Anaesthesia praCtice for Caesarean dElivery Snapshot Study (ACCESS): Protocol and baseline characteristics of registered centres.

BACKGROUND: Specific guidelines to manage caesarean delivery anaesthesia are lacking. A European multicentre study, ACCESS investigates caesarean delivery anaesthesia management in European centres. In order to identify ACCESS participating centres, a registration survey was created. OBJECTIVE: The aim of the current report is to describe the characteristics of ACCESS study participating centres, the rationale for the ACCESS study and the study methodology. DESIGN AND SETTING: The ACCESS study is a European multicentre cross-sectional study to describe anaesthesia management for caesarean delivery (CD) using a snapshot (2-week) design. The ACCESS registration survey gathered: contact details for National Coordinators (NC); Lead Investigators (LI) per centre; centre annual CD volume; expected no. of CD during 2-week snapshot window; centre practice information; data collection language. The ACCESS registration survey was launched July 2022 (Google Forms, Google Inc., Mountain View, CA, USA) and distributed through personal connections, national and international societies, social media networks, during Euroanaesthesia 2023, through the European Society of Anaesthesiology and Intensive Care newsletter. RESULTS: The ACCESS registration survey identified Lead Investigators for 418 centres, in 32 countries, representing an anticipated number of 15,073 CD cases over the planned 12-month study period. A median (range) of 20 (2 to 400) CD cases are anticipated per centre during the 2-week snapshot window. Most 366/418 (87.6%) centres are small, ≤2000 annual CD cases, 42 are medium 2000-5000 cases and 10 are large, ≥5000 annual CD cases. Registered centres reported in 134 (32.0%) centres that anaesthesia for caesarean delivery is performed mostly by a specialist obstetric anaesthesiologist. CONCLUSION: The ACCESS registration survey revealed variability in volume and CD practice as well as training-levels and staffing among European countries. The ACCESS study (https://www.access-study.org/) aims to generate practice data to guide CD anaesthetic management strategies.

Successful contact tracing systems for COVID-19 rely on effective quarantine and isolation.

Models of contact tracing often over-simplify the effects of quarantine and isolation on disease transmission. We develop a model that allows us to investigate the importance of these factors in reducing the effective reproduction number. We show that the reduction in onward transmission during quarantine and isolation has a bigger effect than tracing coverage on the reproduction number. We also show that intuitively reasonable contact tracing performance indicators, such as the proportion of contacts quarantined before symptom onset, are often not well correlated with the reproduction number. We conclude that provision of support systems to enable people to quarantine and isolate effectively is crucial to the success of contact tracing.

A structured model for COVID-19 spread: modelling age and healthcare inequities.

We use a stochastic branching process model, structured by age and level of healthcare access, to look at the heterogeneous spread of COVID-19 within a population. We examine the effect of control scenarios targeted at particular groups, such as school closures or social distancing by older people. Although we currently lack detailed empirical data about contact and infection rates between age groups and groups with different levels of healthcare access within New Zealand, these scenarios illustrate how such evidence could be used to inform specific interventions. We find that an increase in the transmission rates among children from reopening schools is unlikely to significantly increase the number of cases, unless this is accompanied by a change in adult behaviour. We also find that there is a risk of undetected outbreaks occurring in communities that have low access to healthcare and that are socially isolated from more privileged communities. The greater the degree of inequity and extent of social segregation, the longer it will take before any outbreaks are detected. A well-established evidence for health inequities, particularly in accessing primary healthcare and testing, indicates that Maori and Pacific peoples are at a higher risk of undetected outbreaks in Aotearoa New Zealand. This highlights the importance of ensuring that community needs for access to healthcare, including early proactive testing, rapid contact tracing and the ability to isolate, are being met equitably. Finally, these scenarios illustrate how information concerning contact and infection rates across different demographic groups may be useful in informing specific policy interventions.

Mre11 nuclease and C-terminal tail-mediated DDR functions are required for initiating yeast telomere healing.

