Substantially improved gene transfer to interneurons with second-generation glutamate receptor-targeted DART-AAV vectors.

BACKGROUND: Adeno-associated viral vectors (AAVs) are a widely used gene transfer platform in neuroscience. Although naturally AAV serotypes can have preferences for certain tissues, selectivity for particular cell types in the CNS does not exist. Towards interneuron targeting, capsid engineering of AAV2 including display of the designed ankyrin repeat protein (DARPin) 2K19 specific for the glutamate receptor subunit 4 (GluA4) at the N-terminus of the VP2 capsid protein has been established. The resulting AAV-VP2N is highly specific for interneurons, but exhibits rather moderate transduction efficiencies. METHODS: Two alternative insertion sites for 2K19 in the GH2/GH3 loop of capsid proteins VP1 (AAV-VP1L) or VP2 (AAV-VP2L) were exploited to yield second generation GluA4-AAVs. Having packaged reporter genes under ubiquitous promoters, the vectors were characterized for biochemical properties as well as gene delivery into cell lines and rat hippocampal slice cultures. Electrophysiological recordings monitored the functional properties of transduced cells. RESULTS: Compared to AAV-VP2N, the second-generation vectors, especially AAV-VP1L, achieved about 2-fold higher genomic titers as well as a substantially improved GluA4 binding. Improvements in gene transfer activities were 18-fold on GluA4-overexpressing A549 cells and five-fold on rat hippocampal organotypic slice cultures reaching approximately 60 % of all parvalbumin positive interneurons upon a single administration. The spiking behaviour of transduced cells was unaltered and characteristic for a heterogeneous group of interneurons. CONCLUSION: The substantially improved gene transfer activity of the second generation GluA4-targeted AAV combined with low toxicity makes this vector an attractive tool for interneuron-directed gene transfer with unrestricted promotor and transgene choice.

Antifungal lock therapy: an eternal promise or an effective alternative therapeutic approach?

Each year, millions of central venous catheter insertions are performed in intensive care units worldwide. The usage of these indwelling devices is associated with a high risk of bacterial and fungal colonization, leading to the development of microbial consortia, namely biofilms. These sessile structures provide fungal cells with resistance to the majority of antifungals, environmental stress and host immune responses. Based on different guidelines, colonized/infected catheters should be removed and changed immediately in the case of Candida-related central line infections. However, catheter replacement is not feasible for all patient populations. An alternative therapeutic approach may be antifungal lock therapy, which has received high interest, especially in the last decade. This review summarizes the published Candida-related in vitro, in vivo data and case studies in terms of antifungal lock therapy. The number of clinical studies remains limited and further studies are needed for safe implementation of the antifungal lock therapy into clinical practice.

Improvement in Hidradenitis Suppurativa and quality of life in patients treated with adalimumab: Real-world results from the HARMONY Study.

BACKGROUND: Hidradenitis suppurativa (HS), a chronic, recurrent, debilitating skin disease, is characterized by painful, inflammatory, subcutaneous lesions of the axilla, inguinal and anogenital regions. Overall prevalence of HS is ˜1%, and the impact of disease on patient quality of life (QoL) and healthcare resource utilization (HRU) is high. OBJECTIVES: To estimate the real-world effectiveness of adalimumab (Humira®) treatment in patients with moderate-to-severe HS on disease severity, pain, QoL, work productivity and HRU. METHODS: HARMONY (Effectiveness of Adalimumab in Moderate to Severe HidrAdenitis SuppuRativa Patients - a Multi-cOuNtry studY in Real Life Setting) is a multicentre, postmarketing observational study in adult patients with moderate-to-severe HS. Disease severity and QoL parameters were evaluated using validated measures at 12-week intervals over 52 weeks of treatment. The primary endpoint was the proportion of patients achieving a Hidradenitis Suppurativa Clinical Response (HiSCR: ≥50% reduction in abscess and inflammatory nodule count, with no increase in abscess and draining fistula counts relative to baseline) at 12 weeks. Secondary endpoints were HiSCR at 24, 36 and 52 weeks and changes in QoL parameters and work productivity assessments. Analyses were conducted using as-observed data. RESULTS: The proportion of patients reaching the primary HiSCR endpoint was 70.2% (n = 132/188 enrolled) and remained ≥70% until study completion. There were statistically significant (P < 0.0001) reductions in worst and average skin pain. All of the QoL measures evaluated improved significantly (P < 0.0001) by 12 weeks of adalimumab treatment, as did work productivity assessments (P < 0.05), and there was a ˜50% decrease in HRU between baseline and week 52. Adalimumab was well tolerated. CONCLUSIONS: In this real-world setting, adalimumab treatment of moderate-to-severe HS resulted in decreased disease severity and improvements in QoL and productivity. Response to adalimumab was rapid (within 12 weeks) and sustained (52 weeks). No unexpected safety signals were reported.

