Day or overnight transfusion in critically ill patients: does it matter?

BACKGROUND AND OBJECTIVES: The timing of blood administration in critically ill patients is first driven by patients' needs. This study aimed to define the epidemiology and significance of overnight transfusion in critically ill patients. MATERIALS AND METHODS: This is a post hoc analysis of a prospective multicentre observational study including 874 critically ill patients receiving red blood cells, platelets, fresh frozen plasma (FFP) or cryoprecipitate. Characteristics of patients receiving blood only during the day (8 am up until 8 pm) were compared to those receiving blood only overnight (8 pm up until 8 am). Characteristics of transfusion were compared, and factors independently associated with major bleeding were analysed. RESULTS: The 287 patients transfused during the day only had similar severity and mortality to the 258 receiving blood products overnight only. Although bleeding-related admission diagnoses were similar, major bleeding was the indication for transfusion in 12% of patients transfused in daytime only versus 30% of patients transfused at night only (P < 0·001). Similar total amount of blood products were transfused at day and night (2856 versus 2927); however, patients were more likely to receive FFP and cryoprecipitate at night compared with daytime. Overnight transfusion was independently associated with increased odds of major bleeding (odds ratio, 3·16, 95% confidence interval, 2·00-5·01). CONCLUSION: Transfusion occurs evenly across day and night in ICU; nonetheless, there are differences in type of blood products administered that reflect differences in indication. Critically ill patients were more likely to receive blood for major bleeding at night irrespective of admission diagnosis.

Diagnosis and management of thrombotic thrombocytopenic purpura (TTP) in Australia: findings from the first 5 years of the Australian TTP/thrombotic microangiopathy registry.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy (TMA). In 2009, the Australian TTP/TMA registry was established to collect data on patients presenting with TTP/TMA throughout Australia. AIM: To summarise information on the diagnosis and management of patients with TTP collected in the first 5 years (2009-2014) of the Australian TTP registry. METHODS: Registry data from June 2009 to October 2014 were reviewed. RESULTS: Fifty-seven patients were identified with TTP (defined as ADAMTS13 activity <10%), accounting for 72 clinical episodes. ADAMTS13 inhibitor testing was performed in nine out of 57 patients (16%), reflecting the limited availability of accredited testing facilities. Sixty-seven out of 72 episodes were treated with therapeutic plasma exchange (PEx) using cryodepleted plasma (40% of episodes), fresh frozen plasma (36%) or a mixture (22%). Median exposure to plasma products was 55.9 L. PEx was commenced ≥2 days from stated diagnosis in 15% of episodes. Adverse reactions to PEx were common with documented allergic reactions (including life threatening) in 21% of episodes. Adjunctive immunosuppression was documented in 76% of episodes (corticosteroid 71% and rituximab 39%). Platelet transfusion was administered in 15% of episodes. CONCLUSIONS: Data from the Australian TTP/TMA registry suggest a heterogenous approach to the diagnosis and management of TTP in Australia over the assessed period. These observations highlight areas for improvement and standardisation of practice, including comprehensive diagnostic testing, more immediate access to PEx and a more uniform approach to adjunctive immunosuppression and supportive care.

Emergence and diversity of different HIV-1 subtypes in South Africa, 2000-2001.

HIV-1 is a major health problem in South Africa with an average prevalence rate of 29.1% in pregnant women and between 4.9 and 6.1 million people infected. Using env gp120 V3 serotyping and genotyping techniques 410 patient samples were investigated. Most of the samples were obtained from different clinics in the greater Cape Town area of the Western Cape Province in South Africa. These included an academic hospital, state and private clinics, an informal settlement, sex worker cohorts, and the blood transfusion services. RNA was extracted from plasma samples followed by RT-PCR and sequencing of the env gp120 V3 region. Sequence fragments were assembled using Sequencher V4.7 and subsequently codon aligned. Distance calculation, tree construction methods, and bootstrap analysis were implemented using MEGA version 4.0. Viral load measurements indicated that HIV-1 RNA levels from 74 samples were below the assay detection limit. Three hundred thirty-six samples were used for env PCR and sequencing and 320 were assigned to subtypes. The majority of the sequences were subtyped as C (n = 285, 89.0%). Other subtypes detected were subtype A (n = 10, 3.1%); subtype B (n = 22, 6.8%); one each of subtypes F1, G, U, and a CH recombinant. Whether this diversity will have major implications for HIV-1 evolution and vaccine development in this region remains undetermined.