Mre11 is a central factor in creating an optimal substrate for telomerase loading and elongation. We have used a G2/M synchronized telomere-healing assay as a tool to separate different functions of Mre11 that are not apparent in null alleles. An analysis of healing efficiencies of several mre11 alleles revealed that both nuclease and C-terminal mutations led to a loss of healing. Interestingly, trans-complementation of the 49 amino acid C-terminal deletion (DeltaC49) and the D16A mutant, deficient in nuclease activity and partially defective in MRX complex formation, restores healing. DeltaC49 provokes Rad53 phosphorylation after treatment with the radiomimetic agent MMS exclusively through the Tel1 pathway, suggesting that a Tel1-mediated function is initiated through the C-terminal tail.

Telomeres in meiotic recombination: the yeast side story.

The aim of this review is threefold. First, we want to report on recent observations on the role of telomeres in the alignment of homolog and non-homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe and the relationship of early telomere clustering to later recombination events. Second, we compare the similarities and differences between synaptic and asynaptic yeasts. Third, we report on the increasing evidence of the effect of meiosis on telomeric sequences that suggest an induction of a specific form of recombination processes termed telomere rapid deletion.

Oligomeric structure of the Bacillus subtilis cell division protein DivIVA determined by transmission electron microscopy.

DivIVA from Bacillus subtilis is a bifunctional protein with distinct roles in cell division and sporulation. During vegetative growth, DivIVA regulates the activity of the MinCD complex, thus helping to direct cell division to the correct mid-cell position. DivIVA fulfils a quite different role during sporulation in B. subtilis when it directs the oriC region of the chromosome to the cell pole before asymmetric cell division. DivIVA is a 19.5 kDa protein with a large part of its structure predicted to form a tropomyosin-like alpha-helical coiled-coil. Here, we present a model for the quaternary structure of DivIVA, based on cryonegative stain transmission electron microscopy images. The purified protein appears as an elongated particle with lateral expansions at both ends producing a form that resembles a 'doggy-bone'. The particle mass estimated from these images agrees with the value of 145 kDa measured by analytical ultracentrifugation suggesting 6- to 8-mers. These DivIVA oligomers serve as building blocks in the formation of higher order assemblies giving rise to strings, wires and, finally, two-dimensional lattices in a time-dependent manner.

Intrachromatid excision of telomeric DNA as a mechanism for telomere size control in Saccharomyces cerevisiae.

We have previously identified a process in the yeast Saccharomyces cerevisiae that results in the contraction of elongated telomeres to wild-type length within a few generations. We have termed this process telomeric rapid deletion (TRD). In this study, we use a combination of physical and genetic assays to investigate the mechanism of TRD. First, to distinguish among several recombinational and nucleolytic pathways, we developed a novel physical assay in which HaeIII restriction sites are positioned within the telomeric tract. Specific telomeres were subsequently tested for HaeIII site movement between telomeres and for HaeIII site retention during TRD. Second, genetic analyses have demonstrated that mutations in RAD50 and MRE11 inhibit TRD. TRD, however, is independent of the Rap1p C-terminal domain, a central regulator of telomere size control. Our results provide evidence that TRD is an intrachromatid deletion process in which sequences near the extreme terminus invade end-distal sequences and excise the intervening sequences. We propose that the Mre11p-Rad50p-Xrs2p complex prepares the invading telomeric overhang for strand invasion, possibly through end processing or through alterations in chromatin structure.

Sarcolipin, the shorter homologue of phospholamban, forms oligomeric structures in detergent micelles and in liposomes.

The human 31-amino acid integral membrane protein sarcolipin (SLN), which regulates the sarcoplasmic reticulum Ca-ATPase in fast-twitch skeletal muscle, was chemically synthesized. Appropriate synthesis and purification strategies were used to achieve high purity and satisfactory yields of this hydrophobic and poorly soluble protein. Structural and functional properties of SLN were analyzed and compared with the homologous region of human phospholamban (PLB) comprising residues Ala(24)-Leu(52) (PLB-(24-52)), the regulatory protein of the cardiac sarcoplasmic reticulum Ca-ATPase. Circular dichroism spectroscopy showed that SLN is a predominantly alpha-helical protein and that the secondary structure is highly resistant to SDS and thermal denaturation. In this respect SLN is remarkably similar to PLB-(24-52). However, SLN is monomeric in SDS gels, whereas PLB-(24-52) shows a monomer-pentamer equilibrium typical for native PLB. Analytical ultracentrifugation experiments revealed that SLN oligomerizes in the presence of the nonionic detergents octylpolyoxyethylene and octyl glucoside in a concentration-dependent manner. No plateau was observed, and a pentameric state was only reached at much higher protein concentrations compared with PLB-(24-52). Chemical cross-linking showed that also in liposomes SLN has the ability to self-associate to oligomers. PLB-(24-52) specifically oligomerized to pentamers in the presence of octylpolyoxyethylene as well as in liposomes at low protein concentrations. In the presence of octylpolyoxyethylene pentamers were the main oligomeric species, whereas in liposomes monomers and dimers were predominant. Increasing the protein concentration led to self-association of PLB-(24-52) pentamers in the presence of octylpolyoxyethylene. Functional reconstitution of Ca-ATPase with PLB-(24-52) and SLN in liposomes showed that both proteins regulate the Ca-ATPase in a similar manner.

