Evidence for a retroviral insertion in TRPM1 as the cause of congenital stationary night blindness and leopard complex spotting in the horse.

Leopard complex spotting is a group of white spotting patterns in horses caused by an incompletely dominant gene (LP) where homozygotes (LP/LP) are also affected with congenital stationary night blindness. Previous studies implicated Transient Receptor Potential Cation Channel, Subfamily M, Member 1 (TRPM1) as the best candidate gene for both CSNB and LP. RNA-Seq data pinpointed a 1378 bp insertion in intron 1 of TRPM1 as the potential cause. This insertion, a long terminal repeat (LTR) of an endogenous retrovirus, was completely associated with LP, testing 511 horses (χ(2)=1022.00, p<0.0005), and CSNB, testing 43 horses (χ(2)=43, p<0.0005). The LTR was shown to disrupt TRPM1 transcription by premature poly-adenylation. Furthermore, while deleterious transposable element insertions should be quickly selected against the identification of this insertion in three ancient DNA samples suggests it has been maintained in the horse gene pool for at least 17,000 years. This study represents the first description of an LTR insertion being associated with both a pigmentation phenotype and an eye disorder.

Role of complement and Fc{gamma} receptors in the protective activity of the long pentraxin PTX3 against Aspergillus fumigatus

Pentraxin 3 (PTX3) is a soluble pattern recognition molecule playing a nonredundant role in resistance against Aspergillus fumigatus. The present study was designed to investigate the molecular pathways involved in the opsonic activity of PTX3. The PTX3 N-terminal domain was responsible for conidia recognition, but the full-length molecule was necessary for opsonic activity. The PTX3-dependent pathway of enhanced neutrophil phagocytic activity involved complement activation via the alternative pathway; Fc receptor (Fc R) IIA/CD32 recognition of PTX3-sensitized conidia and complement receptor 3 (CR3) activation; and CR3 and CD32 localization to the phagocytic cup. Gene targeted mice (ptx3, FcR common chain, C3, C1q) validated the in vivo relevance of the pathway. In particular, the protective activity of exogenous PTX3 against A fumigatus was abolished in FcR common chain-deficient mice. Thus, the opsonic and antifungal activity of PTX3 is at the crossroad between complement, complement receptor 3-, and Fc R-mediated recognition. Because short pentraxins (eg, C-reactive protein) interact with complement and Fc R, the present results may have general significance for the mode of action of these components of the humoral arm of innate immunity.

Eculizumab in atypical hemolytic uremic syndrome: long-term clinical course and histological findings

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with defective regulation of the alternative complement pathway. The prognosis for patients with aHUS is poor, and plasma exchange represents the first-line therapy. Eculizumab is a humanized monoclonal anti-C5 antibody that prevents the activation of the terminal complement pathway. Here, we report the case of a 9-year-old girl with frequent relapsing aHUS due to heterozygous factor H mutation who was initially treated with plasma exchange three times per week with 150% plasma exchange volume. This treatment frequently caused allergic reactions and school absences. Because any reduction in the frequency of plasma exchange immediately induced relapses of the aHUS, treatment with eculizumab, 600 mg every 2 weeks, was started and plasma exchange completely stopped. On this drug regimen the patient showed no evidence of disease activity during a period of more than 24 months. Renal function improved, proteinuria disappeared, the number of antihypertensive medications could be decreased, and the quality of life increased substantially. The inhibition of the terminal complement pathway by eculizumab was also confirmed by renal biopsy, which showed the absence of thrombotic microangiopathy 2 months after the initiation of eculizumab therapy. This case illustrates the long-term favorable outcome of aHUS with eculizumab treatment.

Long-term clinical and angiographic outcomes of diabetic patients after revascularization with early generation drug-eluting stents

Early generation drug-eluting stents (DESs) reduce restenosis and repeat revascularization procedures. However, the long-term safety and efficacy of early generation DES according to diabetic status are poorly established.

Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries

Click chemistry is a powerful technology for the functionalization of therapeutic proteins with effector moieties, because of its potential for bio-orthogonal, regio-selective, and high-yielding conjugation under mild conditions. Designed Ankyrin Repeat Proteins (DARPins), a novel class of highly stable binding proteins, are particularly well suited for the introduction of clickable methionine surrogates such as azidohomoalanine (Aha) or homopropargylglycine (Hpg), since the DARPin scaffold can be made methionine-free by an M34L mutation in the N-cap which fully maintains the biophysical properties of the protein. A single N-terminal azidohomoalanine, replacing the initiator Met, is incorporated in high yield, and allows preparation of "clickable" DARPins at about 30 mg per liter E. coli culture, fully retaining stability, specificity, and affinity. For a second modification, we introduced a cysteine at the C-terminus. Such DARPins could be conveniently site-specifically linked to two moieties, polyethylene glycol (PEG) to the N-terminus and the fluorophore Alexa488 to the C-terminus. We present a DARPin selected against the epithelial cell adhesion molecule (EpCAM) with excellent properties for tumor targeting as an example. We used these doubly modified molecules to measure binding kinetics on tumor cells and found that PEGylation has no effect on dissociation rate, but slightly decreases the association rate and the maximal number of cell-bound DARPins, fully consistent with our previous model of PEG action obtained in vitro. Our data demonstrate the benefit of click chemistry for site-specific modification of binding proteins like DARPins to conveniently add several functional moieties simultaneously for various biomedical applications.

Short- and long-term cultivation of embryonic and neonatal murine keratinocytes

Studies using cultured cells allow one to dissect complex cellular mechanisms in greater detail than when studying living organisms alone. However, before cultured cells can deliver meaningful results they must accurately represent the in vivo situation. Over the last three to four decades considerable effort has been devoted to the development of culture media which improve in vitro growth and modeling accuracy. In contrast to earlier large-scale, non-specific screening of factors, in recent years the development of such media has relied increasingly on a deeper understanding of the cell's biology and the selection of growth factors to specifically activate known biological processes. These new media now enable equal or better cell isolation and growth, using significantly simpler and less labor-intensive methodologies. Here we describe a simple method to isolate and cultivate epidermal keratinocytes from embryonic or neonatal skin on uncoated plastic using a medium specifically designed to retain epidermal keratinocyte progenitors in an undifferentiated state for improved isolation and proliferation and an alternative medium to support terminal differentiation.

Depicting community perspectives: repeat photography and participatory research as tools for assessing environmental services in Sagarmatha National Park, Nepal

Efforts have been made to provide a scientific basis for using environmental services as a conceptual tool to enhance conservation and improve livelihoods in protected mountain areas (MtPAS). Little attention has been paid to participatory research or locals’ concerns as environmental service (ES) users and providers. Such perspectives can illuminate the complex interplay between mountain ecosystems, environmental services and the determinants of human well-being. Repeat photography, long used in geographical fieldwork, is new as a qualitative research tool. This study uses a novel application of repeat photography as a diachronic photo-diary to examine local perceptions of change in ES in Sagarmatha National Park. Results show a consensus among locals on adverse changes to ES, particularly protection against natural hazards, such as landslides and floods, in the UNESCO World Heritage Site. We argue that our methodology could complement biophysical ecosystem assessments in MtPAS, especially since assessing ES, and acting on that, requires integrating diverse stakeholders’ knowledge, recognizing power imbalances and grappling with complex social-ecological systems.

Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain reveals an unmasking mechanism for receptor activation

Glycoprotein Ib (GPIb) is a platelet receptor with a critical role in mediating the arrest of platelets at sites of vascular damage. GPIb binds to the A1 domain of von Willebrand factor (vWF-A1) at high blood shear, initiating platelet adhesion and contributing to the formation of a thrombus. To investigate the molecular basis of GPIb regulation and ligand binding, we have determined the structure of the N-terminal domain of the GPIb(alpha) chain (residues 1-279). This structure is the first determined from the cell adhesion/signaling class of leucine-rich repeat (LRR) proteins and reveals the topology of the characteristic disulfide-bonded flanking regions. The fold consists of an N-terminal beta-hairpin, eight leucine-rich repeats, a disulfide-bonded loop, and a C-terminal anionic region. The structure also demonstrates a novel LRR motif in the form of an M-shaped arrangement of three tandem beta-turns. Negatively charged binding surfaces on the LRR concave face and anionic region indicate two-step binding kinetics to vWF-A1, which can be regulated by an unmasking mechanism involving conformational change of a key loop. Using molecular docking of the GPIb and vWF-A1 crystal structures, we were also able to model the GPIb.vWF-A1 complex.

