Genomic monitoring to understand the emergence and spread of Usutu virus in the Netherlands, 2016-2018.

Usutu virus (USUV) is a mosquito-borne flavivirus circulating in Western Europe that causes die-offs of mainly common blackbirds (Turdus merula). In the Netherlands, USUV was first detected in 2016, when it was identified as the likely cause of an outbreak in birds. In this study, dead blackbirds were collected, screened for the presence of USUV and submitted to Nanopore-based sequencing. Genomic sequences of 112 USUV were obtained and phylogenetic analysis showed that most viruses identified belonged to the USUV Africa 3 lineage, and molecular clock analysis evaluated their most recent common ancestor to 10 to 4 years before first detection of USUV in the Netherlands. USUV Europe 3 lineage, commonly found in Germany, was less frequently detected. This analyses further suggest some extent of circulation of USUV between the Netherlands, Germany and Belgium, as well as likely overwintering of USUV in the Netherlands.

Towards high quality real-time whole genome sequencing during outbreaks using Usutu virus as example.

Recently, protocols for amplicon based whole genome sequencing using Nanopore technology have been described for Ebola virus, Zika virus, yellow fever virus and West Nile virus. However, there is some debate regarding reliability of sequencing using this technology, which is important for applications beyond diagnosis such as linking lineages to outbreaks, tracking transmission pathways and pockets of circulation, or mapping specific markers. To our knowledge, no in depth analyses of the required read coverage to compensate for the error profile in Nanopore sequencing have been described. Here, we describe the validation of a protocol for whole genome sequencing of USUV using Nanopore sequencing by direct comparison to Illumina sequencing. To that point we selected brain tissue samples with high viral loads, typical for birds which died from USUV infection. We conclude that the low-cost MinION Nanopore sequencing platform can be used for characterization and tracking of Usutu virus outbreaks.

Comparación de las características mecánicas estáticas del fémur aislado de perro, con y sin la colocación de una placa de ortopedia fabricada en polipropileno / Compare the mechanical statistical characteristics of dog femur isolet, with or without placing to an orthopedic píate made of polypropylene

Los objetivos generales fueron: evaluar resistencia a la compresión del fémur derecho aislado y del fémur izquierdo osteotomizado transversalmente fijado con una placa ortopédica fabricada en polipropileno, y comparar las características mecánicas estáticas entre ambos. Este trabajo se realizó mediante la confección de un modelo experimental de carga constituido por un fémur de mamífero (perro) en condiciones aisladas. Se utilizaron los huesos fémur de 13 perros mestizos, adultos jóvenes, de talla mediana a grande. Para los pruebas se utilizó una máquina servohidráulica para ensayos estáticos y dinámicos Amsler Wolpert. Se utilizó para el tratamiento de los datos, el test estadístico de diferencias de medias para muestras pareadas dependientes. Como resultado del análisis de las curvas se obtuvo que: el fémur derecho soportó antes de la falla, entre 4,02 a 8,47 KN de carga (= 6,66 KN) con una deformación de 2,02 a 5,09 mm (= 3,66 mm). En tanto el conjunto fémur izquierdo + placa ortopédica soportó antes de la falla, entre 3,05 a 5,61 KN de carga (= 4,49 KN) con una deformación de 2 a 4.5 mm (= 3 mm). Como conclusión, es posible afirmar que hay diferencias en la resistencia a la carga compresiva entre el fémur derecho y su contralateral izquierdo + placa ortopédica.

The general aims were: to evaluate the resistance to compression of the right femur isolated and that of the left femur transversally osteotomized fixed to an orthopedic plate made of polypropylene and to compare the mechanical statistical characteristics between both. This work was carried out by performing an experimental charge pattern made of a mammal femur (dog) under isolated conditions. Femur bones of 13 hybrid young adult dogs of medium to large size were used. A servohydric engine was used for the Amsler Wolpert static and dynamic essays. The mean difference statistical test for depending paired samples was used for the evaluation of data. As a result of the curves analysis it was obtained that: before failure the right femur underwent a charge of 4.02 to 8.47 KN (= 6.66 KN) with a deformation of 2.02 to 5.09 mm (= 3.66mm) while the charge that the left femur + orthopedic plate underwent before de failure was of 3.05 to 5.61 KN ( = 4.49KN) with a deformation of 2 to 4.5 mm ( = 3mm). As a conclusion it might be stated that there are differences in the resistance to the compressive charge between right femur and its left contra lateral + the orthopedic plate.

