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Introduction: Problem‐solving skills (PSS) help to provide a systematic approach to dealing with and managing complex problems. The overall aim of this study was to assess the acceptability and feasibility of developing and adapting a prison‐based PSS workbook for adults within a medium‐ and low‐secure hospital. Method: We used the Medical Research Council framework in our participatory mixed methods study incorporating an adapted survey (to identify what types of problems people experience in secure hospitals), a series of three interactive workshops (to co‐produce two case study examples for a workbook) and we gathered feedback from patients and hospital staff on the acceptability and feasibility of the workbook. Data from the survey were used to inform the case study examples, and the feedback from patients and hospital staff was descriptively summarised and the results consolidated. Results: In total, 82 (51%) patients took part in the survey; 22 patients and 49 hospital staff provided feedback on the workbook. The survey results indicated that patients regularly experience problems while in the hospital. Patients reported problems relating to restrictions of freedom and boredom. The workshops produced two case studies for the workbooks, with mainly positive patient and staff feedback. More work is required to improve the visual representation of the characters in the case studies, the amount and content of the language and the mechanism of the intervention delivery. Conclusion: The adaptation process proved acceptable and feasible to both patients and staff. The co‐production methodology for the workbook and feedback from patients and staff was an effective way of iteratively refining the materials to ensure that they were both meaningful and acceptable to staff and patients. Subsequent work is required to develop the workbook and evaluate the feasibility of the intervention delivery, recruitment rates, uptake and adherence to the PSS using a randomised controlled trial. Patient or Public Contribution: At each stage of the project consultation with patients and/or hospital staff was involved. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis ( Mtb), the causative agent of TB, remains a serious world health problem owing to limitations of the available drugs and the emergence of resistant strains. In this context, key biosynthetic enzymes from Mtb are attractive targets for the development of new therapeutic drugs. Here, the 1.5 Å resolution crystal structure of Mtb phosphoserine aminotransferase (MtbPSAT) in complex with its cofactor, pyridoxal 5′-phosphate (PLP), is reported. MtbPSAT is an essential enzyme in the biosynthesis of serine and in pathways of one-carbon metabolism. The structure shows that although the Mtb enzyme differs substantially in sequence from other PSAT enzymes, its fold is conserved and its PLP-binding site is virtually identical. Structural comparisons suggest that this site remains unchanged throughout the catalytic cycle. On the other hand, PSAT enzymes are obligate dimers in which the two active sites are located in the dimer interface and distinct differences in the MtbPSAT dimer are noted. These impact on the substrate-binding region and access channel and suggest options for the development of selective inhibitors. [ABSTRACT FROM AUTHOR]

To report time from the onset of symptoms to hospital presentation in Australian and New Zealand patients with subsequently confirmed acute coronary syndrome, and to identify factors associated with prehospital delay time in these patients. Patients with coronary artery disease enrolled in a randomized clinical trial testing an intervention to reduce delay in responding to acute coronary syndrome symptoms had been followed for 24 months. In cases of admission to the ED for possible acute coronary syndrome, medical records were reviewed to determine the diagnosis, prehospital delay time, mode of transport to the hospital and aspirin use before admission. Clinical and demographic data were taken from the trial database. Patients ( n= 140) had an average (SD) age of 67.3 (11.5) years; 36% were female. Two-thirds of patients went to hospital by ambulance and 89.3% had a final diagnosis of unstable angina. The median time from onset of symptoms to arrival at the ED was 2 h and 25 min (interquartile range 1:25-4:59); 12.1% arrived ≤ 1 h and 66% within 4 h. Multiple linear regression analysis showed that use of ambulance (Beta = 0.247, P= 0.012) and younger age (Beta = 0.198, P= 0.043) were independent predictors of shorter delay times. The time from the onset of symptoms to hospital presentation was too long for maximal benefit from treatment in most patients. Further efforts are needed to reduce treatment-seeking delay in response to symptoms of acute coronary syndrome. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a major pathogen that produces a family of 14 staphylococcal superantigen-like (SSL) proteins, which are structurally similar to superantigens but do not stimulate T cells. SSL11 is one member of the family that is found in all staphylococcal strains. Recombinant SSL11 bound to granulocytes and monocytes through a sialic acid-dependent mechanism and was rapidly internalized. SSL11 also bound to sialic acid-containing glycoproteins, such as the Fc receptor for IgA (FcαRI) and P-selectin glycoprotein ligand-1 (PSGL-1), and inhibited neutrophil attachment to a P-selectin-coated surface. Biosensor analysis of two SSL11 alleles binding to sialyl Lewis X [sLex– Neu5Acα2-3Galβ1-4(Fuc1-3)GlcNAc] coupled to bovine serum albumin gave dissociation constants of 0.7 and 7 μm respectively. Binding of SSL11 to a glycan array revealed specificity for glycans containing the trisaccharide sialyllactosamine (sLacNac – Neu5Acα2-3Galβ1-4GlcNAc). A 1.6 Å resolution crystal structure of SSL11 complexed with sLex revealed a discrete binding site in the C-terminal β-grasp domain, with predominant interactions with the sialic acid and galactose residues. A single amino acid mutation in the carbohydrate binding site abolished all SSL11 binding. Thus, SSL11 is a staphylococcal protein that targets myeloid cells by binding sialyllactosamine-containing glycoproteins. [ABSTRACT FROM AUTHOR]