Subdomain-specific localization of CLIMP-63 (p63) in the endoplasmic reticulum is mediated by its luminal alpha-helical segment.

The microtubule-binding integral 63 kD cytoskeleton-linking membrane protein (CLIMP-63; former name, p63) of the rough endoplasmic reticulum (ER) is excluded from the nuclear envelope. We studied the mechanism underlying this ER subdomain-specific localization by mutagenesis and structural analysis. Deleting the luminal but not cytosolic segment of CLIMP-63 abrogated subdomain-specific localization, as visualized by confocal microscopy in living cells and by immunoelectron microscopy using ultrathin cryosections. Photobleaching/recovery analysis revealed that the luminal segment determines restricted diffusion and immobility of the protein. The recombinant full-length luminal segment of CLIMP-63 formed alpha-helical 91-nm long rod-like structures as evident by circular dichroism spectroscopy and electron microscopy. In the analytical ultracentrifuge, the luminal segment sedimented at 25.7 S, indicating large complexes. The complexes most likely arose by electrostatic interactions of individual highly charged coiled coils. The findings indicate that the luminal segment of CLIMP-63 is necessary and sufficient for oligomerization into alpha-helical complexes that prevent nuclear envelope localization. Concentration of CLIMP-63 into patches may enhance microtubule binding on the cytosolic side and contribute to ER morphology by the formation of a protein scaffold in the lumen of the ER.

Properties of bacteriophage T4 baseplate protein encoded by gene 8.

Gene product 8 (gp8, 344 amino acids per monomer) of bacteriophage T4 is one of the baseplate structural proteins. We constructed an expression vector of gp8 and developed a method for purification of recombinant protein. CD spectroscopy showed that gp8 is an alpha/beta type structural protein. Its polypeptide chain consists of nearly 40% beta-structure and 15% alpha-helix. These data agree with results of prediction of secondary structure based on the amino acid sequence of the protein. The sedimentation coefficient under standard conditions (S20,w) is 4.6S. Analytical ultracentrifugation results demonstrated that gp8 in solution has two types of oligomers--dimer and tetramer. The tetramer of gp8 may be included in the wedge (1/6 of the baseplate), and the dimer may be an intermediate product of association.

The effect of nucleotides and mitochondrial chaperonin 10 on the structure and chaperone activity of mitochondrial chaperonin 60.

Mitochondrial chaperonins are necessary for the folding of newly imported and stress-denatured mitochondrial proteins. The goal of this study was to investigate the structure and function of the mammalian mitochondrial chaperonin system. We present evidence that the 60 kDa chaperonin (mt-cpn60) exists in solution in dynamic equilibrium between monomers, heptameric single rings and double-ringed tetradecamers. In the presence of ATP and the 10 kDa cochaperonin (mt-cpn10), the formation of a double ring is favored. ADP at very high concentrations does not inhibit malate dehydrogenase refolding or ATP hydrolysis by mt-cpn60 in the presence of mt-cpn10. We propose that the cis (mt-cpn60)14.nucleotide.(mt-cpn10)7 complex is not a stable species and does not bind ADP effectively at its trans binding site.

Stabilization of short collagen-like triple helices by protein engineering.

