Windsurfing vs kitesurfing:Injuries at the North Sea over a 2-year period

AIM To analyze all windsurfing and kitesurfing(kiteboarding) injuries presented at our coastal hospital over a 2-year period. METHODS Twenty-five windsurfers(21 male; aged 31 ± 8 years) and 32 kitesurfers(23 male; aged 29 ± 11 years) presented at our hospital during the 2-year study period. Various injury data were recorded,including transport to hospital and treatment. After a median follow-up of 16 mo(range,7-33 mo),18 windsurfers(72%) and 26 kitesurfers(81%) completed questionnaires on the trauma mechanisms,the use of protective gear,time spent on windsurfing or kitesurfing,time to return to sports,additional injuries,and chronic disability. RESULTS Most patients sustained minor injuries but severe injuries also occurred,including vertebral and tibial plateau fractures. The lower extremities were affected the most,followed by the head and cervical spine,the upperextremities,and the trunk. The injury rates were 5.2 per 1000 h of windsurfing and 7.0 per 1000 h of kitesurfing(P = 0.005). The injury severity was the same between groups(P = 1.0). Less than 30% of the study population used protective gear. Kitesurfers had a higher number of injuries,and required transport by ambulance,inpatient hospital stay and operative treatment more often than windsurfers,but these differences were not statistically significant(P 0.05). The median time to return to windsurfing and kitesurfing was 5 and 4 wk,respectively(P = 0.79). Approximately one-third of the patients in each group experienced chronic symptoms.CONCLUSION Kitesurfing results in a significantly higher injury rate than windsurfing in the same environmental conditions but the severity of the injuries does not differ.     