Molecular Diversity and Genetic Relatedness of Candida albicans Isolates from Birds in Hungary.

The molecular epidemiology of Candida albicans infections in animals has been rarely studied. In this study, multilocus sequence typing was used to characterise the genetic diversity and population structure of 24 avian origin C. albicans isolates collected from different birds with candidiasis and compared to human isolates. Fourteen diploid sequence types (DSTs) including six new DSTs were determined. Cluster analysis revealed that isolates grouped into 8 clades. Bird isolates mainly belonged to minor clades and Clade 15 with DST 172 was the most common (11 isolates; 45.8%). The remaining isolates were clustered into Clade 7 (5 isolates; 20.8%), Clade 10 (4 isolates; 16.6%), Clade 8 (2 isolates; 8.3%), Clade 4 (1 isolate; 4.2%) and Clade 16 (1 isolate; 4.2%). Unweighted pair group method with arithmetic averages (UPGMA) and eBURST analyses showed that the genetic construction of avian origin C. albicans population is fairly diverse. Although species-specific lineages were not found, some degree of separation in the evolution of bird and human strains could be observed.

In situ labeling of non-accommodating interneurons based on metabolic rates.

Maintaining high frequency firing of narrow action potentials puts a large metabolic load on fast spiking (FS), perisomatic-inhibitory interneurons compared to their slow-spiking, dendrite targeting counterparts. Although the relationship of action potential (AP) firing and metabolism is firmly established, there is no single method to differentiate interneurons in situ based on their firing properties. In this study, we explore a novel strategy to easily identify the metabolically active FS cells among different classes of interneurons. We found that the oxidation of the fluorescent free radical marker 2,7-dichlorodihydrofluorescein (H2DCF) preferentially occurs in interneurons both in slice cultures and acute brain slices. Despite their morphological heterogeneity, almost all DCF-positive (DCF+) neurons belonged to the cluster of non-accommodating FS interneurons. Furthermore, all FS interneurons expressing parvalbumin (PV) both in slice cultures and in acute slices from tdTomato-PVCre transgenic mice were also DCF+. However, only half of the recorded DCF + cells were also PV+, indicating that H2DCF-oxidation occurs in different interneuron classes characterized by non-accomodating AP-firing. Comprehensively enhancing spontaneous neuronal activity led to mitochondrial oxidation of DCF in pyramidal cells as well as interneurons, suggesting that the apparent selectivity towards interneurons represents differences in the underlying metabolic load. While radical-scavenging, inhibition of APs or NO-synthesis, and iron chelation had no effect on the staining pattern, exposure to the complex-I inhibitor, rotenone, prevented interneuronal DCF accumulation. We conclude that H2DCF oxidation is independent of free radicals but correlates with the intensive oxidative energy metabolism and high mitochondrial mass in interneurons sharing the non-accommodating FS phenotype.

Variational principles and nonequilibrium thermodynamics.

Variational principles play a fundamental role in deriving the evolution equations of physics. They work well in the case of non-dissipative evolution, but for dissipative systems, the variational principles are not unique and not constructive. With the methods of modern nonequilibrium thermodynamics, one can derive evolution equations for dissipative phenomena and, surprisingly, in several cases, one can also reproduce the Euler-Lagrange form and symplectic structure of the evolution equations for non-dissipative processes. In this work, we examine some demonstrative examples and compare thermodynamic and variational techniques. Then, we argue that, instead of searching for variational principles for dissipative systems, there is another viable programme: the second law alone can be an effective tool to construct evolution equations for both dissipative and non-dissipative processes. This article is part of the theme issue 'Fundamental aspects of nonequilibrium thermodynamics'.

Synergistic effect of nikkomycin Z with caspofungin and micafungin against Candida albicans and Candida parapsilosis biofilms.

