Elucidation of the K32 Capsular Polysaccharide Structure and Characterization of the KL32 Gene Cluster of Acinetobacter baumannii LUH5549.

Capsular polysaccharide (CPS), isolated from Acinetobacter baumannii LUH5549 carrying the KL32 capsule biosynthesis gene cluster, was studied by sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. The K32 CPS was found to be composed of branched pentasaccharide repeats (K units) containing two residues of ß-D-GalpNAc and one residue of ß-D-GlcpA (ß-D-glucuronic acid) in the main chain and one residue each of ß-D-Glcp and α-D-GlcpNAc in the disaccharide side chain. Consistent with the established CPS structure, the KL32 gene cluster includes genes for a UDP-glucose 6-dehydrogenase (Ugd3) responsible for D-GlcA synthesis and four glycosyltransferases that were assigned to specific linkages. Genes encoding an acetyltransferase and an unknown protein product were not involved in CPS biosynthesis. Whilst the KL32 gene cluster has previously been found in the global clone 2 (GC2) lineage, LUH5549 belongs to the sequence type ST354, thus demonstrating horizontal gene transfer between these lineages.

Neural cell responses to wear debris from metal-on-metal total disc replacements.

PURPOSE: Total disc replacements, comprising all-metal articulations, are compromised by wear and particle production. Metallic wear debris and ions trigger a range of biological responses including inflammation, genotoxicity, cytotoxicity, hypersensitivity and pseudotumour formation, therefore we hypothesise that, due to proximity to the spinal cord, glial cells may be adversely affected. METHODS: Clinically relevant cobalt chrome (CoCr) and stainless steel (SS) wear particles were generated using a six-station pin-on-plate wear simulator. The effects of metallic particles (0.5-50 µm3 debris per cell) and metal ions on glial cell viability, cellular activity (glial fibrillary acidic protein (GFAP) expression) and DNA integrity were investigated in 2D and 3D culture using live/dead, immunocytochemistry and a comet assay, respectively. RESULTS: CoCr wear particles and ions caused significant reductions in glial cell viability in both 2D and 3D culture systems. Stainless steel particles did not affect glial cell viability or astrocyte activation. In contrast, ions released from SS caused significant reductions in glial cell viability, an effect that was especially noticeable when astrocytes were cultured in isolation without microglia. DNA damage was observed in both cell types and with both biomaterials tested. CoCr wear particles had a dose-dependent effect on astrocyte activation, measured through expression of GFAP. CONCLUSIONS: The results from this study suggest that microglia influence the effects that metal particles have on astrocytes, that SS ions and particles play a role in the adverse effects observed and that SS is a less toxic biomaterial than CoCr alloy for use in spinal devices. These slides can be retrieved under Electronic Supplementary Material.

Intermittent fasting in Type 2 diabetes mellitus and the risk of hypoglycaemia: a randomized controlled trial.

AIMS: To establish whether the risk of hypoglycaemia is greater with 2 consecutive days of very-low-calorie diet compared with 2 non-consecutive days of very-low-calorie diet in people with Type 2 diabetes. METHODS: This was a non-blinded randomized parallel group interventional trial of intermittent fasting in adults. The participants had a BMI of 30-45 kg/m2 , Type 2 diabetes treated with metformin and/or hypoglycaemic medications and an HbA1c concentration of 50-86 mmol/mol (6.7-10%). The participants followed a 2092-2510-kJ diet on 2 days per week for 12 weeks. A total of 41 participants were randomized 1:1 to consecutive (n=19) or non-consecutive (n=22) day fasts, of whom 37 (n=18 and n=19, respectively) were included in the final analysis. The primary outcome was difference in the rate of hypoglycaemia between the two study arms. Secondary outcomes included change in diet, quality of life, weight, lipid, glucose and HbA1c levels, and liver function. RESULTS: The mean hypoglycaemia rate was 1.4 events over 12 weeks. Fasting increased the rate of hypoglycaemia despite medication reduction (RR 2.05, 95% CI 1.17 to 3.52). There was no difference between fasting on consecutive days and fasting on non-consecutive days (RR 1.54, 95% CI 0.35 to 6.11). Improvements in weight, HbA1c , fasting glucose and quality of life were experienced by participants in both arms. CONCLUSIONS: In individuals with Type 2 diabetes on hypoglycaemic medications, fasting of any type increased the rate of hypoglycaemia. With education and medication reduction, fewer than expected hypoglycaemic events occurred. Although it was not possible to determine whether fasting on consecutive days increased the risk of hypoglycaemia, an acceptable rate was observed in both arms.