ATP synthase: an electrochemical transducer with rotatory mechanics.

ATP synthase (F0F1-ATPase) uses proton- or sodium-motive force to produce ATP form ADP and P(i). Three lines of experiment have recently demonstrated large-scale intersubunit rotation during ATP hydrolysis by F1. We discuss how ion flow through the membrane-intrinsic portion, F0, may generate torque and how this might be transmitted between stator and rotor to finally expel spontaneously formed ATP from F1 into water.

Added subunit beta of CF1 as well as gamma/delta/epsilon restore photophosphorylation in partially CF1-depleted thylakoids.

We investigated the ability of subunits beta, gamma, delta, and epsilon of CF1, the F1-ATPase of chloroplasts, to interact with exposed CF0 in EDTA-treated, partially CF1-depleted thylakoid membranes. We measured the ability of subunits beta, gamma, delta, and epsilon to stimulate the rate of photophosphorylation under continuous light and, for subunit beta, also the ability to diminish the proton leakage through exposed CF0 by deceleration of the decay of electrochromic absorption transients under flashing light. The greatest effect was caused by subunit beta, followed by gamma/delta/epsilon. Pairwise combinations of gamma, delta, and epsilon or each of these subunits alone were only marginally effective. Subunit gamma from the thermophilic bacterium PS 3 in combination with chloroplast delta and epsilon was as effective as chloroplast gamma. The finding that the small CF1 subunits in concert and the beta subunit by itself specifically interacted with the exposed proton channel CF0, qualifies the previous concept of subunit delta acting particularly as a plug to the open CF0 channel. The interactions between the channel and the catalytic portion of the enzyme seem to involve most of the small, and at least beta of the large subunits.

Subunit delta of H(+)-ATPases: at the interface between proton flow and ATP synthesis.

The ATP synthases in photophosphorylation and respiration are of the F-type with a membrane-bound proton channel, F0, and an extrinsic catalytic portion, F1. The properties of one particular subunit, delta (in chloroplasts and Escherichia coli) and OSCP (in mitochondria), are reviewed and the role of this subunit at the interface between F0 and F1 is discussed. Delta and OSCP from the three sources have in common the molecular mass (approximately 20 kDa), an elongated shape (axial ratio in solution about 3:1), one high-affinity binding site to F1 (Kd approximately 100 nM) plus probably one or two further low-affinity sites. When isolated delta is added to CF1-depleted thylakoid membranes, it can block proton flow through exposed CF0 channels, as do CF1 or CF1(-delta)+ delta. This identifies delta as part of the proton conductor or, alternatively, conformational energy transducer between F0 (proton flow) and F1 (ATP). Hybrid constructs as CF1(-delta)+ E. coli delta and EF1(-delta)+ chloroplast delta diminish proton flow through CF0.CF1(-delta) + E. coli delta does the same on EF0. Impairment of proton leaks either through CF0 or through EF0 causes "structural reconstitution' of ATP synthesis by remaining intact F0F1. Functional reconstitution (ATP synthesis by fully reconstructed F0F1), however, is absolutely dependent on the presence of subunit delta and is therefore observed only with CF1 or CF1(-delta) + chloroplast delta on CF0 and EF1 or EF1(-delta) + E. coli delta on EF0. The effect of hybrid constructs on F0 channels is surprising in view of the limited sequence homology between chloroplast and E. coli delta (36% conserved residues including conservative replacements). An analysis of the distribution of the conserved residues at present does not allow us to discriminate between the postulated conformational or proton-conductive roles of subunit delta.

Complementation of Escherichia coli uncD mutant strains by a chimeric F1-beta subunit constructed from E. coli and spinach chloroplast F1-beta.

ATP-synthesizing F0F1-ATPases are complex enzymes consisting of at least eight different subunits. These subunits are conserved during evolution to a very variable degree ranging in pairwise comparison between, for example, Escherichia coli and spinach chloroplast from 20% to 66% identical residues. It was surprising to find that some of the less well conserved subunits like delta and epsilon could replace their E. coli counterparts, whereas the highly conserved beta subunit, which carries the active site, in the E. coli enzyme could not be substituted by spinach chloroplast beta (Lill et al. (1993) Biochim. Biophys. Acta 1144, 278-284). We constructed a chimeric F1-beta subunit consisting of spinach beta in which the 96 N-terminal amino acids were replaced by the respective residue sequence from E. coli beta. Whereas spinach beta did not complement E. coli uncD mutant strains, the chimeric beta subunit restored growth under conditions of oxidative phosphorylation.

