A CRISPR-Cas9 tool to explore the genetics of Bacillus subtilis phages.

Here, we present pRH030, a new CRISPR-Cas9 tool for the genetic engineering of Bacillus phages and beyond. It is based on the Streptococcus pyogenes cas9 with its native constitutive promoter, tracrRNA, and a gRNA precursor. The constitutive expression of Cas9 was conducive to the inactivation of viral attackers and enhanced phage mutagenesis efficiency up to 100%. The gRNA precursor can be built up to an artificial CRISPR array with up to 5 spacers (target sequences) assembled from ordinary oligonucleotides and directly cloned into pRH030. Required time and resources remain comparable to a single gRNA cloning. These properties make pRH030 an attractive new system for the modification of Bacillus phages and qualify it for research beyond genetic construction.

Comparison of the novel ARCHITECT procalcitonin assay with established procalcitonin assay systems.

AIMS: This study assessed the performance of a new fully automated immunoassay, ARCHITECT B.R.A.H.M.S procalcitonin (PCT), comparing the results with other commercial assays on routine clinical specimens. METHODS: At nine sites from eight countries, precision analysis was carried out on controls by ANOVA. Threshold and linearity were verified according to standard procedures. Comparison of ARCHITECT B.R.A.H.M.S PCT with the Cobas®, LIAISON®, VIDAS® and Kryptor® PCT assays was evaluated using Passing-Bablok and Deming regression analyses. RESULTS: The within-laboratory standard deviation and %CV across all sites ranged from 0.005 to 0.008 and 2.7 to 4.1; 0.040 to 0.212 and 2.1 to 11.7; 1.628 to 4.191 and 2.5-6.3 for the three control levels, respectively. The mean slope (linearity analysis) across all sites ranged from 0.85 to 1.03, with a mean y-intercept ranging from -6.15 to + 1.71 and a correlation coefficient ranging from 0.94 to 1.00. The LoB, LoD, and LoQ claims were verified. Deming regression analysis of 1116 plasma or serum samples with PCT results detected across a dynamic assay range of 0.02-100 µg/l using the ARCHITECT B.R.A.H.M.S PCT assay yielded results of r = 0.989 vs. Roche Cobas®, r = 0.986 vs Kryptor® B.R.A.H.M.S, r = 0.987 vs BioMèrieux VIDAS® and r = 0.972 vs. Diasorin LIAISON®, respectively. Concordance at cut-offs of 0.25 µg/l and 0.50 µg/l were 96.9% and 98.1% with Roche Cobas®, 95.4% and 96.1% with B.R.A.H.M.S Kryptor®, 93.8% and 98.4% with BioMèrieux VIDAS®, and 92.7% and 93.9% with Diasorin LIAISON®. CONCLUSIONS: Compared with other assays, ARCHITECT B.R.A.H.M.S PCT offers excellent precision and low-end sensitivity.

Termination control of electronic phases in oxide thin films and interfaces: LaAlO3/SrTiO3(001).

A wealth of intriguing properties emerge in the seemingly simple system composed of the band insulators LaAlO(3) and SrTiO(3) such as a two-dimensional electron gas, superconductivity and magnetism. In this paper, we review the current insight obtained from first principles calculations on the mechanisms governing the behaviour of thin LaAlO(3) films on SrTiO(3)(001). In particular, we explore the strong dependence of the electronic properties on the surface and interface termination, the finite film thickness, lattice polarization and defects. A further aspect that is addressed is how the electronic behaviour and functionality can be tuned by an SrTiO(3) capping layer, adsorbates and metallic contacts. Lastly, we discuss recent reports on the coexistence of magnetism and superconductivity in this system for what they might imply about the electronic structure of this system.

Fast-track surgery for bilateral total knee replacement.

Bilateral simultaneous total knee replacement (TKR) has been considered by some to be associated with increased morbidity and mortality. Our study analysed the outcome of 150 consecutive, but selected, bilateral simultaneous TKRs and compared them with that of 271 unilateral TKRs in a standardised fast-track setting. The procedures were performed between 2003 and 2009. Apart from staying longer in hospital (mean 4.7 days (2 to 16) versus 3.3 days (1 to 25)) and requiring more blood transfusions, the outcome at three months and two years was similar or better in the bilateral simultaneous TKR group in regard to morbidity, mortality, satisfaction, the range of movement, pain, the use of a walking aid and the ability to return to work and to perform activities of daily living. Bilateral simultaneous TKR can therefore be performed as a fast-track procedure with excellent results.