Structure of the K82 Capsular Polysaccharide from Acinetobacter baumannii LUH5534 Containing a d-Galactose 4,6-Pyruvic Acid Acetal.

Type K82 capsular polysaccharide (CPS) was isolated from Acinetobacter baumannii LUH5534. The structure of a linear tetrasaccharide repeating unit of the CPS was established by sugar analysis along with one- and two-dimensional 1H and 13C NMR spectroscopy. Proteins encoded by the KL82 capsule gene cluster in the genome of LUH5534 were assigned to roles in the synthesis of the K82 CPS. In particular, functions were assigned to two new glycosyltransferases (Gtr152 and Gtr153) and a novel pyruvyltransferase, Ptr5, responsible for the synthesis of d-galactose 4,6-(R)-pyruvic acid acetal.

Non-destructive monitoring of viability in an ex vivo organ culture model of osteochondral tissue.

Organ culture is an increasingly important tool in research, with advantages over monolayer cell culture due to the inherent natural environment of tissues. Successful organ cultures must retain cell viability. The aim of this study was to produce viable and non-viable osteochondral organ cultures, to assess the accumulation of soluble markers in the conditioned medium for predicting tissue viability. Porcine femoral osteochondral plugs were cultured for 20 days, with the addition of Triton X-100 on day 6 (to induce necrosis), camptothecin (to induce apoptosis) or no toxic additives. Tissue viability was assessed by the tissue destructive XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide tetrazolium salt) assay method and LIVE/DEAD® staining of the cartilage at days 0, 6 and 20. Tissue structure was assessed by histological evaluation using haematoxylin & eosin and safranin O. Conditioned medium was assessed every 3-4 days for glucose depletion, and levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP), glycosaminoglycans (GAGs), and matrix metalloproteinase (MMP)-2 and MMP-9. Necrotic cultures immediately showed a reduction in glucose consumption, and an immediate increase in LDH, GAG, MMP-2 and MMP-9 levels. Apoptotic cultures showed a delayed reduction in glucose consumption and delayed increase in LDH, a small rise in MMP-2 and MMP-9, but no significant effect on GAGs released into the conditioned medium. The data showed that tissue viability could be monitored by assessing the conditioned medium for the aforementioned markers, negating the need for tissue destructive assays. Physiologically relevant whole- or part-joint organ culture models, necessary for research and pre-clinical assessment of therapies, could be monitored this way, reducing the need to sacrifice tissues to determine viability, and hence reducing the sample numbers necessary.

The effect of anterior-posterior shear load on the wear of ProDisc-L TDR.

The current wear-testing standard (ISO18192-1) for total disc replacement (TDR) requires only four degrees of freedom (DOF) inputs: axial load, flexion-extension, lateral bending and axial rotation. The study aim was to assess the effect of an additional DOF, anterior-posterior (AP) shear on the wear of the ProDisc-L TDR. A 5DOF simulator was used to test ProDisc-L implants under 4DOF and 5DOF conditions. The 4DOF conditions were defined by ISO18192-1 whilst the 5DOF used ISO18192-1 conditions with the addition of an AP load of +175 and -140 N (anterior and posterior, respectively), extrapolated from in vivo data. The implants were mounted such that the polyethylene insert could be removed for gravimetric measurements. Tests were run using bovine serum (15 g/l protein concentration) as a lubricant for five million cycles (MC), with measurements repeated every 1 MC. The mean wear rate in the 4DOF test was 12.7 +/- 2.1 mg/MC compared to 11.6 +/- 1.2 mg/MC in the 5DOF test. There were marked differences in the wear scars between 4DOF and 5DOF simulations. With 4DOF, wear scars were centralised on the dome of the insert, whilst 5DOF scars were larger, breaching the anterior rim of the dome causing deformation at the edge. The 4DOF wear test showed similar gravimetric wear rates to previously published ISO-tested TDRs. The addition of AP load was found to have no significant effect on the overall wear rate. However, there were pronounced differences in the respective wear scars, which highlights the need for more research in order to understand the factors that influence wear of TDR.

Intermittent fasting 5:2 diet: What is the macronutrient and micronutrient intake and composition?