Over-production, renaturation and reconstitution of delta and epsilon subunits from chloroplast and cyanobacterial F1.

We studied the functioning of chimeric F0F1-ATPases by replacing subunits delta and epsilon of spinach CF1 with their counterparts from Synechocystis sp. PCC 6803. The sequence identities between these subunits are 26 and 41%, respectively. For a systematic approach to such studies and later extension to genetically modified subunits recombinant proteins are required. The genes coding for spinach and Synechocystis delta and epsilon were cloned into pET3 expression vectors and expressed in Escherichia coli. Upon expression at 37 degrees C the recombinant subunits formed inclusion bodies within the host cells except for spinach delta, which was soluble. Synechocystis delta and epsilon could be obtained in soluble form upon expression at 20 degrees C. After purification (and refolding of spinach epsilon) both epsilon subunits inhibited the Ca(2+)-ATPase activity of soluble CF1(- epsilon). Subunits delta and epsilon from both species raised the rate of ATP synthesis in partially CF1-depleted spinach thylakoids when added together with CF1(- delta) or CF1(- delta, epsilon). This showed the functionality of recombinant Synechocystis and spinach delta and epsilon together with spinach alpha 3 beta 3 gamma. The molar excess of epsilon necessary for saturation was higher for Ca(2+)-ATPase inhibition than for reconstitution of photophosphorylation thus pointing to a direct interaction between epsilon and both CF1 and CF0.

Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F1-ATPase subunits.

The genes encoding the five subunits of the F1 portion of the ATPases from both spinach chloroplasts and the cyanobacterium Synechocystis sp. PCC 6803 were cloned into expression vectors and expressed in Escherichia coli. The recombinant subunits formed inclusion bodies within the cells. Each particular subunit was expressed in the respective unc mutant, each unable to grow on non-fermentable carbon sources. The following subunits restored growth under conditions of oxidative phosphorylation: alpha (both sources, cyanobacterial subunit more than spinach subunit), beta (cyanobacterial subunit only), delta (both spinach and Synechocystis), and epsilon (both sources), whereas no growth was achieved with the gamma subunits from both sources. Despite a high degree of sequence homology the large subunits alpha and beta of spinach and cyanobacterial F1 were not as effective in the substitution of their E. coli counterparts. On the other hand, the two smallest subunits of the E. coli ATPase could be more effectively replaced by their cyanobacterial or chloroplast counterparts, although the sequence identity or even similarity is very low. We attribute these findings to the different roles of these subunits in F1: The large alpha and beta subunits contribute to the catalytic centers of the enzyme, a function rendering them very sensitive to even minor changes. For the smaller delta and epsilon subunits it was sufficient to maintain a certain tertiary structure during evolution, with little emphasis on the conservation of particular amino acids.

ATP synthase: a tentative structural model.

Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton- or sodium-motive force across the respective coupling membrane in Archaea, Bacteria and Eucarya. Cation flow through the intrinsic membrane portion of this enzyme (Fo, subunits ab2c9-12) and substrate turnover in the headpiece (F1, subunits alpha3beta3 gammadeltaepsilon) are mechanically coupled by the rotation of subunit gamma in the center of the catalytic hexagon of subunits (alphabeta)3 in F1. ATP synthase is the smallest rotatory engine in nature. With respect to the headpiece alone, it probably operates with three steps. Partial structures of six out of its at least eight different subunits have been published and a 3-dimensional structure is available for the assembly (alphabeta)3gamma. In this article, we review the available structural data and build a tentative topological model of the holoenzyme. The rotor portion is proposed to consist of a wheel of at least nine copies of subunits c, epsilon and a portion of gamma as a spoke, and another portion of gamma as a crankshaft. The stator is made up from a, the transmembrane portion of b2, delta and the catalytic hexagon of (alphabeta)3. As an educated guess, the model may be of heuristic value for ongoing studies on this fascinating electrochemical-to-mechanical-to-chemical transducer.

Reassembly of Synechocystis sp. PCC 6803 F1-ATPase from its over-expressed subunits.