Effect of high-dose preoperative methylprednisolone on pain and recovery after total knee arthroplasty: a randomized, placebo-controlled trial.

BACKGROUND: Total knee arthroplasty (TKA) is associated with severe pain and inflammation despite an extensive multimodal analgesic approach, but the effect of high-dose glucocorticoid administration has not been studied. METHODS: Forty-eight patients undergoing unilateral TKA were included in a randomized, double-blind, placebo-controlled trial receiving preoperative methylprednisolone (MP) 125 mg i.v. or saline. All surgery was performed under lumbar spinal anaesthesia and patients received a standardized, multimodal analgesic regime. The primary endpoint was pain during walking 24 h after surgery, and secondary endpoints were pain at rest, pain upon hip flexion, and pain upon knee flexion. Pain assessments were performed repeatedly for the first 48 h after surgery, in a questionnaire from days 2 to 10, and at follow-up on days 21 and 30. Tertiary endpoints were postoperative nausea and vomiting (PONV), plasma C-reactive protein (CRP) concentrations, fatigue, sleep quality, and rescue analgesic and antiemetic requirements. RESULTS: Pain during walking was significantly lower in the MP group up to 32 h after operation. Overall pain and cumulative pain scores (2-48 h) were lower for all pain assessments (P<0.04). Consumption of rescue oxycodone was lower from 0 to 24 h (P=0.02) and PONV, consumption of ondansetron reduced (P<0.05), and CRP concentrations were lower at 24 h (P<0.000001). Fatigue throughout the day of surgery was lower (P=0.02), but sleep quality was worse on the first night (P=0.002). No side-effects or complications were observed in other respects. CONCLUSIONS: MP 125 mg before surgery improves analgesia and immediate recovery after TKA, even when combined with a multimodal analgesic regime. These findings call for further studies on safety aspects.

Analgesic efficacy of local anaesthetic wound administration in knee arthroplasty: volume vs concentration.

Wound administration of local anaesthetic may be effective for postoperative pain management in knee arthroplasty, but the analgesic efficacy of local anaesthetic in relation to volume vs concentration has not been determined. In a double-blinded trial, 48 patients scheduled for total knee arthroplasty were randomly assigned to receive either a high volume/low concentration solution of ropivacaine (20 ml, 0.5%) or a low volume/high concentration solution of ropivacaine (10 ml, 1%), 6 and 24 h postoperatively through an intracapsular catheter. Pain was assessed for 2 h after administration. Pain was reduced in both groups with ropivacaine administration 24 h postoperatively (p < 0.02), but with no difference in analgesia between groups at all time intervals. No reduction in pain scores was observed with ropivacaine injection 6 h postoperatively. The median (IQR [range]) dose of oxycodone administered was 12.5 (10-19 [0-35]) mg in the high volume/low concentration group, and 20 mg (16-40 [0-65]) mg in the low volume/high concentration group (p = 0.005). In conclusion, intracapsular administration of local anaesthetic may have limited analgesic efficacy with no volume vs concentration relationship after total knee arthroplasty.

Analgesic efficacy of intracapsular and intra-articular local anaesthesia for knee arthroplasty.

The optimal site for wound delivery of local anaesthetic after total knee arthroplasty is undetermined. Sixty patients having total knee arthroplasty received intra-operative infiltration analgesia with ropivacaine 0.2% and were then were randomly assigned to receive either intracapsular or intra-articular catheters with 20 ml ropivacaine 0.5% given at 6 h and again at 24 h, postoperatively. Analgesic efficacy was assessed for 3 h after each injection, using a visual analogue score, where 0 = no pain and 100 = worst pain. There was no statistically significant difference between groups. Maximum pain relief (median (IQR [range])) at rest observed in the 3 h after the 6 and 24 h postoperative injections was 17 (7-31 [0-80]) and 10 (4-27 [0-50]) p = 0.27 for 6-9 h; and 17 (7-33 [0-100]) and 13 (3-25 [0-72]) p = 0.28 for 24-27 h, for intracapsular and intra-articular, respectively. Intracapsular local anaesthetic has similar analgesic efficacy to intra-articular after total knee arthroplasty.