BACKGROUND & AIM: Intermittent fasting (IF) is a dietary intervention that has been investigated as an alternative weight-loss diet due to conventional approaches having poor long-term adherence. However, the macronutrient and micronutrient intake and composition of IF diets have been overlooked. The primary aim of this study was to describe the macronutrient and micronutrient intake of individuals following the 5:2 intermittent fasting diet (IF 5:2). METHODS: Thirty eight overweight and obese participants were included from two previous studies of IF 5:2. The participants selected included 27 males and 11 females, with and without Type 2 Diabetes. The dietary intervention, IF 5:2, consisted of two days per week fasting, either consecutive or non-consecutive, and five days per week of habitual intake. Prospectively completed 4-day estimated food records were used to assess macronutrient and micronutrient intake at baseline and week six. The 4-day records were weighted to give a mean daily intake during IF 5:2. RESULTS: During IF 5:2 the median (25th, 75th quartile) daily macronutrient composition was 22 (19, 24)% from protein, 33 (29, 37)% from fat and 39 (36, 43)% from carbohydrates. The intake (g/d) of carbohydrates and fibre decreased significantly from baseline to week six (p < 0.001) as well as on fasting days compared to non-fasting days (p < 0.001). The intake of calcium, zinc, magnesium and potassium were lower than recommended guidelines. Sodium intake exceeded the suggested daily target. On fasting days, the percent of total energy from protein significantly increased from 21% to 25% (p = 0.02). Despite intake being unrestricted on non-fasting days the energy intake decreased by week six when compared with baseline. CONCLUSION: The composition of IF 5:2 was a high protein, moderate fat, low carbohydrate diet with a low fibre intake. Some micronutrients have lower than recommended intake. However, overall IF 5:2 is a safe acceptable weight-loss diet strategy.

The impact of intra-operative transoesophageal echocardiography on cardiac surgical practice.

The use of transoesophageal echocardiography during cardiac surgery has increased dramatically and it is now widely accepted as a routine monitoring and diagnostic tool. A prospective study was carried out between September 2004 and September 2007, and included all patients in whom intra-operative echocardiography was performed, 2 473 (44%) out of a total of 5 591 cases. Changes to surgery were subdivided into predictable (where echocardiographic examination was planned specifically to guide surgery) and unpredictable (new pathology not diagnosed pre-operatively). A change in the planned surgical procedure was documented in 312 (15%) cases. In 216 (69%) patients the changes were predictable and in 96 (31%) they were unpredictable. The number of predictable changes increased between 2004-5 and 2006-7 (8% vs 13%, p = 0.025). In these cases, intra-operative echocardiography was specifically requested by the surgeon to help determine the operative intervention. This has implications for consent and operative risk, which have yet to be fully determined.

The accuracy and precision of a micro computer tomography volumetric measurement technique for the analysis of in-vitro tested total disc replacements.

Total disc replacements (TDRs) in the spine have been clinically successful in the short term, but there are concerns over long-term failure due to wear, as seen in other joint replacements. Simulators have been used to investigate the wear of TDRs, but only gravimetric measurements have been used to assess material loss. Micro computer tomography (microCT) has been used for volumetric measurement of explanted components but has yet to be used for in-vitro studies with the wear typically less than < 20 mm3 per 10(6) cycles. The aim of this study was to compare microCT volume measurements with gravimetric measurements and to assess whether microCT can quantify wear volumes of in-vitro tested TDRs. microCT measurements of TDR polyethylene cores were undertaken and the results compared with gravimetric assessments. The effects of repositioning, integration time, and scan resolution were investigated. The best volume measurement resolution was found to be +/- 3 mm3, at least three orders of magnitude greater than those determined for gravimetric measurements. In conclusion, the microCT measurement technique is suitable for quantifying in-vitro TDR polyethylene wear volumes and can provide qualitative data (e.g. wear location), and also further quantitative data (e.g. height loss), assisting comparisons with in-vivo and ex-vivo data. It is best used alongside gravimetric measurements to maintain the high level of precision that these measurements provide.

Transposon Tn21, flagship of the floating genome.

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.

Development of specimen-specific finite element models of human vertebrae for the analysis of vertebroplasty.