Polyethylene glycol (molecular weight 400 DA) vehicle improves gene expression of adenovirus mediated gene therapy.

PURPOSE: A significant limitation of adenoviral mediated suicide gene therapy is poor gene distribution in vivo. The choice of vehicle has been demonstrated to affect the level of adenoviral delivered gene transduction. We examined the hypotheses that 1) adenovirus suspended in PEG400 improves gene expression in the naïve canine prostate model, 2) improved transgene expression with PEG400 results in improved tumor control and 3) vehicle affects the initial adenoviral spread from a single intratumor injection. MATERIALS AND METHODS: The magnitude and volume of gene expression were measured 24 hours following intraprostatic injection of adenovirus suspended in PEG400 (12.5% weight per volume) or saline as vehicle. Tumor growth delay was measured in mice bearing human tumor xenografts following the injection of adenovirus in PEG400 and saline. The initial spread of adenovirus was measured by confocal microscopy following a single injection of fluorescently labeled adenoviral particles in human tumor xenografts using each vehicle. RESULTS: Adenovirus suspended in PEG400 provided an average of twice the level of gene expression in the canine prostate and significantly better tumor control relative to saline in preclinical tumor models (p = 0.046 and 0.036, respectively). The initial spread of adenovirus with PEG400 was superior to that of adenovirus in saline and the latter was largely limited to the needle tract. CONCLUSIONS: Adenoviral gene therapy vectors suspended in PEG400 results in improved tumor control because of greater initial adenoviral spread, and the increased volume and magnitude of gene expression in vivo.

Role of norepinephrine in the dorsomedial hypothalamic panic response: an in vivo microdialysis study.

Blockade of gamma-aminobutyric acid-A (GABA(A)) receptors in the dorsomedial hypothalamus (DMH) elicits a panic-like response that includes increases in heart rate (HR), blood pressure (BP), respiration rate (RR), and anxiety. Norepinephrine (NE) has been postulated to be critical in regulating panic and anxiety responses. Therefore, the first study sought to determine changes in extracellular NE levels within the DMH following acute blockade of GABA(A) receptors in the DMH using in vivo microdialysis. Rats were implanted with femoral arterial catheters and microdialysis probes into the DMH. Following recovery, the DMH of conscious rats were perfused with 100, 150, or 200 microM solutions of the GABA(A) receptor antagonist bicuculline methiodide (BMI) via the microdialysis probe. HR and BP responses were recorded and the changes in extracellular levels of NE in the dialysate samples from the DMH were determined by HPLC. Rats receiving BMI injections showed dose-dependent increases in both the extracellular NE levels in the DMH as well as HR and BP. The second study was conducted to test the functional importance of NE in the DMH to the BMI-induced physiological responses. The effects of BMI microinjection into the DMH were measured at baseline and 10 days after local injection of either vehicle or two doses of 6-hydroxydopamine (6-OHDA), a neurotoxin known to lesion NE terminals. There was a significant loss of tissue NE levels as well as BMI-induced HR, BP and RR responses in the 6-OHDA-treated but not vehicle-treated rats. Thus, blockade of GABA(A) receptors in the DMH results in NE release and the presence of NE appears to be necessary for eliciting the physiological components of the panic-like responses in this region.

Knowledge of results for motor learning: relationship between error estimation and knowledge of results frequency.