Recombinant expression of collagens and fragments of collagens is often difficult, as their biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase. Although the use of hydroxyproline-deficient variants offers one possibility to overcome this difficulty, these proteins usually differ markedly in stability when compared with the hydroxyproline-containing analogs. Here, we report a method to stabilize collagen-like peptides by fusing them to the N terminus of the bacteriophage T4 fibritin foldon domain. The isolated foldon domain and the chimeric protein (GlyProPro)(10)foldon were expressed in a soluble form in Escherichia coli. The recombinant proteins and the synthetic (ProProGly)(10) peptide were characterized by circular dichroism (CD) spectroscopy, differential scanning calorimetry, and analytical ultracentrifugation. We show that the foldon domain, which comprises only 27 amino acid residues, forms an obligatory trimer with a high degree of thermal stability. The CD thermal unfolding profiles recorded from foldon are monophasic and completely reversible upon cooling. Similar Van't Hoff and calorimertic enthalpy values of trimer formation indicated a cooperative all-or-none transition. As reported previously, (ProProGly)(10) peptides form collagen triple helices of only moderate stability. When fused to the foldon domain, however, triple helix formation of (GlyProPro)(10) is concentration independent, and the midpoint temperature of the triple helix unfolding is significantly increased. The stabilizing function of the trimeric foldon domain is explained by the close vicinity of its N termini, which induce a high local concentration in the range of 1 M for the C termini of the collagen-like-peptide. Collagen-foldon fusion proteins should be potentially useful to study receptor-collagen interactions.

Divide-and-conquer crystallographic approach towards an atomic structure of intermediate filaments.

Intermediate filaments (IFs) represent an essential component of the cytoskeleton in higher eukaryotic cells. The elementary building block of the IF architecture is an elongated dimer with its dominant central part being a parallel double-stranded alpha-helical coiled coil. Filament formation proceeds via a specific multi-step association of the dimers into the unit-length filaments, which subsequently anneal longitudinally and finally radially compact into mature filaments. To tackle the challenge of a crystallographic structure determination, we have produced and characterised 17 overlapping soluble fragments of human IF protein vimentin. For six fragments ranging in length between 39 and 84 amino acid residues, conditions yielding macroscopic crystals could be established and X-ray diffraction data were collected to the highest resolution limit between 1.4 and 3 A. We expect that solving the crystal structures of these and further fragments will eventually allow us to patch together a molecular model for the full-length vimentin dimer. This divide-and-conquer approach will be subsequently extended to determining the crystal structures of a number of complexes formed by appropriate vimentin fragments, and will eventually allow us to establish the three- dimensional architecture of complete filaments at atomic resolution.

Dissection of a (betaalpha)8-barrel enzyme into two folded halves.

The (betaalpha)8-barrel, which is the most frequently encountered protein fold, is generally considered to consist of a single structural domain. However, the X-ray structure of the imidazoleglycerol phosphate synthase (HisF) from Thermotoga maritima has identified it as a (betaalpha) 8-barrel made up of two superimposable subdomains (HisF-N and HisF-C). HisF-N consists of the four N-terminal (betaalpha) units and HisF-C of the four C-terminal (betaalpha) units. It has been postulated, therefore, that HisF evolved by tandem duplication and fusion from an ancestral half-barrel. To test this hypothesis, HisF-N and HisF-C were produced in Escherichia coli, purified and characterized. Separately, HisF-N and HisF-C are folded proteins, but are catalytically inactive. Upon co-expression in vivo or joint refolding in vitro, HisF-N and HisF-C assemble to the stoichiometric and catalytically fully active HisF-NC complex. These findings support the hypothesis that the (betaalpha)8-barrel of HisF evolved from an ancestral half-barrel and have implications for the folding mechanism of the members of this large protein family.

An intrahelical salt bridge within the trigger site stabilizes the GCN4 leucine zipper.

We previously reported that a helical trigger segment within the GCN4 leucine zipper monomer is indispensable for the formation of its parallel two-stranded coiled coil. Here, we demonstrate that the intrinsic secondary structure of the trigger site is largely stabilized by an intrahelical salt bridge. Removal of this surface salt bridge by a single amino acid mutation induced only minor changes in the backbone structure of the GCN4 leucine zipper dimer as verified by nuclear magnetic resonance. The mutation, however, substantially destabilized the dimeric structure. These findings support the proposed hierarchic folding mechanism of the GCN4 coiled coil in which local helix formation within the trigger segment precedes dimerization.