Effects of the glucocorticoid antagonist, mifepristone, on the consequences of withdrawal from long term alcohol consumption

BACKGROUND: Studies were carried out to test the hypothesis that administration of a glucocorticoid Type II receptor antagonist, mifepristone (RU38486), just prior to withdrawal from chronic alcohol treatment, would prevent the consequences of the alcohol consumption and withdrawal in mice. MATERIALS AND METHODS: The effects of administration of a single intraperitoneal dose of mifepristone were examined on alcohol withdrawal hyperexcitability. Memory deficits during the abstinence phase were measured using repeat exposure to the elevated plus maze, the object recognition test, and the odor habituation/discrimination test. Neurotoxicity in the hippocampus and prefrontal cortex was examined using NeuN staining. RESULTS: Mifepristone reduced, though did not prevent, the behavioral hyperexcitability seen in TO strain mice during the acute phase of alcohol withdrawal (4 hours to 8 hours after cessation of alcohol consumption) following chronic alcohol treatment via liquid diet. There were no alterations in anxiety-related behavior in these mice at 1 week into withdrawal, as measured using the elevated plus maze. However, changes in behavior during a second exposure to the elevated plus maze 1 week later were significantly reduced by the administration of mifepristone prior to withdrawal, indicating a reduction in the memory deficits caused by the chronic alcohol treatment and withdrawal. The object recognition test and the odor habituation and discrimination test were then used to measure memory deficits in more detail, at between 1 and 2 weeks after alcohol withdrawal in C57/BL10 strain mice given alcohol chronically via the drinking fluid. A single dose of mifepristone given at the time of alcohol withdrawal significantly reduced the memory deficits in both tests. NeuN staining showed no evidence of neuronal loss in either prefrontal cortex or hippocampus after withdrawal from chronic alcohol treatment. CONCLUSIONS: The results suggest mifepristone may be of value in the treatment of alcoholics to reduce their cognitive deficits.

Early- and long-term intravascular ultrasound and angiographic findings after bioabsorbable magnesium stent implantation in human coronary arteries

OBJECTIVES: This study aimed to evaluate the degradation rate and long-term vascular responses to the absorbable metal stent (AMS). BACKGROUND: The AMS demonstrated feasibility and safety at 4 months in human coronary arteries. METHODS: The PROGRESS-AMS (Clinical Performance and Angiographic Results of Coronary Stenting) was a prospective, multicenter clinical trial of 63 patients with coronary artery disease who underwent AMS implantation. Angiography and intravascular ultrasound (IVUS) were conducted immediately after AMS deployment and at 4 months. Eight patients who did not require repeat revascularization at 4 months underwent late angiographic and IVUS follow-up from 12 to 28 months. RESULTS: The AMS was well-expanded upon deployment without immediate recoil. The major contributors for restenosis as detected by IVUS at 4 months were: decrease of external elastic membrane volume (42%), extra-stent neointima (13%), and intra-stent neointima (45%). From 4 months to late follow-up, paired IVUS analysis demonstrated complete stent degradation with durability of the 4-month IVUS indexes. The neointima was reduced by 3.6 +/- 5.2 mm(3), with an increase in the stent cross sectional area of 0.5 +/- 1.0 mm(2) (p = NS). The median in-stent minimal lumen diameter was increased from 1.87 to 2.17 mm at long-term follow-up. The median angiographic late loss was reduced from 0.62 to 0.40 mm by quantitative coronary angiography from 4 months to late follow-up. CONCLUSIONS: Intravascular ultrasound imaging supports the safety profile of AMS with degradation at 4 months and maintains durability of the results without any early or late adverse findings. Slower degradation is warranted to provide sufficient radial force to improve long-term patency rates of the AMS.

