Detailed pharmacokinetic characterization of advanced topical acyclovir formulations with IVPT and in vivo Open Flow Microperfusion.

Successful treatment of herpes simplex viruses is currently limited by a lack of effective topical drugs. Commonly used topical acyclovir products only reduce the duration of lesions by a few days. Optimizing topical formulations to achieve an enhanced acyclovir solubility and penetration could increase the efficacy of topically applied acyclovir, but new formulations need to show reliable acyclovir delivery into at least the epidermis/dermis and need to provide sustained acyclovir release for extended time periods. The aim of this study was to compare pharmacokinetic data from in vitro permeation testing (IVPT) and preclinical dermal open flow microperfusion (dOFM) experiments regarding the penetration behavior of different acyclovir formulations relative to the reference product Zovirax® 5% cream. Four test formulations that delivered the best penetration data in IVPT were further tested using continuous dOFM in vivo dermal sampling. The use of dOFM identified one of the four tested formulations to perform significantly better than the other three tested formulations and the reference product. In vivo dOFM data showed differences in the dermal acyclovir concentration that had not been detected by using IVPT. Improved acyclovir delivery to the dermis was likely achieved by the new formulation that uses a much lower drug load compared to the reference product. This optimized formulation was able to achieve a dermal concentration similar to oral application and can thus provide the opportunity of more efficacious topical HSV-1 treatment with less side effects than oral systemic treatment.

Dermal open flow microperfusion for PK-based clinical bioequivalence studies of topical drug products.

Topically applied drug products have experienced an extraordinary price increase in the United States, mostly due to a lack of generic products. Generic drug development is hindered by high costs and risks associated with clinical endpoint studies required to show bioequivalence (BE) of prospective generic products relative to their reference products. There is a continued need for cost- and time-efficient alternatives to clinical endpoint studies to determine BE of topically applied dermal drug products. Cutaneous PK-based BE studies present such an alternative and dOFM (dermal open flow microperfusion) has already been successfully used in several verifications studies to show an accurate and sensitive assessment of the rate and extent at which drugs become available in the skin. dOFM technology is discussed as well as the dOFM setup of clinical pilot and main studies to achieve BE assessment with a minimum number of participants and an outlook is given on the use of dOFM technology for other drug products.

The effect of spermidine on autoimmunity and beta cell function in NOD mice.

Spermidine is a natural polyamine which was shown to prolong lifespan of organisms and to improve cardiac and cognitive function. Spermidine was also reported to reduce inflammation and modulate T-cells. Autophagy is one of the mechanisms that spermidine exerts its effect. Autophagy is vital for ß-cell homeostasis and autophagy deficiency was reported to lead to exacerbated diabetes in mice. The effect of spermidine in type 1 diabetes pathogenesis remains to be elucidated. Therefore, we examined the effect of spermidine treatment in non-obese diabetic (NOD) mice, a mouse model for type 1 diabetes. NOD mice were given untreated or spermidine-treated water ad libitum from 4 weeks of age until diabetes onset or 35 weeks of age. We found that treatment with 10 mM spermidine led to higher diabetes incidence in NOD mice despite unchanged pancreatic insulitis. Spermidine modulated tissue polyamine levels and elevated signs of autophagy in pancreas. Spermidine led to increased proportion of pro-inflammatory T-cells in pancreatic lymph nodes (pLN) in diabetic mice. Spermidine elevated the proportion of regulatory T-cells in early onset mice, whereas it reduced the proportion of regulatory T-cells in late onset mice. In summary spermidine treatment led to higher diabetes incidence and elevated proportion of T-cells in pLN.

miRNAs as Regulators of the Early Local Response to Burn Injuries.