Subunits alpha, beta, and gamma of the F1-part of cyanobacterial F0F1-ATPase have been cloned into expression vectors. Over-expressed subunit beta was found soluble in the cytoplasmic fraction of Escherichia coli cells under appropriate culture and induction conditions and was purified from cell extracts. Recombinant alpha and gamma subunits precipitated into inclusion bodies and had to be solubilized, purified and refolded. The correct folding and functional integrity of the alpha and beta subunits was monitored by their ability to bind nucleotides. Active cyanobacterial F1-ATPase was assembled from its purified subunits alpha, beta, gamma, delta and epsilon. The reassembled enzyme reconstituted ATP synthesis in F1-depleted thylakoid membranes of Synechocystis sp. PCC 6803 and hydrolyzed ATP.

Three-stepped rotation of subunits gamma and epsilon in single molecules of F-ATPase as revealed by polarized, confocal fluorometry.

The proton translocating ATP synthase is conceived as a rotatory molecular engine. ATP hydrolysis by its headpiece, CF1, drives the rotation of subunit gamma relative to the hexagonally arranged large subunits, (alphabeta)3. We investigated transition states of the rotatory drive by polarized confocal fluorometry (POCOF) as applied to single molecules of engineered, immobilized and load-free spinach-CF1. We found that the hydrolysis of ATP caused the stepped and sequential progression of subunit gamma through three discrete angular positions, with the transition states of gamma being too shortlived for detection. We also observed the stepped motion of epsilon, whereas delta was immobile as (alphabeta)3.

F-ATPase: specific observation of the rotating c subunit oligomer of EF(o)EF(1).

The rotary motion in response to ATP hydrolysis of the ring of c subunits of the membrane portion, F(o), of ATP synthase, F(o)F(1), is still under contention. It was studied with EF(o)EF(1) (Escherichia coli) using microvideography with a fluorescent actin filament. To overcome the limited specificity of actin attachment through a Cys-maleimide couple which might have hampered the interpretation of previous work, we engineered a 'strep-tag' sequence into the C-terminal end of subunit c. It served (a) to purify the holoenzyme and (b) to monospecifically attach a fluorescent actin filament to subunit c. EF(o)EF(1) was immobilized on a Ni-NTA-coated glass slide by the engineered His-tag at the N-terminus of subunit beta. In the presence of MgATP we observed up to five counterclockwise rotating actin filaments per picture frame of 2000 microm(2) size, in some cases yielding a proportion of 5% rotating over total filaments. The rotation was unequivocally attributable to the ring of subunit c. The new, doubly engineered construct serves as a firmer basis for ongoing studies on torque and angular elastic distortions between F(1) and F(o).

Purification and characterization of the inhibitory subunit (delta) of the ATP-synthase from Micrococcus luteus.

Subunit delta was isolated from the ATP-synthase from Micrococcus luteus strain (ATCC 4698). delta, in the case of M. luteus F0F1-ATPase, acts as an inhibitor of ATP hydrolysis and thus resembles subunits in E. coli and chloroplast ATP-synthase. After treatment with 1.5 M LiCl the ATP-synthase dissociated, and subsequently subunit delta (27 kDa) was purified by hydrophobic interaction chromatography. Inhibition of ATP-synthase lacking delta by addition of delta showed non-competitive kinetics with a Ki of approximately 5.9 nM. Subunit epsilon from chloroplast F1, which corresponds functionally to the M. luteus F0F1-delta, and chloroplast delta were tested for ATPase inhibitory activity by addition to the partially delta-depleted ATP-synthase from M. luteus. CF1-epsilon inhibited M. luteus ATP-synthase up to 80%, whereas CF1-delta did not show any influence.

Inter-subunit rotation and elastic power transmission in F0F1-ATPase.

ATP synthase (F-ATPase) produces ATP at the expense of ion-motive force or vice versa. It is composed from two motor/generators, the ATPase (F1) and the ion translocator (F0), which both are rotary steppers. They are mechanically coupled by 360 degrees rotary motion of subunits against each other. The rotor, subunits gamma(epsilon)C10-14, moves against the stator, (alphabeta)3delta(ab2). The enzyme copes with symmetry mismatch (C3 versus C10-14) between its two motors, and it operates robustly in chimeric constructs or with drastically modified subunits. We scrutinized whether an elastic power transmission accounts for these properties. We used the curvature of fluorescent actin filaments, attached to the rotating c ring, as a spring balance (flexural rigidity of 8.10(-26) N x m2) to gauge the angular profile of the output torque at F0 during ATP hydrolysis by F1. The large average output torque (56 pN nm) proved the absence of any slip. Angular variations of the torque were small, so that the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the three-fold stepping and high activation barrier (>40 kJ/mol) of the driving motor (F1) itself, the rather constant output torque seen by F0 implied a soft elastic power transmission between F1 and F0. It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate under load of the two counteracting and stepping motors/generators.

