A novel nanoluciferase transgenic reporter measures proteinuria in zebrafish.

The zebrafish is an important animal system for modeling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable molecular and morphological homology with the human nephron. A key clinical indicator of kidney disease is proteinuria, but a high-throughput readout of proteinuria in the zebrafish is currently lacking. To remedy this, we used the Tol2 transposon system to generate a transgenic zebrafish line that uses the fabp10a liver-specific promoter to over-express a nanoluciferase molecule fused with the D3 domain of Receptor-Associated Protein (a type of molecular chaperone) which we term NL-D3. Using a luminometer, we quantified proteinuria in NL-D3 zebrafish larvae by measuring the intensity of luminescence in the embryo medium. In the healthy state, NL-D3 is not excreted, but when embryos were treated with chemicals that affected either proximal tubular reabsorption (cisplatin, gentamicin) or glomerular filtration (angiotensin II, Hanks Balanced Salt Solution, Bovine Serum Albumin), NL-D3 is detected in fish medium. Similarly, depletion of several gene products associated with kidney disease (nphs1, nphs2, lrp2a, ocrl, col4a3, and col4a4) also induced NL-D3 proteinuria. Treating col4a4 depleted zebrafish larvae (a model of Alport syndrome) with captopril reduced proteinuria in this system. Thus, our findings validate the use of the NL-D3 transgenic zebrafish as a robust and quantifiable proteinuria reporter. Hence, given the feasibility of high-throughput assays in zebrafish, this novel reporter will permit screening for drugs that ameliorate proteinuria, thereby prioritizing candidates for further translational studies.

Basement membrane defects in CD151-associated glomerular disease.

BACKGROUND: CD151 is a cell-surface molecule of the tetraspanin family. Its lateral interaction with laminin-binding integrin ɑ3ß1 is important for podocyte adhesion to the glomerular basement membrane (GBM). Deletion of Cd151 in mice induces glomerular dysfunction, with proteinuria and associated focal glomerulosclerosis, disorganisation of GBM and tubular cystic dilation. Despite this, CD151 is not routinely screened for in patients with nephrotic-range proteinuria. We aimed to better understand the relevance of CD151 in human kidney disease. METHODS: Next-generation sequencing (NGS) was used to detect the variant in CD151. Electron and light microscopy were used to visualise the filtration barrier in the patient kidney biopsy, and immunoreactivity of patient red blood cells to anti-CD151/MER2 antibodies was performed. Further validation of the CD151 variant as disease-causing was performed in zebrafish using CRISPR-Cas9. RESULTS: We report a young child with nail dystrophy and persistent urinary tract infections who was incidentally found to have nephrotic-range proteinuria. Through targeted NGS, a novel, homozygous truncating variant was identified in CD151, a gene rarely reported in patients with nephrotic syndrome. Electron microscopy imaging of patient kidney tissue showed thickening of GBM and podocyte effacement. Immunofluorescence of patient kidney tissue demonstrated that CD151 was significantly reduced, and we did not detect immunoreactivity to CD151/MER2 on patient red blood cells. CRISPR-Cas9 depletion of cd151 in zebrafish caused proteinuria, which was rescued by injection of wild-type CD151 mRNA, but not CD151 mRNA containing the variant sequence. CONCLUSIONS: Our results indicate that a novel variant in CD151 is associated with nephrotic-range proteinuria and microscopic haematuria and provides further evidence for a role of CD151 in glomerular disease. Our work highlights a functional testing pipeline for future analysis of patient genetic variants. A higher resolution version of the Graphical abstract is available as Supplementary information.

Survivorship and long-term outcome of a consecutive series of 200 Scandinavian Total Ankle Replacement (STAR) implants.