The authors of the present study investigated the apparent contradiction between early and more recent views of knowledge of results (KR), the idea that how one is engaged before receiving KR may not be independent of how one uses that KR. In a 2 ×: 2 factorial design, participants (N = 64) practiced a simple force-production task and (a) were required, or not required, to estimate error about their previous response and (b) were provided KR either after every response (100%) or after every 5th response (20%) during acquisition. A no-KR retention test revealed an interaction between acquisition error estimation and KR frequencies. The group that received 100% KR and was required to error estimate during acquisition performed the best during retention. The 2 groups that received 20% KR performed less well. Finally, the group that received 100% KR and was not required to error estimate during acquisition performed the poorest during retention. One general interpretation of that pattern of results is that motor learning is an increasing function of the degree to which participants use KR to test response hypotheses (J. A. Adams, 1971; R. A. Schmidt, 1975). Practicing simple responses coupled with error estimation may embody response hypotheses that can be tested with KR, thus benefiting motor learning most under a 100% KR condition. Practicing simple responses without error estimation is less likely to embody response hypothesis, however, which may increase the probability that participants will use KR to guide upcoming responses, thus attenuating motor learning under a 100% KR condition. The authors conclude, therefore, that how one is engaged before receiving KR may not be independent of how one uses KR.

NH2-terminal cleavage of xenopus fibroblast growth factor 3 is necessary for optimal biological activity and receptor binding.

Fibroblast growth factor 3 (FGF3) was originally identified as the mouse proto-oncogene Int-2, which is activated by proviral insertion in tumors induced by mouse mammary tumor virus. To facilitate the biological characterization of the ligand, we have analyzed its homologue in Xenopus laevis, XFGF3. Here we confirm that the X. laevis genome contains two distinct FGF3 alleles, neither of which is capable of encoding the NH2-terminally extended forms specified by the mouse and human FGF3 genes. Unlike the mammalian proteins, XFGF3 is efficiently secreted as a Mr 31,000 glycoprotein, gp31, which undergoes proteolytic cleavage to produce an NH2-terminally truncated product, gp27. Processing removes a segment of 18 amino acids immediately distal to the signal peptide that is not present in the mammalian homologues. By inserting an epitope-tag adjacent to the cleavage site, we show that a substantial amount of the gp27 is generated intracellularly, although processing can also occur in the extracellular matrix. Two residues are also removed from the COOH terminus. To compare the biological properties of the different forms, cDNAs were constructed that selectively give rise to the larger, gp31, or smaller, gp27, forms of XFGF3. As judged by their ability to cause morphological transformation of NIH3T3 cells, their mitogenicity on specific cell types, and their affinity for the IIIb and IIIc isoforms of Xenopus FGF receptors, gp27 has a much higher biological activity than gp31. Sequence comparison revealed an intriguing similar cleavage motif immediately downstream of the signal peptide cleavage site in the NH2-terminus of mouse and human FGF3. Analysis of secreted mutant mouse FGF3 confirmed an additional NH2-terminal processing at the corresponding sequence motif. NH2-terminal trimming of Xenopus and mammalian FGF3s may therefore be a prerequisite of optimal biological activity.

The utility of transesophageal echocardiography in determining the source of arterial embolization.

Arterial embolism is frequently of a cardiac source. Arterial-arterial and paradoxical embolization also occurs. Failure to identify the origin may subject the patient to an important series of events. Herein we describe seven cases in which transesophageal echocardiography (TEE) was uniquely valuable in identifying the source or mechanism and in which conventional echocardiography and aortography were nondiagnostic. We conducted a chart review of patients with arterial emboli definitively diagnosed after undergoing TEE. Seven patients (mean age 68 years) were included in the study. Peripheral embolization occurred in four patients, visceral embolization occurred in one, and two experienced cerebrovascular events. Five patients had transthoracic echocardiography and six had aortography; none of these identified the source of embolization. All were diagnosed by TEE. Mobile aortic thrombus was the primary source in three patients, paradoxical embolization occurred in two, and two others had a combination of findings. Two patients received operative management with one mortality, and five received nonoperative management. The source of arterial emboli remains obscure in some patients. TEE can be valuable in identifying sources or mechanisms of embolization when angiography and conventional echocardiography are negative.

Cysteine-rich fibroblast growth factor receptor alters secretion and intracellular routing of fibroblast growth factor 3.