Iron uptake by humans depends on the ability of the serum protein transferrin (Tf) to bind iron as Fe3+ with high affinity but reversibly. Iron release into cells occurs through receptor-mediated endocytosis, aided by the lower endosomal pH of about 5.5. The protonation of a hydrogen-bonded pair of lysines, Lys206 and Lys296, adjacent to the N-lobe iron site of Tf has been proposed to create a repulsive interaction that stimulates domain opening and iron release. The crystal structures of two mutants, K206E (in which Lys206 is mutated to Glu) and K206E/K296E (in which both lysines are mutated to Glu), have been determined. The K206E structure (2.6 Å resolution; R = 0.213, Rfree = 0.269) shows that a salt bridge is formed between Glu206 and Lys296, thus explaining the drastically slower iron release by this mutant. The K206E/K296E double-mutant structure (2.8 Å resolution; R = 0.232, Rfree = 0.259) shows that the Glu296 side chain moves away from Glu206, easing any repulsive interaction and instead interacting with the iron ligand His249. The evident conformational flexibility is consistent with an alternative model for the operation of the dilysine pair in iron release in which it facilitates concerted proton transfer to the tyrosine ligand Tyr188 as one step in the weakening of iron binding. [ABSTRACT FROM AUTHOR]

Large-scale structural genomics centres rely heavily on robotics to ensure that maximum throughput is achieved. However, the size and cost of these approaches is out of the reach of most academic structural biology efforts. A major challenge for such groups is to adapt current high-throughput schemes to a reasonable scale with the resources available. A flexible medium-throughput approach has been developed that is suitable for typical academic research groups. Following nested PCR, targets are routinely cloned into two Gateway expression vectors (pDESTIS for an N-terminal GST tag and pDEST17 for an N-terminal His tag). Expression of soluble recombinant protein in Escherichia coli is rapidly assessed in 96-well format. An eight-probe sonicator is utilized and a six- buffer lysis screen was incorporated to enhance solubility. Robotics is reserved for crystallization, since this is the key bottleneck for crystallography. Screening proteins with a 480- condition protocol using a Cartesian nanolitre-dispensing robot has increased crystallization success markedly, with an overall success rate (structures solved out of proteins screened) of 19%. The methods are robust and economical - with the exception of the crystallization robot, investment in additional equipment has been minimal at US$9000. All protocols are designed for individuals so that graduate students and postdoctoral fellows gain expertise in every aspect of the structural pipeline, from cloning to crystallization. [ABSTRACT FROM AUTHOR]

Bacterial resistance to the aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, APH(2")-Ib, has been cloned and the protein (comprising 299 amino-acid residues) expressed in Escherichia coil, purified and crystallized in the presence of 16%(w/v) PEG 3350 and gentamicin. The crystals belong to the monoclinic space group P21, with approximate unit-cell parameters a = 79.7, b = 58.8, c = 81.4 Å, β = 98.4°, and preliminary X-ray diffraction analysis is consistent with the presence of two molecules in the asymmetric unit. Synchrotron diffraction data to approximately 2.65 Å resolution were collected from a native APH(2")-Ib crystal at beamline BL9-2 at SSRL (Stanford, CA, USA). Selenium-substituted crystals have also been produced and structure determination is proceeding. [ABSTRACT FROM AUTHOR]

The enzymes of the shikimate pathway are attractive targets for new-generation antimicrobial agents. The first step of this pathway is catalysed by 3-deoxy- D-arabino-heptulosonate-7-phosphate (DAH7P) synthase and involves the condensation of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form DAH7P. DAH7P synthases have been classified into two apparently evolutionarily unrelated types and whereas structural data have been obtained for the type I DAH7P synthases, no structural information is available for their type II counterparts. The type II DAH7P synthase from Mycobacterium tuberculosis was co-expressed as native and selenomethionine-substituted protein with the Escherichia coli chaperonins GroEL and GroES in EL coli, purified and crystallized. Native crystals of M. tuberculosis DAH7P synthase belong to space group P3121 or P3221 and diffract to 2.5 Å, with unit-cell parameters a = b = 203.61, c = 66.39 Å. There are either two or three molecules in the asymmetric unit. Multiwavelength anomalous diffraction (MAD) phasing using selenomethionine-substituted protein is currently under way. [ABSTRACT FROM AUTHOR]