AIMS: We report the long-term clinical and radiological outcomes of a consecutive series of 200 total ankle arthroplasties (TAAs, 184 patients) at a single centre using the Scandinavian Total Ankle Replacement (STAR) implants. PATIENTS AND METHODS: Between November 1993 and February 2000, 200 consecutive STAR prostheses were implanted in 184 patients by a single surgeon. Demographic and clinical data were collected prospectively and the last available status was recorded for further survival analysis. All surviving patients underwent regular clinical and radiological review. Pain and function were assessed using the American Orthopaedic Foot and Ankle Society (AOFAS) hindfoot scoring system. The principal endpoint of the study was failure of the implant requiring revision of one or all of the components. Kaplan-Meier survival curves were generated with 95% confidence intervals and the rate of failure calculated for each year. RESULTS: A total of 84 patients (87 ankles) were alive by the end of this study. Of the surviving 84 patients (87 ankles; rheumatoid arthritis (RA), n = 40; OA, n = 47), 45 were women and 39 were men, with a mean age of 54 years (18 to 72 years) at the time of surgery. A total of 32 implants failed (16%), requiring revision surgery. The mean time to revision was 80 months (2 to 257). The implant survival at 15.8 years, using revision as an endpoint, was 76.16% (95% confidence interval (CI) 64.41 to 87.91). We found a steady but low decrease in survival over the study period. The mean AOFAS score improved from 28 (10 to 52) preoperatively to 61 (20 to 90) at long-term follow-up. CONCLUSION: STAR prostheses in the United Kingdom have now been largely superseded by newer design TAAs, potentially with improved characteristics and surgical techniques. The long-term survivorship for the STAR prosthesis can provide a benchmark for these later designs of ankle arthroplasty.

'Concord' grapevine nutritional status and chlorosis rank associated with fungal and bacterial root zone microbiomes.

Leaf chlorosis in vineyards is associated with reduced crop yields and quality. While iron (Fe) is understood to play a crucial role in chlorosis, total plant and soil Fe are not always indicative of chlorosis in grapevines. Physiology of chlorosis in vineyards has been well-studied, but the soil microbial consequences of and contributions to chlorosis have received little attention. We used next-generation sequencing (NGS) to examine the bacterial and fungal communities associated with grapevines demonstrating varying degrees of visual chlorosis symptoms. Additionally, chemical analyses of soils and grape leaves were used to explore the influence of plant nutritional status and soil chemistry on microbial community composition. Finally, factors influencing bacterial community composition were correlated with predicted bacterial community function. Leaf tissue magnesium (leaf Mg) concentrations and chlorosis rank were correlated with bacterial community composition as determined via dbRDA (distance-based Redundancy Analysis) using Bray-Curtis dissimilarities. Non-metric multidimensional scaling (NMDS) revealed a significant correlation between fungal community composition and soil Fe and pH, along with leaf N, Mg, and Ca (mg.kg-1). Chlorosis rank was moderately correlated with KEGG Orthology (KO) terms associated with nitrogen (N) and carbon (C) metabolism in soils, while leaf Mg was associated with a spectrum of KO terms including glycosphingolipid biosynthesis, glycan degradation, transporters, and porphyrin and chlorophyll metabolism. Additionally, abundance of many bacterial operational taxonomic units was significantly correlated with leaf Mg, including those from the following orders: Rhodobacterales, Acidobacteriales, Opitutales, Sphingomonadales, Burkholderiales, Saprospirales, and Flavobacteriales. Our findings suggest grapevine chlorosis is interrelated with soil microbial community structure and function, plant nutrition, and soil chemistry.

Post-Myocardial Infarction T-tubules Form Enlarged Branched Structures With Dysregulation of Junctophilin-2 and Bridging Integrator 1 (BIN-1).