Expression of the cysteine-rich fibroblast growth factor (FGF) receptor (CFR) in COS-1 cells strongly inhibits the secretion of co-expressed FGF3. By using a column retention assay and affinity chromatography, we demonstrate that at physiological salt concentrations FGF3 binds with strong affinity to CFR in vivo and in vitro. Furthermore, to show that FGF3 binds to CFR in vivo, truncation mutants of CFR with changed subcellular distributions were shown to cause a similar redistribution of FGF3. Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the Golgi apparatus, we show here that in COS-1 cells a substantial proportion of CFR is secreted. This is due to a carboxyl-terminal proteolytic cleavage that releases the intraluminal portion of the protein for secretion. However, the apparent size of the integral membrane and secreted CFR appears similar, since the loss of protein mass is balanced by a gain of complex carbohydrates. The released CFR is associated with the extracellular matrix through its affinity for glycosaminoglycans. These findings show that CFR can modulate the secretion of FGF3 and may control its biological activity by regulating its secretion.

Percutaneous transluminal angioplasty of infrainguinal vessels.

In the last decade, percutaneous angioplasty (PTA) has been used with increasing frequency to treat infrainguinal atherosclerotic lesions. In hopes of better delineating the role of PTA, we undertook a retrospective analysis of infrainguinal PTA in one hospital over a 7-year period. The charts of all patients receiving infrainguinal PTA from 1989 to 1996 were reviewed. Demographics, site and type of lesion, and results of treatment were recorded. Survival curves were plotted using the Kaplan-Meier method following current Society of Vascular Surgery/International Society for Cardiovascular Surgery (SVS/ISCVS) guidelines. Differences in times to first failure were tested using the log rank method. Failures were documented by duplex ultrasound. All patients requiring repeat intervention underwent contrast angiography. In selected patients with stenotic lesions <3 cm, infrainguinal PTA may be an appropriate initial treatment modality. However, 5-year patency rates are significantly lower than those achieved by saphenous vein grafting. The efficacy of the procedure is markedly decreased when used to treat arterial stenoses >3 cm in length as well as occlusions, and surgical revascularization may be a more appropriate initial therapeutic procedure.

A note on state individual differences in accuracy of response imagery: aftereffects and reminiscence.

In two sessions, separated by 7 days, subjects imagined themselves performing a tracking task under a massed practice schedule. After conditions of interpolated rest and no-rest, which were counterbalanced across sessions, subjects actually performed the tracking task. During imagery practice, subjects verbally reported the temporal component of the task. The temporal accuracy of verbal reports varied widely across subjects, but not within subjects. Furthermore, a performance gain was demonstrated as a function of interpolated rest versus no-rest (reminiscence effect). Finally, the accuracy of verbal reports predicted imagery aftereffects, but not reminiscence effects.

Ethanol and negative feedback regulation of mesolimbic dopamine release in rats.

The objectives of this study were to examine the relationship between somatodendritic and terminal field dopamine (DA) release following manipulation of DA D2 receptors in the ventral tegmental area (VTA), systemic administration of ethanol, and inhibition of DA uptake in the nucleus accumbens (ACB). Perfusion of 5, 25 and 100 microM quinpirole (a D2 agonist), or sulpiride (a D2 antagonist) through the microdialysis probe in the VTA produced dose-related decreases or increases, respectively, in the extracellular levels of DA in both the VTA and ACB of adult Wistar rats. The IP administration of 2-3 g/kg ethanol produced a sustained increase in the extracellular levels of DA (150-200% of baseline) in the ACB for at least 2 h after injection, whereas only a transient increase was observed in the VTA. Local perfusion of the ACB with 100 microM GBR12909, a DA uptake inhibitor, elevated the extracellular levels of DA in the ACB to approximately 400% of baseline, but decreased the extracellular levels of DA in the VTA to approximately 50% of baseline. Overall, the results suggest that (a) there is an association between somatodendritic and terminal field DA release when D2 cell body autoreceptors in the VTA are manipulated, (b) elevating synaptic levels of DA in the terminal field activates a long-loop negative feedback system to the VTA, and (c) different mechanisms may be mediating the actions of ethanol on DA neuronal activity and terminal DA release.

Does dextran 40 improve the early patency of autogenous infrainguinal bypass grafts?