Antifungal lock therapy has received significant interest in the last few years because the frequently usage of intravascular devices is associated with an increasing number of catheter-related bloodstream infections caused by Candida species. Antifungal combinations with synergistic interaction can be a good choice for antifungal lock therapy; therefore, interactions were examined between two echinocandins (caspofungin and micafungin) and the chitin synthesis inhibitor nikkomycin Z against Candida albicans and C. parapsilosis biofilms. Susceptibility was evaluated using the XTT-based checkerboard microdilution method, while the nature of interactions was assessed by calculating fractional inhibitory concentration indices and using the Bliss independence model. Mathematic-based evaluations were supplemented with fluorescent LIVE/DEAD viability assay. The results obtained by statistical interaction analyses correlated well with the viability assay. The tested echinocandins with nikkomycin Z caused an extended cell death and the structure of the biofilm was sparse compared to the control, especially for C. albicans. The findings support the simultaneous usage of nikkomycin Z and caspofungin or micafungin in alternative therapies such as the antifungal lock therapy. SIGNIFICANCE AND IMPACT OF THE STUDY: Antifungal lock therapy can be a potential therapeutic approach to eradicate the intraluminal Candida biofilms; however, there is no approved lock strategy against fungal species so far. The results of this study provide valuable evidence that nikkomycin Z acts synergistically in combination with caspofungin or micafungin against biofilms. In addition, this synergy was more pronounced for micafungin combined with nikkomycin Z. Therefore, nikkomycin Z can be considered as a potential agent in antifungal lock therapy especially with micafungin against C. albicans or C. parapsilosis biofilms.

Activity of exogenous tyrosol in combination with caspofungin and micafungin against Candida parapsilosis sessile cells.

AIMS: Fungal quorum-sensing molecules may have an inhibitory effect as adjuvant against Candida biofilms. Therefore, in vitro activity of caspofungin and micafungin was evaluated against Candida parapsilosis biofilms in the presence of tyrosol. METHODS AND RESULTS: Interactions were assessed using fractional inhibitory concentration index (FICI) determination, metabolic activity-based time-kill experiments and scanning electron microscopy (SEM). Tyrosol caused 1-16-fold and 2-32-fold decrease in median caspofungin and micafungin MICs respectively. Based on FICI, synergy was observed in isolates 27001 and 17820 with caspofungin and 27001 with micafungin. In time-kill experiments, the metabolic activity reduction was higher for micafungin compared to caspofungin at 24 h especially when used in 64 and 256 mg l-1 concentrations. In the case of micafungin, the 256 mg l-1  + 1 mmol l-1 combination caused significantly higher decrease in metabolic activity compared to the corresponding concentration alone (256 mg l-1 ) at 24 h (P < 0·05). SEM confirmed the higher killing effect of tested echinocandins with tyrosol. CONCLUSIONS: Considerable metabolic activity reduction and cell damage was detected for combinations especially in the case of micafungin. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings support the development of new alternative therapeutic strategies against C. parapsilosis biofilms.

Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs.

Dynamic alterations in flavin adenine dinucleotide (FAD) fluorescence permit insight into energy metabolism-dependent changes of intramitochondrial redox potential. Monitoring FAD fluorescence in living tissue is impeded by photobleaching, restricting the length of microfluorimetric recordings. In addition, photodecomposition of these essential electron carriers negatively interferes with energy metabolism and viability of the biological specimen. Taking advantage of pulsed LED illumination, here we determined the optimal excitation settings giving the largest fluorescence yield with the lowest photobleaching and interference with metabolism in hippocampal brain slices. The effects of FAD bleaching on energy metabolism and viability were studied by monitoring tissue pO2 , field potentials and changes in extracellular potassium concentration ([K+ ]o ). Photobleaching with continuous illumination consisted of an initial exponential decrease followed by a nearly linear decay. The exponential decay was significantly decelerated with pulsed illumination. Pulse length of 5 ms was sufficient to reach a fluorescence output comparable to continuous illumination, whereas further increasing duration increased photobleaching. Similarly, photobleaching increased with shortening of the interpulse interval. Photobleaching was partially reversible indicating the existence of a transient nonfluorescent flavin derivative. Pulsed illumination decreased FAD photodecomposition, improved slice viability and reproducibility of stimulus-induced FAD, field potential, [K+ ]o and pO2 changes as compared to continuous illumination.

Models in research of pharmacoresistant epilepsy: present and future in development of antiepileptic drugs.

Currently available antiepileptic drugs (AEDs) were developed to suppress seizure activity but less for prevention of epileptogenesis or for treatment of epileptogenic encephalopathies. Despite considerable efforts towards pharmacological control of seizures, about 30 % of epileptic patients do not achieve complete seizure control, and these numbers are even higher in patients suffering from partial seizures - a common form of epilepsy in adults. The mechanisms behind drug-resistance are far from being understood. Likely several unrelated mechanisms might lead in concert to reduced efficacy of the AEDs. Consequently, there is a need for predictive biomarkers of susceptibility to pharmacoresistant seizures and for new therapies interfering with epileptogenesis and preventing development of drug-resistance instead of merely suppressing seizures. This also necessitates the design of novel in vitro and in vivo epilepsy models that would better mimic the progressive nature of epilepsy and resemble the state of a chronic epileptic tissue. In this review we discuss current theories of drug-resistance and give a short summary of the epilepsy models that are frequently used for testing AEDs. We will also highlight caveats of the different models and consider novel approaches to overcome these difficulties. Finally we give a short outlook on unconventional therapies interfering with epileptogenesis as well as with drug delivery and retention.