Parallel electron-hole bilayer conductivity from electronic interface reconstruction.

The perovskite SrTiO3-LaAlO3 structure has advanced to a model system to investigate the rich electronic phenomena arising at polar oxide interfaces. Using first principles calculations and transport measurements we demonstrate that an additional SrTiO3 capping layer prevents atomic reconstruction at the LaAlO3 surface and triggers the electronic reconstruction at a significantly lower LaAlO3 film thickness than for the uncapped systems. Combined theoretical and experimental evidence (from magnetotransport and ultraviolet photoelectron spectroscopy) suggests two spatially separated sheets with electron and hole carriers, that are as close as 1 nm.

Analgesic efficacy of subcutaneous local anaesthetic wound infiltration in bilateral knee arthroplasty: a randomised, placebo-controlled, double-blind trial.

BACKGROUND: High-volume wound local infiltration analgesia is effective in knee arthroplasty, but the analgesic efficacy of subcutaneous wound infiltration has not been evaluated. METHODS: In a randomised, double-blind, placebo-controlled trial in 16 patients undergoing bilateral knee arthroplasty with high-volume local infiltration analgesia in the deeper layers, saline or ropivacaine 2 mg/ml was infiltrated into the subcutaneous part of the wound in each knee along with the placement of multi-fenestrated catheters in the subcutaneous wound layers in both knees. Pain was assessed for 6 h post-operatively and for 3 h after a bolus injection given through the catheter 24 h post-operatively. RESULTS: Visual analogue scale (VAS) pain scores were significantly lower from the knee infiltrated with ropivacaine compared with the knee infiltrated with saline in the subcutaneous layer of the wound, at rest (P<0.02), with flexion of the knee (P<0.04) and when the leg was straight and elevated (P<0.04). Twenty-four hours post-operatively, a decline in the VAS pain scores was observed in both groups, with no statistically significant difference between injection of ropivacaine or saline in the subcutaneously placed catheters (P>0.05). CONCLUSION: As part of a total wound infiltration analgesia intraoperative subcutaneous infiltration with ropivacaine in bilateral total knee arthroplasty is effective in early post-operative pain management, while a post-operative subcutaneous bolus administration through a multiholed catheter 24 h post-operatively did not show improved analgesia compared with the administration of saline.

A CdSe quantum dot based resonant cavity light-emitting diode showing single line emission up to 90 K.

A II-VI wide-bandgap resonant cavity light-emitting diode is presented. The active region consists of CdSe quantum dots embedded in ZnSSe/MgS barriers, resulting in improved quantum efficiency at elevated temperatures. The resonant cavity is formed by a 14-period bottom distributed Bragg reflector and the semiconductor to air interface on top of the structure. Temperature dependent micro-electroluminescence measurements reveal emission of a single quantum dot up to 90 K. The turn-on voltages are 6 V at 4 K and 4 V at room temperature. These results are promising for the realization of green surface-emitting devices in general, and especially for an electrically driven prospective single photon source operating at room temperature.

Subacute pain and function after fast-track hip and knee arthroplasty.

In a well-defined fast-track setup for total hip and knee arthroplasty, with a multimodal analgesic regimen consisting of intra-operative local anaesthetic infiltration and oral celecoxib, gabapentin and paracetamol for 6 days postoperatively, we conducted a prospective, consecutive, observational study. The purpose was to describe the prevalence and intensity of subacute postoperative pain and opioid related side effects, use of analgesics and functional ability 1-10 and 30 days postoperatively. Fast-track total hip and knee arthroplasty with early discharge (< 3 days) resulted in acceptable levels of pain and postoperative nausea and vomiting with concomitant low use of opioids in > 95% of patients after discharge before day 10 after total hip arthroplasty. However, after total knee arthroplasty 52% patients reported moderate pain (VAS 30-59 mm), and 16% severe pain (VAS > or = 60 mm) when walking 1 month after surgery with a concomitant increase in the use of strong opioids. These results emphasise the need for improvement in analgesia after discharge following total knee arthroplasty, to facilitate rehabilitation.

Percutaneous intramyocardial stem cell injection in patients with acute myocardial infarction: first-in-man study.