BACKGROUND: Heart failure is a common secondary complication following a myocardial infarction (MI), characterized by impaired cardiac contraction and t-tubule (t-t) loss. However, post-MI nano-scale morphological changes to the remaining t-ts are poorly understood. METHOD AND RESULTS: We utilized a porcine model of MI, using a nonlethal microembolization method to generate controlled microinfarcts. Using serial block face scanning electron microscopy, we report that post-MI, after mild left-ventricular dysfunction has developed, t-ts are not only lost in the peri-infarct region, but also the remnant t-ts form enlarged, highly branched disordered structures, containing a dense intricate inner membrane. Biochemical and proteomics analyses showed that the calcium release channel, ryanodine receptor 2 (RyR2), abundance is unchanged, but junctophilin-2 (JP2), important for maintaining t-t trajectory, is depressed (-0.5×) in keeping with the t-ts being disorganized. However, immunolabeling shows that populations of RyR2 and JP2 remain associated with the remodeled t-ts. The bridging integrator 1 protein (BIN-1), a regulator of tubulogensis, is upregulated (+5.4×), consistent with an overdeveloped internal membrane system, a feature not present in control t-ts. Importantly, we have determined that t-ts, in the remote region, are narrowed and also contain dense membrane folds (BIN-1 is up-regulated +3.4×), whereas the t-ts have a radial organization comparable to control JP2 is upregulated +1.7×. CONCLUSIONS: This study reveals previously unidentified remodeling of the t-t nano-architecture in the post-MI heart that extends to the remote region. Our findings highlight that targeting JP2 may be beneficial for preserving the orientation of the t-ts, attenuating the development of hypocontractility post-MI.

Abuse Potential and Pharmacodynamic Characteristics of Oral and Intranasal Eluxadoline, a Mixed µ- and κ-Opioid Receptor Agonist and δ-Opioid Receptor Antagonist.

Drugs with µ-opioid receptor (OR) activity can be associated with abuse and misuse. The peripherally acting mixed µ-OR and κ-OR agonist and δ-OR antagonist eluxadoline is approved in the United States for the treatment of irritable bowel syndrome with diarrhea. In two separate crossover studies, we evaluated the oral and intranasal abuse potential of eluxadoline versus placebo and the active control oxycodone. Healthy recreational opioid users received eluxadoline 100, 300, and 1000 mg, oxycodone 30 and 60 mg, and placebo (oral study), or eluxadoline 100 and 200 mg, oxycodone 15 and 30 mg, and placebos matched to eluxadoline and oxycodone (intranasal study). In the oral study, Drug Liking Visual Analog Scale (VAS) peak (maximum) effect (Emax) score (primary endpoint) was significantly greater with eluxadoline 300 and 1000 mg versus placebo, but scores were significantly lower versus oxycodone. Following intranasal insufflation of eluxadoline, Drug Liking VAS Emax scores were not statistically different versus placebo, and were significantly lower versus oxycodone. Across other subjective measures, eluxadoline was generally similar to or disliked versus placebo. Pupillometry indicated no or minimal central effects with oral and intranasal eluxadoline, respectively. Adverse events of euphoric mood were reported with oral and intranasal eluxadoline but at a far lower frequency versus oxycodone. These data demonstrate that eluxadoline has less abuse potential than oxycodone in recreational opioid users.

Eluxadoline for Irritable Bowel Syndrome with Diarrhea.