Impact of environmental and genetic factors on the scale shape of zebrafish, Danio rerio (Hamilton 1822): a geometric morphometric study.

Intraspecific morphological variability may reflect either genetic divergence among groups of individuals or response of individuals to environmental circumstances within the frame of phenotypic plasticity. Several studies were able to discriminate wild fish populations based on their scale shape. Here we examine whether the variations in the scale shape in fish populations could be related to genetic or environmental factors, or to both of them. In the first experiment, two inbred lines of zebrafish, Danio rerio (Hamilton 1822) reared under identical environmental conditions were compared. Secondly, to find out what effect environmental factors might have, offsprings were divided into two groups and reared on different diets for 12 weeks. Potential recovery of scales from an environmental effect was also assessed. Experimental groups could successfully be distinguished according to the shape of scales in both experiments, and the results showed that both genetic and environmental factors may notably influence scale shape. It was concluded that scale shape analysis might be used as an explanatory tool to detect potential variability of environmental influences impacting genetically homogeneous groups of fish. However, due to its sensitivity to environmental heterogeneity, the applicability of this technique in identifying intraspecific stock membership of fish could be limited.

Biofilm development in fixed bed biofilm reactors: experiments and simple models for engineering design purposes.

Biofilm development in a fixed bed biofilm reactor system performing municipal wastewater treatment was monitored aiming at accumulating colonization and maximum biofilm mass data usable in engineering practice for process design purposes. Initially a 6 month experimental period was selected for investigations where the biofilm formation and the performance of the reactors were monitored. The results were analyzed by two methods: for simple, steady-state process design purposes the maximum biofilm mass on carriers versus influent load and a time constant of the biofilm growth were determined, whereas for design approaches using dynamic models a simple biofilm mass prediction model including attachment and detachment mechanisms was selected and fitted to the experimental data. According to a detailed statistical analysis, the collected data have not allowed us to determine both the time constant of biofilm growth and the maximum biofilm mass on carriers at the same time. The observed maximum biofilm mass could be determined with a reasonable error and ranged between 438 gTS/m(2) carrier surface and 843 gTS/m(2), depending on influent load, and hydrodynamic conditions. The parallel analysis of the attachment-detachment model showed that the experimental data set allowed us to determine the attachment rate coefficient which was in the range of 0.05-0.4 m d(-1) depending on influent load and hydrodynamic conditions.

Treatment of traumatic corneal abrasions: a three-arm, prospective, randomized study.

PURPOSE: To compare three different treatment modalities for traumatic corneal abrasions. METHODS: We conducted a prospective, randomized, masked, three-arm clinical study of patients presenting with superficial corneal foreign bodies. Treatment modalities were: (1) pressure patching with ofloxacin ointment (patch group, PG, n = 18), (2) therapeutic contact lens with ofloxacin eye drops (contact lens group, CLG, n = 20) and (3) ofloxacin ointment alone (ointment group, OG, n = 28). Primary outcome measure was the difference of the mean corneal abrasion area between the three groups at 3 different time points (baseline, day 1 and day 7). RESULTS: A total of 66 patients were included in the study over a period of 2 years. Mean initial corneal abrasion area was 3.6 ± 3.4 mm² in the PG, 4.2 ± 4.0 mm² in the CLG and 3.7 ± 3.1 mm² in the OG (p = 0.875). Differences in corneal abrasion area at any time point were not statistically significant (abrasion area decrease from presentation to day 1 was 3.4 ± 3.3 mm² in the PG, 4.1 ± 4.0 mm² in the CLG and 3.5 ± 3.1 mm² in the OG, p = 0.789). The epithelium was healed in all patients at day 7. CONCLUSIONS: Treating traumatic corneal abrasions by pressure patching, a bandage contact lens or ointment alone was equal in reducing the abrasion area or reducing pain. According to our results the treatment of choice for traumatic abrasions may be adapted to the needs and preferences of the patient.

Adrenergic modulation of sharp wave-ripple activity in rat hippocampal slices.