BACKGROUND: Clinical studies on intracoronary stem cell infusion in patients with acute myocardial infarction (AMI) have shown promising results for left ventricular ejection fraction (LVEF). However, preclinical studies have shown that intramyocardial cell injection is better than the intracoronary approach. OBJECTIVE: To test safety and feasibility of intramyocardial cell injection and left ventricular electromechanical mapping (EMM) early after AMI. DESIGN: On day 10.5 (5) (mean (SD)) after AMI and percutaneous coronary intervention with stent implantation (culprit lesion: 15 LAD, 3 circumflex and 2 right coronary arteries) 20 patients (mean (SD) 60.4 (11.4) years) received bone marrow derived mononuclear cells in the low-voltage area using EMM-guided percutaneous intramyocardial injection. EMM and coronary angiography were performed in 15 patients at 6-months' follow-up. Echocardiography, recording of laboratory data and clinical assessment (6-month and 12-month follow-up) were carried out in all 20 patients. RESULTS: None of the patients showed periprocedural complications. Three patients received an implantable cardioverter-defibrillator for primary prevention of sudden cardiac death and 6 (30%) patients showed in-stent restenosis. One patient underwent bypass surgery owing to chronic stent occlusion after 6 months. 2.0 (0.6)x10(8) cells, including 1.0 (0.3)x10(6) CD45(dim)/CD34(hi) stem cells, were injected in each patient. EMM showed a mean (SD) improvement from a baseline unipolar voltage of 45.5 (14.3)% to 59.3 (19.8)% of normal voltage (p = 0.002) and reduction of the low-voltage area from 28.7 (12.1)% to 20.3 (13.5)%; (p = 0.016). During the 12-month follow-up, the left ventricular ejection fraction (LVEF) improved from 40.8 (6.9)% to 47.1 (10.6)%; (p = 0.037). CONCLUSION: Left ventricular EMM and percutaneous intramyocardial cell injection in patients with AMI was shown to be a safe procedure. It is associated with improved LVEF and electromechanical parameters after 12-months' follow-up. TRIAL REGISTRATION NUMBER: Eudra-CT-No 2005-003629-19.

High-volume infiltration analgesia in total knee arthroplasty: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: High-volume infiltration analgesia may be effective with a low risk of side effects in hip and knee arthroplasty. The present placebo-controlled study was carried out to evaluate the analgesic effect of high-volume infiltration analgesia in bilateral total knee arthroplasty, along with a detailed description of the infiltration technique. METHODS: In a randomized, double-blind, placebo-controlled trial in 12 patients undergoing bilateral knee arthroplasty, saline or high-volume (170 ml) ropivacaine (0.2%) with epinephrine was infiltrated around each knee, with repeated doses administered through an intra-articular catheter for 24 h and pain and opioid requirements assessed for 48 h in a fast-track setting. RESULTS: Pain at rest and during movement was significantly reduced for up to 32 h with the high-volume local anesthetic infiltration technique. No major side effects were observed. The median hospital stay was 4 days. CONCLUSION: High-volume infiltration analgesia is effective in knee arthroplasty and, due to its simplicity, may be preferable compared with other analgesic techniques in knee arthroplasty.

Identification of methicillin-resistant Staphylococcus aureus (MRSA): Comparison of a new molecular genetic test kit (GenoType MRSA) with standard diagnostic methods.

Results obtained from 188 isolates of staphylococci using standard diagnostic methods for identifying MRSA were compared with those achieved with a newly available molecular genetic test kit, the GenoType, Version 1, MRSA (Hain Lifescience GmbH, Nehren, Germany). The GenoType MRSA detects the mecA gene and, in addition, a highly specific sequence for Staphylococcus aureus (S. aureus) by polymerase chain reaction (PCR) and reverse hybridization. There was a 100% overall correlation between the results of conventional and molecular genetic testing. 143 isolates were tested positive for MRSA, 10 isolates were identified as oxacillin-sensitive Staphylococcus aureus strains (MSSA), and 35 isolates were coagulase-negative staphylococci of various species. However, five of the 143 MRSA strains yielded ambiguous results with the first line standard tests and therefore required additional testing leading to delay of definitive diagnosis. As expected, mecA could not only be detected in MRSA strains, but also in coagulase-negative staphylococci. The reliable identification as S. aureus from the same isolate is therefore an essential prerequisite for MRSA diagnosis. The GenoType MRSA fulfills this requirement by parallel detection of a S. aureus-specific sequence and the mecA gene. Molecular genetic testing with the GenoType MRSA kit needs much less time than conventional microbiological methods. Therefore genetic testing provides not only a considerable advantage with respect to reliability but also to speed.