Background Effective and safe treatments are needed for patients who have irritable bowel syndrome (IBS) with diarrhea. We conducted two phase 3 trials to assess the efficacy and safety of eluxadoline, a new oral agent with mixed opioid effects (µ- and κ-opioid receptor agonist and δ-opioid receptor antagonist), in patients with IBS with diarrhea. Methods We randomly assigned 2427 adults who had IBS with diarrhea to eluxadoline (at a dose of 75 mg or 100 mg) or placebo twice daily for 26 weeks (IBS-3002 trial) or 52 weeks (IBS-3001 trial). The primary end point was the proportion of patients who had a composite response of decrease in abdominal pain and improvement in stool consistency on the same day for at least 50% of the days from weeks 1 through 12 and from weeks 1 through 26. Results For weeks 1 through 12, more patients in the eluxadoline groups (75 mg and 100 mg) than in the placebo group reached the primary end point (IBS-3001 trial, 23.9% with the 75-mg dose and 25.1% with the 100-mg dose vs. 17.1% with placebo; P=0.01 and P=0.004, respectively; IBS-3002 trial, 28.9% and 29.6%, respectively, vs. 16.2%; P<0.001 for both comparisons). For weeks 1 through 26, the corresponding rates in IBS-3001 were 23.4% and 29.3% versus 19.0% (P=0.11 and P<0.001, respectively), and the corresponding rates in IBS-3002 were 30.4% and 32.7% versus 20.2% (P=0.001 and P<0.001, respectively). The most common adverse events associated with 75 mg of eluxadoline and 100 mg of eluxadoline, as compared with placebo, were nausea (8.1% and 7.5% vs. 5.1%), constipation (7.4% and 8.6% vs. 2.5%), and abdominal pain (5.8% and 7.2% vs. 4.1%). Pancreatitis developed in 5 (2 in the 75-mg group and 3 in the 100-mg group) of the 1666 patients in the safety population (0.3%). Conclusions Eluxadoline is a new therapeutic agent that reduced symptoms of IBS with diarrhea in men and women, with sustained efficacy over 6 months in patients who received the 100-mg dose twice daily. (Funded by Furiex Pharmaceuticals, an affiliate of Allergan; IBS-3001 and IBS-3002 ClinicalTrials.gov numbers, NCT01553591 and NCT01553747 , respectively.).

Three-dimensional structure of the intercalated disc reveals plicate domain and gap junction remodeling in heart failure.

The intercalated disc (ICD) orchestrates electrochemical and mechanical communication between neighboring cardiac myocytes, properties that are perturbed in heart failure (HF). Although structural data from transmission electron microscopy two-dimensional images have provided valuable insights into the domains forming the ICD, there are currently no three-dimensional (3D) reconstructions for an entire ICD in healthy or diseased hearts. Here, we aimed to understand the link between changes in protein expression in an ovine tachypacing-induced HF model and ultrastructural remodeling of the ICD by determining the 3D intercalated disc architecture using serial block face scanning electron microscopy. In the failing myocardium there is no change to the number of ICDs within the left ventricle, but there is an almost doubling of the number of discs with a surface area of <1.0 × 10(8)µm(2) in comparison to control. The 3D reconstructions further revealed that there is remodeling of the plicate domains and gap junctions with vacuole formation around and between the contributing membranes that form the ICDs in HF. Biochemical analysis revealed upregulation of proteins involved in stabilizing the adhesive and mechanical properties consistent with the morphological changes. Our studies here have shown that in tachypacing-induced HF mechanical stresses are associated with both structural and molecular alterations. To our knowledge, these data together provide novel, to our knowledge, insights as to how remodeling at the molecular and structural levels leads to impaired intercellular communication.

Effect of uptake transporters OAT3 and OATP1B1 and efflux transporter MRP2 on the pharmacokinetics of eluxadoline.

The effects of OATP1B1, OAT3, and MRP2 on the pharmacokinetics of eluxadoline, an oral, locally active, opioid receptor agonist/antagonist being developed for treatment of IBS-d were assessed in vivo. Coadministration of a single 200 mg dose of eluxadoline with cyclosporine, and probenecid increased eluxadoline systemic exposure [AUC(0-inf) ] by 4.4- and 1.4-fold, respectively, whereas peak exposure (Cmax ) increased 6.2-fold and 1.3-fold, respectively. Cyclosporine had little effect on renal clearance (CLren ) of eluxadoline whereas probenecid reduced CLren by nearly 50%. These study results suggested that sinusoidal OATP1B1-mediated hepatic uptake of eluxadoline (during first-pass and systemic extraction) plays a major role in its absorption and disposition, whereas OAT3-mediated basolateral uptake in the proximal renal tubules and MRP2-mediated canalicular and renal tubular apical efflux play only minor roles in its overall disposition. All treatments were safe and well tolerated.