Catheter ablation of persistent and long-standing persistent atrial fibrillation. Strategies and results

Catheter ablation for paroxysmal atrial fibrillation is a meanwhile established therapy option, which is most frequently performed using radiofrequency ablation. Mid-term success rate of 70 % are achievable with a single ablation procedure. However, the mechanistics of persistent atrial fibrillation are less well understood and catheter ablation is a far more challenging procedure. Different ablation approaches are being performed to treat persistent atrial fibrillation ranging from sole pulmonary vein isolation to additional ablation of fractionated electrograms aiming for termination of atrial fibrillation. Thus far, it has not been investigated which strategy is most successful in treating persistent atrial fibrillation. After extended ablation of atrial fibrillation, occurrence of organized atrial arrhythmias is not uncommon and can be successfully ablated. These consecutive arrhythmias can be considered as a next step towards stable sinus rhythm after repeat ablation. Improvement of mapping methods as well as a better understanding of mechanisms of atrial fibrillation may increase success rate of catheter ablation of persistent atrial fibrillation and may also help to improve success rate of these complex procedures.

Terminal short arm domains of basement membrane laminin are critical for its self-assembly

Laminin self-assembles into large polymers by a cooperative two-step calcium-dependent mechanism (Yurchenco, P. D., E. C. Tsilibary, A. S. Charonis, and H. Furthmayr. 1985. J. Biol. Chem. 260:7636-7644). The domain specificity of this process was investigated using defined proteolytically generated fragments corresponding to the NH2-terminal globule and adjacent stem of the short arm of the B1 chain (E4), a complex of the two short arms of the A and B2 chains attached to the proximal stem of a third short arm (E1'), a similar complex lacking the globular domains (P1'), and the distal half of the long arm attached to the adjacent portion of the large globule (E8). Polymerization, followed by an increase of turbidity at 360 nm in neutral isotonic TBS containing CaCl2 at 35 degrees C, was quantitatively inhibited in a concentration-dependent manner with laminin fragments E4 and E1' but not with fragments E8 and P1'. Affinity retardation chromatography was used for further characterization of the binding of laminin domains. The migration of fragment E4, but not of fragments E8 and P1', was retarded in a temperature- and calcium-dependent fashion on a laminin affinity column but not on a similar BSA column. These data are evidence that laminin fragments E4 and E1' possess essential terminal binding domains for the self-aggregation of laminin, while fragments E8 and P1' do not. Furthermore, the individual domain-specific interactions that contribute to assembly are calcium dependent and of low affinity.

4-year clinical outcomes and predictors of repeat revascularization in patients treated with new-generation drug-eluting stents: a report from the RESOLUTE All-Comers trial (A Randomized Comparison of a Zotarolimus-Eluting Stent With an Everolimus-Eluting Stent for Percutaneous Coronary Intervention)

OBJECTIVES The aim of the study was to investigate 4-year outcomes and predictors of repeat revascularization in patients treated with the Resolute zotarolimus-eluting stent (R-ZES) (Medtronic, Minneapolis, Minnesota) and XIENCE V everolimus-eluting stent (EES) (Abbott Vascular, Abbott Park, Illinois) in the RESOLUTE (A Randomized Comparison of a Zotarolimus-Eluting Stent With an Everolimus-Eluting Stent for Percutaneous Coronary Intervention) All-Comers trial. BACKGROUND Data on long-term outcomes of new-generation drug-eluting stents are limited, and predictors of repeat revascularization due to restenosis and/or progression of disease are largely unknown. METHODS Patients were randomly assigned to treatment with the R-ZES (n = 1,140) or the EES (n = 1,152). We assessed pre-specified safety and efficacy outcomes at 4 years including target lesion failure and stent thrombosis. Predictors of revascularization at 4 years were identified by Cox regression analysis. RESULTS At 4 years, the rates of target lesion failure (15.2% vs. 14.6%, p = 0.68), cardiac death (5.4% vs. 4.7%, p = 0.44), and target vessel myocardial infarction (5.3% vs. 5.4%, p = 1.00), clinically-indicated target lesion revascularization (TLR) (7.0% vs. 6.5%, p = 0.62), and definite/probable stent thrombosis (2.3% vs. 1.6%, p = 0.23) were similar with the R-ZES and EES. Independent predictors of TLR were age, insulin-treated diabetes, SYNTAX (Synergy between PCI with Taxus and Cardiac Surgery) score, treatment of saphenous vein grafts, ostial lesions, and in-stent restenosis. Independent predictors of any revascularization were age, diabetes, previous percutaneous coronary intervention, absence of ST-segment elevation myocardial infarction, smaller reference vessel diameter, SYNTAX score, and treatment of left anterior descending, right coronary artery, saphenous vein grafts, ostial lesions, or in-stent restenosis. CONCLUSIONS R-ZES and EES demonstrated similar safety and efficacy throughout 4 years. TLR represented less than one-half of all repeat revascularization procedures. Patient- and lesion-related factors predicting the risk of TLR and any revascularization showed considerable overlap. (A Randomized Comparison of a Zotarolimus-Eluting Stent With an Everolimus-Eluting Stent for Percutaneous Coronary Intervention [RESOLUTE-AC]; NCT00617084).