Ion-beam analysis of CuInSe2 solar cells deposited on polyimide foil.

CuInSe(2) (CIS) solar cells deposited on polyimide foil by the Solarion company in a web-coater-based process using sputter and evaporation techniques were investigated in the ion beam laboratory LIPSION of the University of Leipzig by means of Rutherford backscattering spectrometry (RBS) and particle-induced X-ray emission (PIXE) using high-energy broad ion beams and microbeams. From these measurements the composition of the absorber and the lateral homogeneity and film thicknesses of the individual layers could be determined on the basis of some reasonable assumptions. For the first time, quantitative depth profiling of the individual elements was performed by microPIXE measurements on a beveled section prepared by ion-beam etching of a CIS solar cell. Within the CIS absorber layer no significant concentration-depth gradients were found for Cu, In, and Se, in contrast with results from secondary neutral mass spectrometry (SNMS) depth profiling, which was applied to the same samples for comparison. Furthermore, both PIXE and SNMS showed the presence of a remarkable amount of Cd from the CdS buffer layer in the underlying absorber.

[Multiple organ failure caused by extraadrenal pheochromocytoma]. / Multiorgansvigt på baggrund af ekstraadrenalt faeokromocytom.

Extraadrenal phaeochromocytomas are very rare tumours, commonly associated with hypertensive paroxysms. We report a 20 year-old man with unsuspected extraadrenal phaeochromocytoma, who at admission presented with septicaemia and adult respiratory distress syndrome, suggesting pneumonia.

A conserved structural element in horse and mouse IGF2 genes binds a methylation sensitive factor.

The equine IGF2 gene has been cloned and characterised. It spans a 9 kb region, which is substantially less than the corresponding human gene. Three coding exons and three untranslated leader exons, all highly homologous to those in other species, were identified. Downstream of the polyadenylation site in exon 6, a dinucleotide repeat sequence was identified. Three putative promoters (P1-P3) were localised in the 5' region of the gene. RNase protection analysis revealed two active promoters in fetal tissues, P2 and P3, whereas P3 was the only promoter active in adult tissues. This represents a transcriptional pattern different from that in humans or rodents. A novel structural element, an inverted repeat, is predicted in the 3' region of the IGF2 gene. This repeat is conserved between species and located in a region which is differentially methylated in the human and mouse genes and might therefore be involved in the imprinting mechanism. The inverted repeat acquires a stem-loop structure in vitro with a hybrid A/B-DNA conformation in the stem area. Both in horse and mouse, a methylation-sensitive protein binds this structure with a strong requirement for the loop area. Furthermore, the protein might be developmentally regulated.

Transcriptional regulation and biological significance of the insulin like growth factor II gene.

The insulin like growth factors I and II are the most ubiquitous in the mammalian embryo. Moreover they play a pivotal role in the development and growth of tumours. The bioavailability of these growth factors is regulated on a transcriptional as well as on a posttranslational level. The expression of non-signalling receptors as well as binding proteins does further tune the local concentration of IGFs. This paper aims at reviewing how the transcription of the IGF genes is regulated. The biological significance of these control mechanisms will be discussed.

FISH mapping of the IGF2 gene in horse and donkey-detection of homoeology with HSA11.

Three genomic subclones derived from a phage clone containing the equine IGF2 gene were used to FISH map the gene on horse (ECA) and donkey (EAS) metaphase chromosomes. The gene mapped on ECA 12q13 band and is the first locus mapped to this horse chromosome. In donkey the gene mapped very terminal on the long arm of one small submetacentric chromosome that shows almost identical DAPI-banding pattern with ECA12. This is the first locus mapped in donkey genome. Cross species chromosome painting of equine metaphase chromosomes with human Chromosome (Chr) 11-specific probe showed homoeology of this human chromosome with ECA12 and ECA7. The novel ECA12 comparative painting results are thus in accordance with the localization of the equine IGF2 gene. Comparison of the hitherto known physical locations of IGF2 in different species, viz. human, cattle, sheep, horse, and donkey, shows that this gene tends to maintain a terminal location on the chromosome arm.