Three-dimensional reconstruction of cardiac sarcoplasmic reticulum reveals a continuous network linking transverse-tubules: this organization is perturbed in heart failure.

RATIONALE: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation-contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure. OBJECTIVE: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. METHODS AND RESULTS: Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collapsed t-ts, patches of SR tangling, and disorder with rearrangement of the mitochondria. CONCLUSIONS: We provide the first high-resolution 3D structure of the SR network showing that it forms a cell-wide communication pipeline facilitating Ca(2+) diffusion, buffering, and synchronicity. The distribution of the SR within the cell is related to interspecies differences in excitation-contraction coupling, and we report the first detailed analysis of SR remodeling as a result of heart failure.

Microtopography of the eye surface of the crab Carcinus maenas: an atomic force microscope study suggesting a possible antifouling potential.

Marine biofouling causes problems for technologies based on the sea, including ships, power plants and marine sensors. Several antifouling techniques have been applied to marine sensors, but most of these methodologies are environmentally unfriendly or ineffective. Bioinspiration, seeking guidance from natural solutions, is a promising approach to antifouling. Here, the eye of the green crab Carcinus maenas was regarded as a marine sensor model and its surface characterized by means of atomic force microscopy. Engineered surface micro- and nanotopography is a new mechanism found to limit biofouling, promising an effective solution with much reduced environmental impact. Besides giving a new insight into the morphology of C. maenas eye and its characterization, our study indicates that the eye surface probably has antifouling/fouling-release potential. Furthermore, the topographical features of the surface may influence the wettability properties of the structure and its interaction with organic molecules. Results indicate that the eye surface micro- and nanotopography may lead to bioinspired solutions to antifouling protection.

Eluxadoline benefits patients with irritable bowel syndrome with diarrhea in a phase 2 study.

BACKGROUND & AIMS: Simultaneous agonism of the µ-opioid receptor and antagonism of the δ-opioid receptor can reduce abdominal pain and diarrhea in patients with irritable bowel syndrome with diarrhea (IBS-D) without constipating side effects. We evaluated the efficacy and safety of a minimally absorbed, µ-opioid receptor agonist and δ-opioid receptor antagonist (eluxadoline) in a phase 2 study in patients with IBS-D. METHODS: We randomly assigned 807 patients to groups that received oral placebo twice daily or 5, 25, 100, or 200 mg oral eluxadoline for 12 weeks. The primary end point was clinical response at week 4, defined by a mean reduction in daily pain score from baseline of ≥ 30%, and of at least 2 points on 0-10 scale, as well as a stool consistency score of 3 or 4 on the Bristol Stool Scale (1-7) for at least 66% of daily diary entries during that week. RESULTS: Significantly more patients receiving 25 mg (12.0%) or 200 mg (13.8%) eluxadoline met the primary end point of clinical response than patients given placebo (5.7%; P < .05). Patients receiving eluxadoline at 100 mg and 200 mg also had greater improvements in bowel movement frequency and urgency, global symptoms, quality of life, and adequate relief assessments (P < .05). Additionally, patients receiving 100 mg (28.0%) or 200 mg (28.5%) eluxadoline were significantly more likely than those receiving placebo (13.8%; P < .005) to meet the US Food and Drug Administration response end point during the full 12 weeks of the study. Eluxadoline was well tolerated with a low incidence of constipation. CONCLUSIONS: In a phase 2 study of the mixed µ-opioid receptor agonist/δ-opioid receptor antagonist eluxadoline vs placebo in patients with IBS-D, patients given eluxadoline were significantly more likely to be clinical responders, based on a composite of improvement in abdominal pain and stool consistency. Further study of eluxadoline is warranted to assess its potential as a treatment for IBS-D.