The aim of this study was to determine the accuracy of specimen-specific finite element models of untreated and cement-augmented vertebrae by direct comparison with experimental results. Eleven single cadaveric vertebrae were imaged using micro computed tomography (microCT) and tested to failure in axial compression in the laboratory. Four of the specimens were first augmented with PMMA cement to simulate a prophylactic vertebroplasty. Specimen-specific finite element models were then generated using semi-automated methods. An initial set of three untreated models was used to determine the optimum conversion factors from the image data to the bone material properties. Using these factors, the predicted stiffness and strength were determined for the remaining specimens (four untreated, four augmented). The model predictions were compared with the corresponding experimental data. Good agreement was found with the non-augmented specimens in terms of stiffness (root-mean-square (r.m.s.) error 12.9 per cent) and strength (r.m.s. error 14.4 per cent). With the augmented specimens, the models consistently overestimated both stiffness and strength (r.m.s. errors 65 and 68 per cent). The results indicate that this method has the potential to provide accurate predictions of vertebral behaviour prior to augmentation. However, modelling the augmented bone with bulk material properties is inadequate, and more detailed modelling of the cement region is required to capture the bone-cement interactions if the models are to be used to predict the behaviour following vertebroplasty.

Recovery rate data for silicon nitride nanoparticle isolation using sodium polytungstate density gradients.

The average recovery rate of silicon nitride nanoparticles isolated from serum using the method detailed in previous article "A novel method for isolation and recovery of ceramic nanoparticles and metal wear debris from serum lubricants at ultra-low wear rate" (Lal et al., 2016) [1] was tested gravimetrically by weighing particles doped into serum before and after the isolation process. An average recovery rate of approximately 89.6% (± 7.1 SD) was achieved.

Development and optimisation data of a tissue digestion method for the isolation of orthopaedic wear particles.

The data contained within this article relate to several enzymatic tissue digestion experiments which were performed to produce an optimised protocol for the digestion of tissue samples. The digestion experiments involved a total of four different digestion protocols. The first protocol involved digestion with proteinase K, without the use of glycine. The second protocol involved digestion with proteinase K in the presence of glycine. The third protocol consisted of proteinase K digestion in the presence of glycine, with more frequent enzyme replenishment. The final protocol was similar to the third protocol but included a papain digestion stage prior to digestion with proteinase K. The data contained within this article are photographs of tissue samples which were captured at key stages of the four protocols and written descriptions based on visual observation of the tissue samples, which document the appearance of the tissue digests.

Validation of a novel particle isolation procedure using particle doped tissue samples.

A novel particle isolation method for tissue samples was developed and tested using particle-doped peri-articular tissues from ovine cadavers. This enabled sensitivity of the isolation technique to be established by doping tissue samples of 0.25 g with very low particle volumes of 2.5 µm3 per sample. Image analysis was used to verify that the method caused no changes to particle size or morphologies.

Recovery of low volumes of wear debris from rat stifle joint tissues using a novel particle isolation method.

Less than optimal particle isolation techniques have impeded analysis of orthopaedic wear debris in vivo. The purpose of this research was to develop and test an improved method for particle isolation from tissue. A volume of 0.018 mm3 of clinically relevant CoCrMo, Ti-6Al-4V or Si3N4 particles was injected into rat stifle joints for seven days of in vivo exposure. Following sacrifice, particles were located within tissues using histology. The particles were recovered by enzymatic digestion of periarticular tissue with papain and proteinase K, followed by ultracentrifugation using a sodium polytungstate density gradient. Particles were recovered from all samples, observed using SEM and the particle composition was verified using EDX, which demonstrated that all isolated particles were free from contamination. Particle size, aspect ratio and circularity were measured using image analysis software. There were no significant changes to the measured parameters of CoCrMo or Si3N4 particles before and after the recovery process (KS tests, p > 0.05). Titanium particles were too few before and after isolation to analyse statistically, though size and morphologies were similar. Overall the method demonstrated a significant improvement to current particle isolation methods from tissue in terms of sensitivity and efficacy at removal of protein, and has the potential to be used for the isolation of ultra-low wearing total joint replacement materials from periprosthetic tissues. STATEMENT OF SIGNIFICANCE: This research presents a novel method for the isolation of wear particles from tissue. Methodology outlined in this work would be a valuable resource for future researchers wishing to isolate particles from tissues, either as part of preclinical testing, or from explants from patients for diagnostic purposes. It is increasingly recognised that analysis of wear particles is critical to evaluating the safety of an orthopaedic device.

The biomechanical response of spinal cord tissue to uniaxial loading.