Norepinephrine (NE) has been shown to facilitate learning and memory by modulating synaptic plasticity in the hippocampus in vivo. During memory consolidation, transiently stored information is transferred from the hippocampus into the cortical mantle. This process is believed to depend on the generation of sharp wave-ripple complexes (SPW-Rs), during which previously stored information might be replayed. Here, we used rat hippocampal slices to investigate neuromodulatory effects of NE on SPW-Rs, induced by a standard long-term potentiation (LTP) protocol, in the CA3 and CA1. NE (10-50 µM) dose-dependently and reversibly suppressed the generation of SPW-Rs via activation of α1 adrenoreceptors, as indicated by the similar effects of phenylephrine (100 µM). In contrast, the unspecific ß adrenoreceptor agonist isoproterenol (2 µM) significantly increased the incidence of SPW-Rs. Furthermore, ß adrenoreceptor activation significantly facilitated induction of both LTP and SPW-Rs within the CA3 network. Suppression of SPW-Rs by NE was associated with a moderate hyperpolarization in the majority of CA3 pyramidal cells and with a reduction of presynaptic Ca(2+) uptake in the stratum radiatum. This was indicated by activity-dependent changes in [Ca(2+) ](o) and Ca(2+) fluorescence signals, by changes in the paired pulse ratio of evoked EPSPs and by analysis of the coefficient of variance. In the presence of NE, repeated high frequency stimulation (high-frequency stimulation (HFS)) failed to induce SPW-Rs, although SPW-Rs appeared following washout of NE. Together, our data indicate that the NE-mediated suppression of hippocampal SPW-Rs depends on α1 adrenoreceptor activation, while their expression and activity-dependent induction is facilitated via ß1-adrenoreceptors.

[Spontaneous perforation of a retroperitoneal duodenal diverticulum]. / Perforation spontanée d'un diverticule rétropéritonéal du duodénum.

Spontaneous perforation is a rare complication of a retroperitoneal duodenal diverticulum; diagnosis and treatment are both difficult. We report a case of perforated duodenal diverticulitis diagnosed by CT, which required surgical intervention after failure of medical treatment.

Complex decongestive physiotherapy decreases capillary fragility in lipedema.

Lipedema is a disproportional obesity featuring frequent hematoma formation due to even minor traumatic injuries. On the basis of clinical observations, complete decongestive physiotherapy diminishes the incidence of hematomas due to minor injuries beyond leg volume reduction. Hematoma development may be caused by altered capillary resistance (CR) or altered capillary fragility (CF). We measured capillary fragility (CF) before and after complex decongestive physiotherapy (CDP) to examine, whether CDP could reduce CF. 38 women with lipedema were included in the study. Twenty-one (21) patients were treated with CDP and 17 using exclusively moisturizers as the control group. CDP comprised once daily manual lymph drainage, intermittent pneumatic compression and multilayered short-stretch bandaging performed throughout a 5-day-course. CF was evaluated with the vacuum suction method (VSM) using Parrot's angiosterrometer in both groups. Decongestive therapy resulted in a significant reduction of the number of petechiae while no change was detected within the control group. Complete decongestive physiotherapy significantly reduced CF in patients with lipedema and this reduction may lead to reduced hematoma formation.

Kinetics of autothermal thermophilic aerobic digestion - application and extension of Activated Sludge Model No 1 at thermophilic temperatures.

The application of an ASM1-based mathematical model for the modeling of autothermal thermophilic aerobic digestion is demonstrated. Based on former experimental results the original ASM1 was extended by the activation of facultative thermophiles from the feed sludge and a new component, the thermophilic biomass was introduced. The resulting model was calibrated in the temperature range of 20-60 degrees C. The temperature dependence of the growth and decay rates in the model is given in terms of the slightly modified Arrhenius and Topiwala-Sinclair equations. The capabilities of the calibrated model in realistic ATAD scenarios are demonstrated with a focus on autothermal properties of ATAD systems at different conditions.

[Combined bipolar radiofrequency and cementoplasty of bone metastases]. / Radiofréquence bipolaire et cimentoplastie dans le traitement des métastases osseuses.

Bone metastases are the most common cause of pain in cancer patients. Pain management in cancer patients, often revealing the disease and always present at advanced stages, is an important and difficult task. Pain is not always properly controlled by high doses of specific medication, radiation therapy or chemotherapy. When these therapies do not provide adequate pain relief, percutaneous neurolysis, infiltrations, alcoholizations and cementoplasty may be considered. More recently RF ablation has been proposed. On weight-bearing bones, RF can be combined with acrylic cement injection. The authors present here this very effective new technique which is complementary to classical pain management techniques.