 kcal/mol x K, respectively. The positive value of DeltaDeltaS is probably due to a decrease in the entropy of uncomplexed alanine4Aib relative to the wild-type peptide. The positive value of DeltaDeltaH: degrees is unexpected and is probably due to favorable interactions formed in uncomplexed alanine4Aib. This study addresses the thermodynamic and structural consequences of a replacement of alanine by Aib both in the unfolded and complexed states in proteins.

Thermodynamic and structural studies of cavity formation in proteins suggest that loss of packing interactions rather than the hydrophobic effect dominates the observed energetics.

The hydrophobic effect is widely believed to be an important determinant of protein stability. However, it is difficult to obtain unambiguous experimental estimates of the contribution of the hydrophobic driving force to the overall free energy of folding. Thermodynamic and structural studies of large to small substitutions in proteins are the most direct method of measuring this contribution. We have substituted the buried residue Phe8 in RNase S with alanine, methionine, and norleucine. Binding thermodynamics and structures were characterized by titration calorimetry and crystallography, respectively. The crystal structures of the RNase S F8A, F8M, and F8Nle mutants indicate that the protein tolerates the changes without any main chain adjustments. The correlation of structural and thermodynamic parameters associated with large to small substitutions was analyzed for nine mutants of RNase S as well as 32 additional cavity-containing mutants of T4 lysozyme, human lysozyme, and barnase. Such substitutions were typically found to result in negligible changes in DeltaC(p)() and positive values of both DeltaDeltaH degrees and DeltaDeltaS of folding. Enthalpic effects were dominant, and the sign of DeltaDeltaS is the opposite of that expected from the hydrophobic effect. Values of DeltaDeltaG degrees and DeltaDeltaH degrees correlated better with changes in packing parameters such as residue depth or occluded surface than with the change in accessible surface area upon folding. These results suggest that the loss of packing interactions rather than the hydrophobic effect is a dominant contributor to the observed energetics for large to small substitutions. Hence, estimates of the magnitude of the hydrophobic driving force derived from earlier mutational studies are likely to be significantly in excess of the actual value.

Protein stabilization through phage display.

RNase S consists of two proteolytic fragments of RNase A, residues 1-20 (S20) and residues 21-124 (S pro). A 15-mer peptide (S15p) with high affinity for S pro was selected from a phage display library. Peptide residues that are buried in the structure of the wild type complex are conserved in S15p though there are several changes at other positions. Isothermal titration calorimetry studies show that the affinity of S15p is comparable to that of the wild type peptide at 25 degrees C. However, the magnitudes of DeltaH(o) and DeltaC(p) are lower for S15p, suggesting that the thermal stability of the complex is enhanced. In agreement with this prediction, at pH 6, the T(m) of the S15p complex was found to be 10 degrees C higher than that of the wild type complex. This suggests that for proteins where fragment complementation systems exist, phage display can be used to find mutations that increase protein thermal stability.

A thermodynamic coupling mechanism for the disaggregation of a model peptide substrate by chaperone secB.

Molecular chaperones prevent protein aggregation in vivo and in vitro. In a few cases, multichaperone systems are capable of dissociating aggregated state(s) of substrate proteins, although little is known of the mechanism of the process. SecB is a cytosolic chaperone, which forms part of the precursor protein translocation machinery in Escherichia coli. We have investigated the interaction of the B-chain of insulin with chaperone SecB by light scattering, pyrene excimer fluorescence, and electron spin resonance spectroscopy. We show that SecB prevents aggregation of the B-chain of insulin. We show that SecB is capable of dissociating soluble B-chain aggregates as monitored by pyrene fluorescence spectroscopy. The kinetics of dissociation of the B-chain aggregate by SecB has been investigated to understand the mechanism of dissociation. The data suggests that SecB does not act as a catalyst in dissociation of the aggregate to individual B-chains, rather it binds the small population of free B-chains with high affinity, thereby shifting the equilibrium from the ensemble of the aggregate toward the individual B-chains. Thus SecB can rescue aggregated, partially folded/misfolded states of target proteins by a thermodynamic coupling mechanism when the free energy of binding to SecB is greater than the stability of the aggregate. Pyrene excimer fluorescence and ESR methods have been used to gain insights on the bound state conformation of the B-chain to chaperone SecB. The data suggests that the B-chain is bound to SecB in a flexible extended state in a hydrophobic cleft on SecB and that the binding site accommodates approximately 10 residues of substrate.