The spinal cord is an integral component of the spinal column and is prone to physical injury during trauma or more long-term pathological insults. The development of computational models to simulate the cord-column interaction during trauma is important in developing a proper understanding of the injury mechanism. Such models would be invaluable in seeking both preventive strategies that reduce the propensity for injury and identifying specific treatment regimes. However, these developments are hampered by the limited information available on the structural and mechanical properties of this soft tissue owing to the difficulty in handling this material in a cadaveric situation. The purpose of the present paper is to report the rapid deterioration in the quality of the tissues once excised, which provides a further challenge to the successful elucidation of the structural properties of the tissue. In particular, the tangent modulus of the tissue is seen to increase sharply over a period of 72 h.

Wear simulation of a polyethylene-on-metal cervical total disc replacement under different concentrations of bovine serum lubricant.

Metal-on-polyethylene total disc replacements have been an alternative to spinal fusion in the lumbar spine under certain indications for more than a decade. Recently, cervical total disc replacement has also become an alternative to cervical fusion. Knowledge acquired from years of in vitro simulator studies on other joint replacements has highlighted the risks associated with premature wear due to unforeseen adverse clinical conditions and the effect of particulate debris on surrounding natural tissues. Having no evidence of the type and composition of the lubricating fluid that will result after spinal arthroplasty, a study on the effects of lubricant serum concentration was undertaken. The wear rate was shown to be inversely proportional to protein content of the serum over a range of 50%-3% bovine serum to water concentration.

A novel method for isolation and recovery of ceramic nanoparticles and metal wear debris from serum lubricants at ultra-low wear rates.

UNLABELLED: Ceramics have been used to deliver significant improvements in the wear properties of orthopaedic bearing materials, which has made it challenging to isolate wear debris from simulator lubricants. Ceramics such as silicon nitride, as well as ceramic-like surface coatings on metal substrates have been explored as potential alternatives to conventional implant materials. Current isolation methods were designed for isolating conventional metal, UHMWPE and ceramic wear debris. In this paper, we describe a methodology for isolation and recovery of ceramic or ceramic-like coating particles and metal wear particles from serum lubricants under ultra-low and low wear performance. Enzymatic digestion was used to digest the serum proteins and sodium polytungstate was used as a novel density gradient medium to isolate particles from proteins and other contaminants by ultracentrifugation. This method demonstrated over 80% recovery of particles and did not alter the size or morphology of ceramic and metal particles during the isolation process. STATEMENT OF SIGNIFICANCE: Improvements in resistance to wear and mechanical damage of the articulating surfaces have a large influence on longevity and reliability of joint replacement devices. Modern ceramics have demonstrated ultra-low wear rates for hard-on-hard total hip replacements. Generation of very low concentrations of wear debris in simulator lubricants has made it challenging to isolate the particles for characterisation and further analysis. We have introduced a novel method to isolate ceramic and metal particles from serum-based lubricants using enzymatic digestion and novel sodium polytungstate gradients. This is the first study to demonstrate the recovery of ceramic and metal particles from serum lubricants at lowest detectable in vitro wear rates reported in literature.

Origins of the mobile gene cassettes found in integrons.

Many of the acquired antibiotic resistance genes found in enterobacteria and pseudomonads are part of small mobile elements known as gene cassettes, and other genes are also likely to be found in cassettes. The origins of the genes and the recombination sites that make up cassettes are not known, but recent analyses of available data suggest that cassettes may be ancient structures, and some hypotheses for how they are formed can now be examined.

The solution structure of the F42A mutant of human interleukin 2.

Interleukin 2 (IL-2) is one of the major cytokines produced by T lymphocytes in response to antigen. It is a potent growth and differentiation factor for several cell-types and is structurally related to the four-helix bundle family of cytokines. Mutation of residue Phe42 to Ala abolishes binding to the alpha chain of the tri-partite IL-2 receptor. The three-dimensional structure of the F42A mutant IL-2 has been calculated by two dimensional NMR methods and compared to a structure of wild-type IL-2 determined by X-ray crystallography. The overall topology of the two structures is the same. The main differences between the structures are within the ill-defined loops connecting the helices and the region of the protein that is believed to interact with the alpha-chain of the receptor. Thus, the mutation of Phe42 to Ala does not perturb the overall three-dimensional structure of IL-2, and does not appear to change the putative binding sites for the beta and gamma chains of the receptor. The structural differences observed in this mutant suggest that the replacement of Phe42 with Ala causes the re-orientation of neighbouring side-chains that are also involved in binding the alpha-chain of the receptor.