Interaction of Ceftobiprole with the Low-Affinity PBP 5 of Enterococcus faecium

Ceftobiprole is a new cephalosporin that exhibits a high level of affinity for methicillin-resistant Staphylococcus aureus PBP 2a. It was reported that ceftobiprole did not interact with a mutated form of the low-affinity protein Enterococcus faecium PBP 5 (PBP 5fm) that, when overexpressed, confers a β-lactam resistance phenotype to the bacterium. Our results show that ceftobiprole binds to unmutated PBP 5fm to form a stable acyl-enzyme and that ceftobiprole is able to efficiently kill a penicillin-resistant Enterococcus faecium strain that produces this protein.β-Lactam antibiotics (penicillins, cephalosporins, carbapenems, and monobactams) are the most frequently prescribed antibacterial agents used to fight serious bacterial infections. They inactivate the membrane-bound d,d-transpeptidases essential for peptidoglycan synthesis by forming with them stable acyl-enzymes (9). This explains why these enzymes are generally designated penicillin-binding proteins (PBPs) (17, 19). In Gram-positive cocci, the resistance to β-lactam antibiotics is primarily conferred by the presence or the overproduction of a low-affinity PBP. In enterococci, among which is the opportunistic human pathogen Enterococcus faecium (3), the resistance to penicillin may be associated with overproduction of the intrinsic low-affinity protein PBP 5 (PBP 5fm) or to other alterations affecting PBP 5fm (15).Ceftobiprole (BPR) (BAL9141) is a novel broad-spectrum cephalosporin that is active against Gram-positive and Gram-negative bacterial groups, including methicillin-resistant Staphylococcus aureus (MRSA) (2, 6, 10, 12) and Enterococcus faecalis (1). It has been demonstrated to be a good inhibitor of the S. aureus low-affinity protein PBP 2a (6, 8). A previous study reported that ceftobiprole had poor inhibitory activity against β-lactam-resistant E. faecium and that ceftobiprole did not bind to the mutated, low-affinity PBP 5fm protein isolated from such a strain (10). To better understand the difference between these two low-affinity PBPs with respect to ceftobiprole, we have characterized the interaction between an unmutated form of PBP 5fm and ceftobiprole.To perform this study, we used the penicillin-sensitive Enterococcus faecium strain D63 (benzylpenicillin MIC = 5 μg/ml and ampicillin MIC = 15 μg/ml) and its laboratory-derived penicillin-resistant strain D63r (benzylpenicillin MIC = 70 μg/ml and ampicillin MIC = 125 μg/ml) (21). The PBP profile of the latter strain exhibits a 6-fold increase in quantity of PBP 5fm (21). The ceftobiprole MICs determined by the microdilution method (4) for E. faecium D63r and D63 were 8 and 2 μg/ml, respectively. These values contrasted with those reported by other workers, who have found that most ampicillin-resistant E. faecium clinical isolates were resistant to ceftobiprole (13). They concluded that ceftobiprole was ineffective against ampicillin-resistant enterococcal strains.To further study the killing effects of benzylpenicillin and ceftobiprole on E. faecium D63r, we exposed exponentially growing cultures of both the sensitive and the resistant strains to increasing concentrations of antibiotic corresponding to 1 and 4 times the respective MICs (Fig. ​(Fig.1)1) (18). For concentrations higher than their MICs, benzylpenicillin and ceftobiprole show killing effects (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Time-kill curves for resistant E. faecium D63r and susceptible E. faecium D63 in the presence of benzylpenicillin or ceftobiprole. The MICs for D63r were 70 μg/ml for benzylpenicillin and 8 μg/ml for ceftobiprole. The MICs for D63 were 5 μg/ml for benzylpenicillin and 2 μg/ml for ceftobiprole. The surviving bacteria were counted after 0, 4, and 24 h of incubation at 37°C by subculturing serial dilutions (at least 10-fold, to minimize drug carryover).Our study was completed by the determination of the 50% inhibitory concentration (IC₅₀) values for ceftobiprole, benzylpenicillin, cefepime, and ceftazidime for the different PBPs of E. faecium D63r by using purified membrane preparations and fluorescent ampicillin (20, 21). Membrane preparations (300 μg of proteins) were first incubated (20 min at 37°C) with increasing concentrations of ceftobiprole and next incubated (with a saturating concentration of 25 μM fluorescent ampicillin) for an hour at 37°C. The titration of the PBPs by ceftobiprole (Fig. ​(Fig.22 and Table ​Table1)1) revealed that at a 2-fold MIC, all high-molecular-mass PBPs are inhibited by the antibiotic (Fig. ​(Fig.2)2) and that the low-molecular-mass protein PBP 6, which acts as a d,d-carboxypeptidase (7), is not affected. The high-molecular-mass protein PBP 2 is the most sensitive to ceftobiprole (IC₅₀ = 0.2 μg/ml). PBPs 1 and 3 show similar IC₅₀s (<1 μg/ml), whereas the low-affinity proteins PBP 5fm, PBP 4, and PBP 5* possess slightly higher values (≥1 μg/ml). This pattern of inhibition is completely different from that obtained with benzylpenicillin, for which the resistant protein PBP 5fm is the most insensitive PBP. The IC₅₀ for ceftobiprole on purified soluble PBP 5fm (sPBP 5fm) gives results similar to those observed for the membrane preparations (0.7 μg/ml). Note that PBP 4 and PBP 5* exhibit very similar IC₅₀ profiles (Table ​(Table1).1). A similar observation was made for PBP 4 and PBP 4* in Enterococcus hirae. PBP 4* was shown to be produced by a proteolytic cleavage of the 60 N-terminal amino acid residues of PBP 4 (11). It is very likely that, in the E. faecium membranes used here, PBP 5* was the result of an N-terminal truncation of PBP 4.Open in a separate windowFIG. 2.Relative affinities of ceftobiprole (A) and benzylpenicillin (B) for E. faecium D63r PBPs. The affinities of ceftobiprole and other β-lactams for PBPs were analyzed by a competition assay using fluorescent ampicillin. Membrane proteins were prepared from D63r and D63. Nonlabeled antibiotics were incubated with membrane proteins at 37°C for 20 min, followed by the addition of fluorescent ampicillin during 1-h incubation. The membrane fractions were subjected to SDS-PAGE and fluorography.

TABLE 1.