PURPOSE: We determined whether the administration of dextran 40 would increase the early (30-day) patency of autogenous infrainguinal bypass grafts. METHODS: During a 4-year period, 244 patients undergoing 273 autogenous infrainguinal bypass grafts were prospectively enrolled into and completed this study. Patients were randomized into two groups; one of the groups received a 72-hour infusion of dextran 40 after surgery, and the other did not. Comparisons were made between those patients who did and did not receive dextran 40 with respect to risks factors, demographics, and early graft patency. RESULTS: One hundred twenty-six procedures were accompanied by the use of dextran; 147 were not. There was no significant difference between the two groups with respect to patient age, gender, perioperative risk factors, indication for surgery, or location of bypass graft (popliteal vs tibial). Among those patients receiving dextran, there were eight early occlusions (6.4%) and four deaths (3.2%); 89.7% of the patients were alive with patent grafts 30 days after surgery. In the group not receiving dextran, there were 10 early occlusions (6.8%) and 3 deaths (2%); 90.5% of the patients were alive with patent grafts 30 days after surgery. There was no significant difference between the two groups with respect to rate of early occlusion (p = 1.00), death (p = 0.71), or 30-day patency (p = 0.84). CONCLUSIONS: The administration of dextran 40 does not increase the early patency of autogenous infrainguinal bypass grafts. Its routine use during these procedures cannot be recommended.

(Non)Issues of infinite regress in modeling motor behavior.

In the past, infinite regress criticisms that have been raised about models of motor behavior have been reserved for executive-type models (e.g., Beek & Meijer, 1988). On the basis of Gödel's (1930/1986) proof that an algorithm cannot prove its own validity, the authors reason that executive- as well as self-organized-type explanatory models of motor behavior have infinite regress difficulties. The conclusion offered in the present article is that judgments on a model's theoretical importance should be based not on issues of infinite regress but on other relevant characteristics, such as its propensity for falsification (Popper, 1959).

GABA(A) receptor blockade in the anterior ventral tegmental area increases extracellular levels of dopamine in the nucleus accumbens of rats.

Previously, it was shown that microinfusion of the GABA(A) antagonist picrotoxin into the anterior ventral tegmental area (VTA) is reinforcing. It was hypothesized that this reinforcing effect of picrotoxin in the anterior VTA is mediated, at least in part, by the activation of the mesoaccumbens dopamine (DA) system. The objective of the present study was to determine if blockade of GABA(A) receptors in the anterior VTA can increase extracellular levels of DA in the nucleus accumbens (ACB), using an in vivo microdialysis technique in freely moving rats. Concentrations of picrotoxin (40, 80, and 160 microM) that had previously been shown to produce a reinforcing effect increased the extracellular levels of DA and its major metabolites in the ACB. The increased extracellular DA levels induced by intra-VTA injection of picrotoxin was markedly attenuated by coadministration with the GABA(A) agonist muscimol, whereas intra-VTA injection of muscimol alone did not have an apparent effect on extracellular DA levels in the ACB. Microinjection of another GABA(A) antagonist, bicuculline, into the anterior VTA also increased the extracellular release of DA in the ACB. These results suggest that DA neurons projecting from the anterior VTA to the ACB are tonically inhibited by GABA through its actions at the GABA(A) receptors.

Response imagery: aftereffects and reminiscence.

In two experiments, subjects imagined themselves performing a tracking task under a massed practice schedule. After interpolated rest or no rest, subjects actually performed the criterion task. Some subjects' imagery was augmented with sounds that matched the temporal characteristics of the criterion task. These subjects produced greater aftereffects than subjects who imaged without augmentation or subjects provided with imagery augmentation matching a variation of the criterion. Reminiscence (performance gain attributed to interpolated rest) was demonstrated with imagery, except when the accompanying augmentation was faster than the criterion.

Serotonin-3 receptor and ethanol-stimulated somatodendritic dopamine release.

The effects of local application of the 5-HT3 receptor agonist, 1-(m-chlorophenyl)-biguanide (CPBG), and i.p. administration of ethanol on the extracellular levels of dopamine (DA) in the ventral tegmental area (VTA) were studied using in vivo microdialysis. Adult female Wistar rats were implanted with microdialysis probes in the VTA at least 24 h before each experiment. Stable extracellular levels of DA (101 +/- 9 fmol/20 min) were established before initiating the experiments. Application of 10-250 microM CPBG through the microdialysis probe dose-dependently enhanced the extracellular concentrations of DA but did not alter the levels of either 3,4-dihydroxyphenylacetic acid or homovanillic acid in the dialysate. The effects of CPBG were reversible and dependent upon Ca2+. Co-perfusion with the 5-HT3 receptor antagonist, 3-tropanyl-indole-3-carboxylate (ICS 205-930), inhibited the effects of CPBG on enhancing extracellular DA levels. The i.p. administration of 2 g/kg ethanol significantly (p < 0.005) enhanced the levels of DA to 150% of baseline values; this ethanol-induced increase was prevented by local perfusion with 100 microM ICS 205-930. These results suggest that 5-HT3 receptors in the VTA are involved in regulating the somatodendritic release of DA and in mediating the stimulatory effects of ethanol on this neuronal system.