Role of mixed ion channel effects in the cardiovascular safety assessment of the novel anti-MRSA fluoroquinolone JNJ-Q2.

BACKGROUND AND PURPOSE: JNJ-Q2, a novel broad-spectrum fluoroquinolone with anti-methicillin-resistant Staphylococcus aureus activity, was evaluated in a comprehensive set of non-clinical and clinical cardiovascular safety studies. The effect of JNJ-Q2 on different cardiovascular parameters was compared with that of moxifloxacin, sparfloxacin and ofloxacin. Through comparisons with these well-known fluoroquinolones, the importance of effects on compensatory ion channels to the cardiovascular safety of JNJ-Q2 was investigated. EXPERIMENTAL APPROACH: JNJ-Q2 and comparator fluoroquinolones were evaluated in the following models/test systems: hERG-transfected HEK293 cells sodium channel-transfected CHO cells, guinea pig right atria, arterially perfused rabbit left ventricular wedge preparations and in vivo studies in anaesthetized guinea pigs, anaesthetized and conscious telemetered dogs, and a thorough QT study in humans. KEY RESULTS: The trend for effects of JNJ-Q2 on Tp-Te, QT, QRS and PR intervals in the non-clinical models and the plateau in QTc with increasing plasma concentration in humans are consistent with offsetting sodium and calcium channel activities that were observed in the non-clinical studies. These mixed ion channel activities result in the less pronounced or comparable increase in QTc interval for JNJ-Q2 compared with moxifloxacin and sparfloxacin despite its greater in vitro inhibition of I(Kr). CONCLUSIONS AND IMPLICATIONS: Based on the non-clinical and clinical cardiovascular safety assessment, JNJ-Q2 has a safe cardiovascular profile for administration in humans with comparable or reduced potential to prolong QT intervals, compared with moxifloxacin. The results demonstrate the importance of compensatory sodium and calcium channel activity in offsetting potassium channel activity for compounds with a fluoroquinolone core.

Summary of Pharmacokinetics and Tissue Distribution of a Broad-Spectrum Fluoroquinolone, JNJ-Q2.

This article summarizes key pharmacokinetic properties of JNJ-Q2, a broad-spectrum fluoroquinolone, being developed for acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia. Two randomized placebo-controlled studies and one open-label study are presented: single and multiple ascending-dose studies evaluating intravenous (IV) and oral pharmacokinetics, absolute bioavailability accumulation, and lung penetration. Fifty-seven participants received JNJ-Q2, which was safe and well tolerated. The half-life was 13 to 20 hours, clearance (CLtot ) was 4.41 to 6.22 L/h, volume of distribution (Vd ) was 86 to 148 L, renal clearance (CLren ) was 0.58 L/h, and the fraction excreted in urine (%Fe) was 12%. Accumulation ratio was 1.63, and absolute bioavailability was 0.65. The lung penetration study demonstrated substantial concentration of JNJ-Q2 in epithelial lining fluid and alveolar macrophages with ratios to free plasma of 50.6 and 156.9, respectively, at 6 hours postdose. JNJ-Q2 doses under consideration for future clinical trials are 150 mg for IV and 250-mg tablets for oral administration.

Randomized, double-blind, phase II, multicenter study evaluating the safety/tolerability and efficacy of JNJ-Q2, a novel fluoroquinolone, compared with linezolid for treatment of acute bacterial skin and skin structure infection.