Inhibition of PBPs from E. faecium D63r and D63

LIP5 Interacts with Aquaporin 2 and Facilitates Its Lysosomal Degradation

Vasopressin binding to the V2 receptor in renal principal cells leads to activation of protein kinase A, phosphorylation of aquaporin 2 (AQP2) at Ser256, and the translocation of AQP2 to the apical membrane, resulting in concentration of the urine. In contrast, phorbol ester–induced activation of protein kinase C pathway leads to ubiquitination of AQP2 at Lys270 and its internalization to multivesicular bodies, where it is targeted for lysosomal degradation or stored for recycling. Because little is known about the regulation of AQP2 trafficking, we used the carboxy-terminal tail of constitutively nonphosphorylated AQP2 (S256A) as a bait for interacting proteins in a yeast two-hybrid assay. We isolated lysosomal trafficking regulator–interacting protein 5 (LIP5) and found that LIP5 interacted with the proximal carboxy-terminal tail (L230-D243) of AQP2 in vitro but not with AQP3 or AQP4, which are also expressed in principal cells. Immunohistochemistry revealed that LIP5 co-localized with AQP2 in principal cells. LIP5 binding occurred independent of the state of Ser256 phosphorylation or Lys270 ubiquitination. LIP5 has been shown to facilitate degradation of the EGF receptor; here, LIP5 seemed to bind this receptor. Knockdown of LIP5 in mouse renal cells (mpkCCD) reduced the phorbol ester–induced degradation of AQP2 approximately two-fold. In summary, LIP5 binds cargo proteins and, considering the role of LIP5 in protein sorting to multivesicular bodies, plays a role in the degradation of AQP2, possibly by reducing the formation of late endosomes.Tight regulation of the translocation of aquaporin 2 (AQP2) water channels to and from the apical membrane of renal collecting duct cells by the antidiuretic hormone arginine vasopressin (AVP) is fundamental for water homeostasis. Upon hypernatremia or hypovolemia, binding of AVP to its type 2 receptor (V2R) increases intracellular calcium and cAMP concentrations, which activate and tether protein kinase A (PKA) to AQP2-containing vesicles, resulting in phosphorylation of AQP2 and other proteins.^1–7 Consequently, these vesicles dock and fuse with the apical membrane, rendering principal cells water permeable.Regulated translocation of AQP2 to and from the apical membrane suggests the existence of proteins interacting with cytosolic segments of AQP2. Only the amino-terminal and carboxyl-terminal tails (N- and C-tails, respectively) of AQP2 extend well into the cytosol, and the C-tail of AQP2 has been shown to have an important role in its apical sorting: First, Ser256 in the AQP2 C-tail is phosphorylated in vivo by AVP stimulation, and studies in both cell and animal models revealed that this phosphorylation event is essential for AQP2 translocation to the plasma membrane.^8–13 Second, all AQP2 mutants encoded in families with a dominant inheritance of nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to AVP, are missorted as a result of mutations in the C-tail.^12,14–19 In addition, the AQP2 C-tail is mono-ubiquitinated at Lys270 upon AVP removal or PKC activation, which enhances endocytosis and degradation of AQP2.²⁰So far, the Rap1 GTPase-activating protein Spa1, heat-shock protein 70, and Myelin and Lymphocyte Associated Protein (MAL) are the only proteins known to bind the AQP2 C-tail, potentially playing physiologic roles in AQP2 trafficking^21–23; therefore, to gain more insight in the proteins and mechanisms involved in the regulation of AQP2, we used yeast two-hybrid assays to screen a mouse kidney cDNA library for proteins interacting with the C-tail of AQP2. We found the lysosomal trafficking regulator (LYST) interacting protein 5 (LIP5; Swiss-Prot entry {"type":"entrez-protein","attrs":{"text":"Q9CR26","term_id":"30580371","term_text":"Q9CR26"}}Q9CR26; corresponding gene name DRG-1) to interact specifically with AQP2. Interestingly, LIP5 is reported to function in multivesicular body (MVB) formation,²⁴ although a direct interaction of LIP5 with MVB cargo proteins has never been reported. Subsequent analyses revealed the LIP5-binding region within AQP2 and the role of LIP5 in AQP2 regulation.     

Molecular karyotyping of patients with unexplained mental retardation by SNP arrays: A multicenter study

Genomic microarrays have been implemented in the diagnosis of patients with unexplained mental retardation. This method, although revolutionizing cytogenetics, is still limited to the detection of rare de novo copy number variants (CNVs). Genome‐wide single nucleotide polymorphism (SNP) microarrays provide high‐resolution genotype as well as CNV information in a single experiment. We hypothesize that the widespread use of these microarray platforms can be exploited to greatly improve our understanding of the genetic causes of mental retardation and many other common disorders, while already providing a robust platform for routine diagnostics. Here we report a detailed validation of Affymetrix 500k SNP microarrays for the detection of CNVs associated to mental retardation. After this validation we applied the same platform in a multicenter study to test a total of 120 patients with unexplained mental retardation and their parents. Rare de novo CNVs were identified in 15% of cases, showing the importance of this approach in daily clinical practice. In addition, much more genomic variation was observed in these patients as well as their parents. We provide all of these data for the scientific community to jointly enhance our understanding of these genomic variants and their potential role in this common disorder. Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

VEGF‐D deficiency in mice does not affect embryonic or postnatal lymphangiogenesis but reduces lymphatic metastasis