Planning reaches by evaluating stored postures.

This article describes a theory of the computations underlying the selection of coordinated motion patterns, especially in reaching tasks. The central idea is that when a spatial target is selected as an object to be reached, stored postures are evaluated for the contributions they can make to the task. Weights are assigned to the stored postures, and a single target posture is found by taking a weighted sum of the stored postures. Movement is achieved by reducing the distance between the starting angle and target angle of each joint. The model explains compensation for reduced joint mobility, tool use, practice effects, performance errors, and aspects of movement kinematics. Extensions of the model can account for anticipation and coarticulation effects, movement through via points, and hierarchical control of series of movements.

Detection and subtyping of human herpesvirus-8 in renal transplant patients before and after remission of Kaposi's sarcoma.

BACKGROUND: Kaposi's sarcoma (KS) is a complication of renal transplantation. If the human herpesvirus-8 (HHV-8) causes KS, the virus should be present in all KS lesions and be drastically reduced or cleared from involved tissue on remission of the KS. METHODS: Fourteen renal transplant patients with cutaneous KS, including autopsy material from two cases, were investigated for the presence of HHV-8. A second skin biopsy was taken from 11 survivors, after remission of KS, from normal skin in the same anatomical region as the first biopsy. Remission was induced by reduction or cessation of immunosuppression. A peripheral blood sample was collected simultaneously with the repeat biopsy. A nested polymerase chain reaction assay was used to detect HHV-8 DNA in the biopsy tissue and peripheral blood mononuclear cells followed by direct sequencing of polymerase chain reaction product to detect any nucleotide changes. RESULTS: HHV-8 DNA was detected in all the cutaneous KS and all the visceral KS samples, as well as a number of KS-free organs including the thyroid, salivary gland, and myocardium that have not been described before. Mutations in the viral DNA could be demonstrated in all patients. The mutations found were related more to that seen in AIDS-KS cases than that found in African endemic KS cases. HHV-8 sequences could be detected in follow-up frozen skin biopsies of five patients but were negative in the equivalent formalin-fixed specimens. Viral DNA was also detected in 2 of 11 peripheral blood mononuclear cell samples collected at the time of the follow-up skin biopsies. CONCLUSION: Reduction or withdrawal of immunosuppression allows the immune system to recover sufficiently to reduce viral replication with subsequent viral persistence and low grade viral replication that coincides with clinical remission of the KS lesions. This provides further evidence for the important etiological role played by HHV-8 in the pathogenesis of posttransplant KS.

Identification of env subtypes in fourteen HIV type 1 isolates from south Africa.

PIP: All subtypes of HIV-1 have been identified in Africa. The envelope glycoprotein of the HIV-1 is the most variable region of the virus, with the third variable region, including the V3 loop, a major target of vaccine research. This paper reports the identification of the HIV-1 subtypes present in 14 viral strains, isolated between 1984 and 1992 in South Africa. HIV-1 strains were isolated routinely at Tygerberg Hospital in the Western Cape region, with genomic DNA isolated from virus-infected cultures. After presenting the distance calculations and describing the tree constructions and bootstrap analysis, the authors emphasize the need for ongoing molecular epidemiological analysis of HIV-1 subtypes to track the current epidemic in South Africa. More rapid methods will facilitate subtyping to monitor the circulation and spread of HIV-1 subpopulations in the country.^ieng

Characterization and phylogenetic analysis of South African HIV-1 subtype C accessory genes.

To acquire new knowledge about the genetic diversity and potential impact on vaccine strategies of HIV-1 subtype C in South Africa, we have characterized the vif, vpr, and vpu genes of 15 isolates. Phylogenetic analysis of the genomic fragment encompassing these genes revealed subtype C subclusters, suggesting close relatedness with subtype C strains from other geographic locations and excluded isolation of South African strains. The putative T155 phosphorylation site in the C terminal of Vif was absent in all subtype C sequences. Variation in the predicted amino acid sequences of the three genes further showed strong correlation with other subtype C sequences.