The Scheduling of Knowledge of Results.

The experiments outlined in this article were performed so that the acquisition effects of KR scheduling on no-KR retention could be determined. In Experiment 1, the group that alternated between 12 KR and 12 no-KR responses produced better retention than both the group that alternated between 6 KR and 6 no-KR responses and an all-KR group. The partial KR group that performed the best on retention also received the least number of reversals from KR to no-KR responses, however. In Experiment 2, when acquisition KR reversals ere held constant for partial KR groups, groups that received either random KR scheduling or all KR produced similar and better retention that groups who received blocked KR scheduling. These results were reconciled with KR frequency experiments by proposing that memory processes invoked by KR protocols decrease from KR frequency, to reversal, to scheduling conditions.

The treatment of hand ischemia by arterial ligation and upper extremity bypass after angioaccess surgery.

BACKGROUND: Hand ischemia remains an uncommon but potentially devastating complication of angioaccess operations. In the past, fistula ligation, arterial banding, and graft lengthening have been recommended as treatment with varying degrees of success. The procedure of arterial ligation and upper extremity bypass has recently been proposed as a more physiologic alternative method of treating this problem. STUDY DESIGN: The records of six patients with hand ischemia after hemodialysis access surgery who were treated with arterial ligation and revascularization were reviewed. Indications for operation, patient demographics, and risk factors were noted. The clinical results of surgery as well as fistula and bypass graft patency were recorded. RESULTS: All six patients demonstrated marked improvement in the perfusion of the affected hand. Five of six patients had complete resolution of their symptoms, while one continues to have a mild residual numbness of the hand. All fistulas and arterial bypass grafts remain patent at a follow-up period of two to 18 months (mean, 7.4 months). There were no perioperative complications or deaths. CONCLUSIONS: The technique of arterial ligation and upper extremity bypass provides a consistent and reliable method of correcting hand ischemia after angioaccess surgery. We believe it is the procedure that is most likely to alleviate the clinical symptoms of hand ischemia without jeopardizing the long-term function of the hemodialysis fistula.

Selective use of the intensive care unit after nonaortic arterial surgery.

PURPOSE: The purpose of this study was to determine whether the institution of a clinical protocol combining 6 hours of recovery room observation and guidelines for intensive care unit (ICU) admission would allow selected patients to be safely transferred directly to a surgical floor after nonaortic arterial reconstruction. METHODS: After a clinical pathway was formed, 134 consecutive patients undergoing 154 nonaortic arterial operations were prospectively enrolled in this study. Patients requiring ICU care and the responsible factors were identified. Comparisons of risk factors and demographics were made between those patients who did and did not require ICU care. RESULTS: Twelve (7.8%) patients spent a total of 27 days in the ICU (range 1 to 11 days). As per our guidelines four patients were transferred to the ICU for invasive monitoring, and four were sent to the ICU because of refractory hemodynamic instability or arrhythmia in the postanesthetic recovery room. An additional four patients were transferred to the ICU after having been on the surgical floor for 24 to 72 hours because of the following perioperative complications: prolonged chest pain (one), pneumonia (one), heart failure (one), and graft occlusion requiring a urokinase infusion. Patients admitted to the ICU were more likely to have heart disease (p = 0.02) and to have had an operation other than carotid endarterectomy (p = 0.04) than those who were not. The 30-day mortality rate was 1.4%. CONCLUSIONS: The implementation of a clinical protocol similar to the one used in this study will allow many patients undergoing nonaortic vascular surgery to avoid the use of the ICU. This approach will conserve hospital and financial resources without adversely affecting patient morbidity and mortality rates.