Vascular endothelial growth factor‐D (VEGF‐D) is one of the two ligands of the VEGFR‐3 receptor on lymphatic endothelial cells. Gene‐silencing studies in mice and Xenopus tadpoles recently showed that the role of endogenous VEGF‐D in lymphatic development is moderate. By contrast, exogenous VEGF‐D is capable of stimulating lymphangiogenesis. Nonetheless, its endogenous role in pathological conditions remains largely unknown. Hence, we reassessed its role in disease, using Vegf‐d^null mice. Vegf‐d^null mice were generated that, under physiological conditions, displayed normal embryonic and postnatal lymphangiogenesis and lymphatic remodelling, efficient lymphatic functioning and normal health. Vegf‐d^null mice also reponded normally in models of skin wound healing and healing of infarcted myocardium, despite enhanced expression of VEGF‐D in these models in wild‐type mice. In contrast, Vegf‐d^null mice displayed reduced peritumoral lymphangiogenesis and lymph node metastasis in an orthotopic pancreatic tumour model. Together, our data indicate that endogenous VEGF‐D in mice is dispensible for lymphangiogenesis during development, in postnatal and adult physiology and in several pathological conditions, but significantly contributes to lymphatic metastasis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Qualitative and quantitative assessment of degeneration of cervical intervertebral discs and facet joints

Degeneration of intervertebral discs and facet joints is one of the most frequently encountered spinal disorders. In order to describe and quantify degeneration and evaluate a possible relationship between degeneration and biomechanical parameters, e.g., the intervertebral range of motion and intradiscal pressure, a scoring system for degeneration is mandatory. However, few scoring systems for the assessment of degeneration of the cervical spine exist. Therefore, two separate objective scoring systems to qualitatively and quantitatively assess the degree of cervical intervertebral disc and facet joint degeneration were developed and validated. The scoring system for cervical disc degeneration consists of three variables which are individually scored on neutral lateral radiographs: “height loss” (0–4 points), “anterior osteophytes” (0–3 points) and “endplate sclerosis” (0–2 points). The scoring system for facet joint degeneration consists of four variables which are individually scored on neutral computed tomography scans: “hypertrophy” (0–2 points), “osteophytes” (0–1 point), “irregularity” on the articular surface (0–1 point) and “joint space narrowing” (0–1 point). Each variable contributes with varying importance to the overall degeneration score (max 9 points for the scoring system of cervical disc degeneration and max 5 points for facet joint degeneration). Degeneration of 20 discs and facet joints of 20 patients was blindly assessed by four raters: two neurosurgeons (one senior and one junior) and two radiologists (one senior and one junior), firstly based on first subjective impression and secondly using the scoring systems. Measurement errors and inter- and intra-rater agreement were determined. The measurement error of the scoring system for cervical disc degeneration was 11.1 versus 17.9% of the subjective impression results. This scoring system showed excellent intra-rater agreement (ICC = 0.86, 0.75–0.93) and excellent inter-rater agreement (ICC = 0.78, 0.64–0.88). Surgeons as well as radiologists and seniors as well as juniors obtained excellent inter- and intra-rater agreement. The measurement error of the scoring system for cervical facet joint degeneration was 20.1 versus 24.2% of the subjective impression results. This scoring system showed good intra-rater agreement (ICC = 0.71, 0.42–0.89) and fair inter-rater agreement (ICC = 0.49, 0.26–0.74). Both scoring systems fulfilled the criteria for recommendation proposed by Kettler and Wilke. Our scoring systems can be reliable and objective tools for assessing cervical disc and facet joint degeneration. Moreover, the scoring system of cervical disc degeneration was shown to be experience- and discipline-independent. An erratum to this article can be found at     

Anterior cervical hypertrichosis and mental retardation

Anterior cervical hypertrichosis or hairy throat is a rare dysmorphic sign described in a total of 19 patients so far. The association with a number of additional features has been reported, including mental retardation. We report on another patient with this condition who also had moderate mental retardation, mildly dysmorphic facial features, obesity, hypermetropia and additional hair anomalies (low dorsal hair line on the neck, lumbosacral hypertrichosis). Karyotype and array comparative genomic hybridization analysis at 1 Mb resolution were normal.     

Measuring clinical pathway adherence

As clinical pathway adoption continues worldwide, it is necessary to establish adherence measurement methods in order to understand the difficulties and results of implementation. Adherence measurement literature mostly provides binary measurements of adherence to guidelines regarding individual medical activities over patient groups. The resulting measurements are of limited value in view of the pathways actually followed by individual patients. We develop and test dynamic programming formulations for adherence measurement in clinical pathways – based on partially ordered data in medical records and pathway definitions. With these new methods at hand, we analyze clinical pathway adherence at the Cardiovascular Center of Maastricht University Medical Center.