Von Willebrand Factor Improves Risk Prediction in Addition to N-Terminal Pro-B-type Natriuretic Peptide in Patients Referred to Coronary Angiography and Signs and Symptoms of Heart Failure and Preserved Ejection Fraction.

BACKGROUND Heart failure with preserved ejection fraction (HFpEF) represents a growing health burden associated with substantial mortality and morbidity. Consequently, risk prediction is of highest importance. Endothelial dysfunction has been recently shown to play an important role in the complex pathophysiology of HFpEF. We therefore aimed to assess von Willebrand factor (vWF), a marker of endothelial damage, as potential biomarker for risk assessment in patients with HFpEF. METHODS AND RESULTS Concentrations of vWF were assessed in 457 patients with HFpEF enrolled as part of the LUdwigshafen Risk and Cardiovascular Health (LURIC) study. All-cause mortality was observed in 40% of patients during a median follow-up time of 9.7 years. vWF significantly predicted mortality with a hazard ratio (HR) per increase of 1 SD of 1.45 (95% confidence interval, 1.26-1.68; P<0.001) and remained a significant predictor after adjustment for age, sex, body mass index, N-terminal pro-B-type natriuretic peptide (NT-proBNP), renal function, and frequent HFpEF-related comorbidities (adjusted HR per 1 SD, 1.22; 95% confidence interval, 1.05-1.42; P=0.001). Most notably, vWF showed additional prognostic value beyond that achievable with NT-proBNP indicated by improvements in C-Statistic (vWF×NT-proBNP: 0.65 versus NT-proBNP: 0.63; P for comparison, 0.004) and category-free net reclassification index (37.6%; P<0.001). CONCLUSIONS vWF is an independent predictor of long-term outcome in patients with HFpEF, which is in line with endothelial dysfunction as potential mediator in the pathophysiology of HFpEF. In particular, combined assessment of vWF and NT-proBNP improved risk prediction in this vulnerable group of patients.

Evolving biomarkers improve prediction of long-term mortality in patients with stable coronary artery disease: the BIO-VILCAD score.

OBJECTIVE Algorithms to predict the future long-term risk of patients with stable coronary artery disease (CAD) are rare. The VIenna and Ludwigshafen CAD (VILCAD) risk score was one of the first scores specifically tailored for this clinically important patient population. The aim of this study was to refine risk prediction in stable CAD creating a new prediction model encompassing various pathophysiological pathways. Therefore, we assessed the predictive power of 135 novel biomarkers for long-term mortality in patients with stable CAD. DESIGN, SETTING AND SUBJECTS We included 1275 patients with stable CAD from the LUdwigshafen RIsk and Cardiovascular health study with a median follow-up of 9.8 years to investigate whether the predictive power of the VILCAD score could be improved by the addition of novel biomarkers. Additional biomarkers were selected in a bootstrapping procedure based on Cox regression to determine the most informative predictors of mortality. RESULTS The final multivariable model encompassed nine clinical and biochemical markers: age, sex, left ventricular ejection fraction (LVEF), heart rate, N-terminal pro-brain natriuretic peptide, cystatin C, renin, 25OH-vitamin D3 and haemoglobin A1c. The extended VILCAD biomarker score achieved a significantly improved C-statistic (0.78 vs. 0.73; P = 0.035) and net reclassification index (14.9%; P < 0.001) compared to the original VILCAD score. Omitting LVEF, which might not be readily measureable in clinical practice, slightly reduced the accuracy of the new BIO-VILCAD score but still significantly improved risk classification (net reclassification improvement 12.5%; P < 0.001). CONCLUSION The VILCAD biomarker score based on routine parameters complemented by novel biomarkers outperforms previous risk algorithms and allows more accurate classification of patients with stable CAD, enabling physicians to choose more personalized treatment regimens for their patients.