Hemoglobin (Hb) Bart's is present in the red blood cells of millions of people worldwide who suffer from α-thalassemia. α-Thalassemia is a disease in which there is a deletion of one or more of the four α-chain genes, and excess γ and β chains spontaneously form homotetramers. The γ4 homotetrameric protein known as Hb Bart's is a stable species that exhibits neither a Bohr effect nor heme-heme cooperativity. Although Hb Bart's has a higher O2 affinity than either adult (α2β2) or fetal (α2γ2) Hbs, it has a lower affinity for O2 than HbH (β4). To better understand the association and ligand binding properties of the γ4 tetramer, we have solved the structure of Hb Bart's in two different oxidation and ligation states. The crystal structure of ferrous carbonmonoxy (CO) Hb Bart's was determined by molecular replacement and refined at 1.7 Å resolution ( R = 21.1%, Rfree = 24.4%), and that of ferric azide (N3−) Hb Bart's was similarly determined at 1.86 Å resolution ( R = 18.4%, Rfree = 22.0%). In the carbonmonoxy-Hb structure, the CO ligand is bound at an angle of 140°, and with an unusually long Fe-C bond of 2.25 Å. This geometry is attributed to repulsion from the distal His63 at the low pH of crystallization (4.5). In contrast, azide is bound to the oxidized heme iron in the methemoglobin crystals at an angle of 112°, in a perfect orientation to accept a hydrogen bond from His63. Compared to the three known quaternary structures of human Hb (T, R, and R2), both structures most closely resemble the R state. Comparisons with the structures of adult Hb and HbH explain the association and dissociation behaviour of Hb homotetramers relative to the heterotetrameric Hbs. [ABSTRACT FROM AUTHOR]

A variety of human haemoglobins (Hbs) are produced at different stages of human development, including three embryonic Hbs, foetal Hb and adult Hb. All are heterotetramers. During crystallization experiments on human embryonic Hb Portland (ζ2γ2), it was discovered by crystallographic and biochemical analysis that the homotetramer Hb Bart's (γ4) preferentially crystallizes from ζ2γ2 solutions below pH 5. This results from dissociation of Hb Portland into γ2 dimers and ζ monomers and has interesting implications for subunit interactions and tetramer stability in Hbs. It also makes possible a full crystallographic analysis of Hb Bart's, which is of considerable medical significance because of its presence in the red blood cells of millions of people worldwide who suffer from α-thalassaemia. [ABSTRACT FROM AUTHOR]

Methylmalonyl-CoA epimerase (MMCE) is an enzyme that inter-converts the R and S epimers of methylmalonyl-CoA in the pathway that links propionyl-CoA with succinyl-CoA. This is used for both biosynthetic and degradative processes, including the breakdown of odd-numbered fatty acids and some amino acids. The enzyme has been expressed in Escherichia coli both as the native enzyme and as its selenomethionine (SeMet) derivative. Crystals of both forms have been obtained by vapour diffusion using monomethylether PEG 2000 as precipitant. The native MMCE crystals are orthorhombic, with unit-cell parameters a = 56.0, b = 114.0, c 156.0 Å, and the SeMet- MMCE crystals are monoclinic, with unit-cell parameters a = 43.6, b = 78.6, c = 89.4 Å, β = 92.0°; both diffract to better than 2.8 Å resolution. [ABSTRACT FROM AUTHOR]

Human bile-salt dependent lipase (BSDL), secreted into both the digestive tract and human milk, is integral to the effective absorption of dietary lipids. In attempts to obtain crystals suitable for high-resolution X-ray crystallographic studies, various forms of the enzyme have been crystallized, including native and desialidated human milk BSDL and both intact recombinant BSDL and a truncated form lacking the heavily glycosylated C-terminal repeat region. Trigonal crystals of native BSDL, with unit-cell parameters a = b = 90.0, c = 156.1 Å, were obtained using 15-20%(w/v) PEG 8000 as precipitant. These crystals diffract to 3.5 A along the unique axis, but to only 5-7 A in orthogonal directions. Crystals of recombinant truncated BSDL grown from 15-20%(w/v) PEG 6000 are ortho- rhombic, space group P212121, with unit-cell parameters a = 59.2, b = 90.0, c = 107.7 Å, and diffract to 2.6 Å resolution. These are suitable for structural analysis by X-ray crystallography. [ABSTRACT FROM AUTHOR]

A correction to the article "The crystal structure of staphylococcal superantigen-like protein 11 in complex with sialyl Lewis X reveals the mechanism for cell binding and immune inhibition," that was published in 2007 issue, is presented.