JNJ-Q2 is a fluoroquinolone with broad coverage including methicillin-resistant Staphylococcus aureus (MRSA). A double-blind, multicenter, phase II noninferiority study treated 161 patients for 7 to 14 days, testing the efficacy of JNJ-Q2 (250 mg, twice a day [BID]) versus linezolid (600 mg, BID) in patients with acute bacterial skin and skin structure infections (ABSSSI). The prespecified criterion for noninferiority was 15%. Primary intent-to-treat analysis was unable to declare noninferiority, as the risk difference lower bound of the 95% confidence interval between treatments was 19% at 36 to 84 h postrandomization for the composite end point of lesion assessment and temperature. Prespecified clinical cure rates 2 to 14 days after completion of therapy were similar (83.1% for JNJ-Q2 versus 82.1% for linezolid). Post hoc analyses revealed that JNJ-Q2 was statistically noninferior to linezolid (61.4% versus 57.7%, respectively; P = 0.024) based on the 2010 FDA guidance, which defines treatment success as lack of lesion spread and afebrile status within 48 to 72 h postrandomization. Despite evidence of systemic disease, <5% of patients presented with fever, suggesting fever is not a compelling surrogate measure of systemic disease resolution for this indication. Nausea and vomiting were the most common adverse events. Of the patients, 86% (104/121) had S. aureus isolated from the infection site; 63% of these were MRSA. The results suggest JNJ-Q2 shows promise as an effective treatment for ABSSSI, demonstrating (i) efficacy for early clinical response (i.e., lack of spread of lesions and absence of fever at 48 to 72 h), and (ii) cure rates for ABSSSI pathogens (especially MRSA) consistent with the historical literature.

A national survey of instrument sharpening guidelines.

Most orthopaedic procedures involve high-speed tools, which can cause thermal necrosis of bone. Blunt instruments are likely to generate more heat, thereby increasing the chances of thermal necrosis. This could greatly compromise the outcome of even the best prosthesis in good surgical hands. In today's age a lot of emphasis is laid on high performance and longevity of the implant. Therefore, it is worthwhile that we consider the relevance and importance of instrument sharpening. We carried out a survey across hospitals in UK to find out whether they follow any guidelines or protocol with regards to instrument sharpening. Upto 75% of hospitals did not follow any guideline or policy, while 20% of hospitals carried out some sort of quality checks on instruments. This implies that there could be a significant risk of suboptimal outcome due to unnecessary osteonecrosis that can affect most operative orthopaedic interventions, particularly joint arthroplasties. Blunt instruments are too common in orthopaedic theatres and this problem needs to be addressed. Our study brings to light the fact that there seems to be no consensus on this issue and there is theoretically a significant risk of suboptimal outcome because of unnecessary osteonecrosis. This potentially may affect the long and short-term results even in good hands and with excellent prosthesis (cemented or uncemented).

Challenges and solutions of remote sensing at offshore wind energy developments.

Radar is becoming an important tool used to gather data on bird and bat activity at proposed and existing land-based wind energy sites. Radar will likely play an even more important role at the increasing development of wind energy offshore, given both the lack of knowledge about bird and bat activity offshore and the increased difficulty in obtaining offshore information. Most radar studies to date have used off-the-shelf or modified marine radars. However, there are several issues that continue to hinder the potential usefulness of radar at wind energy sites, with offshore sites providing a particular suite of challenges. We identify these challenges along with current or developing solutions.

The effects of salinity on the Manila clam (Ruditapes philippinarum) using the neutral red retention assay with adapted physiological saline solutions.

This study investigated the internal osmotic regulatory capabilities of the Manila clam (Ruditapes philippinarum) following in vivo exposure to a range of salinities. A second objective was to measure the health status of the Manila clam following exposure to different salinities using the neutral red retention (NRR) assay, and to compare results using a range of physiological saline solutions (PSS). On exposure to seawater of differing salinities, the Manila clam followed a pattern of an osmoconformer, although they seemed to partially regulate their circulatory haemolytic fluids to be hyperosmotic to the surrounding aqueous environment. Significant differences were found when different PSS were used, emphasizing the importance of using a suitable PSS to reduce additional osmotic stress. Using PSS in the NRR assay that do not exert additional damage to lysosomal membrane integrity will help to more accurately quantify the effects of exposure to pollutants on the organism(s) under investigation.