In burn injuries, risk factors and limitations to treatment success are difficult to assess clinically. However, local cellular responses are characterized by specific gene-expression patterns. MicroRNAs (miRNAs) are single-stranded, non-coding RNAs that regulate mRNA expression on a posttranscriptional level. Secreted through exosome-like vesicles (ELV), miRNAs are intracellular signalers and epigenetic regulators. To date, their role in the regulation of the early burn response remains unclear. Here, we identified 43 miRNAs as potential regulators of the early burn response through the bioinformatics analysis of an existing dataset. We used an established human ex vivo skin model of a deep partial-thickness burn to characterize ELVs and miRNAs in dermal interstitial fluid (dISF). Moreover, we identified miR-497-5p as stably downregulated in tissue and dISF in the early phase after a burn injury. MiR-218-5p and miR-212-3p were downregulated in dISF, but not in tissue. Target genes of the miRNAs were mainly upregulated in tissue post-burn. The altered levels of miRNAs in dISF of thermally injured skin mark them as new biomarker candidates for burn injuries. To our knowledge, this is the first study to report miRNAs altered in the dISF in the early phase of deep partial-thickness burns.

Implementation status of morbidity and mortality conferences in Austrian hospitals-A cross-sectional national survey study.

INTRODUCTION: Morbidity and mortality conferences (M&MCs) are an instrument for learning from past complications, unexpected follow-ups and deaths in hospitals and are important for improving patient safety. However, there are currently no quantitative data on the implementation of M&MCs in Austria. The aim of the study was to determine the status quo of the M&MCs in Austria. MATERIALS AND METHODS: A national cross-sectional study was conducted by means of a survey of 982 chief physicians of surgical disciplines, internal medicine, anesthesiology, intensive care, gynecology/obstetrics and pediatrics. The questionnaire focused on overall goals, structure and procedures of hospital M&MCs. RESULTS: Of the 982 contacted chief physicians, 314 (32.0%) completed the survey. Almost two thirds of the respondents, i.e. 203 (64.7%), had already implemented M&MCs. Of the 111 chief physicians who had not yet introduced M&MCs, 62 (55.9%) were interested in introducing such conferences in the future. Of the 203 respondents that had implemented M&MCs, 100 stated that their M&MC could be improved. They reported issues with "shame and blame" culture, hierarchical structures, too little knowledge about the capability of M&MC and, in particular, time constraints. Overall, the participating chief physicians showed that they are striving to improve their existing M&MCs. DISCUSSION/CONCLUSION: While we found a relatively high number of already implemented M&MCs we also identified a large heterogeneity in the format of the M&MCs. A highly structured M&MC including guidelines, checklists or templates does not only considerably improve its outcome but can also alleviate the main limiting factor which is the lack of time.

A novel human ex vivo skin model to study early local responses to burn injuries.

Burn injuries initiate numerous processes such as heat shock response, inflammation and tissue regeneration. Reliable burn models are needed to elucidate the exact sequence of local events to be able to better predict when local inflammation triggers systemic inflammatory processes. In contrast to other ex vivo skin culture approaches, we used fresh abdominal skin explants to introduce contact burn injuries. Histological and ultrastructural analyses confirmed a partial-thickness burn pathology. Gene expression patterns and cytokine production profiles of key mediators of the local inflammation, heat shock response, and tissue regeneration were analyzed for 24 h after burn injury. We found significantly increased expression of factors involved in tissue regeneration and inflammation soon after burn injury. To investigate purely inflammation-mediated reactions we injected lipopolysaccharide into the dermis. In comparison to burn injury, lipopolysaccharide injection initiated an inflammatory response while expression patterns of heat shock and tissue regeneration genes were unaffected for the duration of the experiment. This novel ex vivo human skin model is suitable to study the local, early responses to skin injuries such as burns while maintaining an intact overall tissue structure and it gives valuable insights into local mechanisms at the very beginning of the wound healing process after burn injuries.

Internet-Based Health Information-Seeking Behavior of Students Aged 12 to 14 Years: Mixed Methods Study.

BACKGROUND: Many children and adolescents are surrounded by smartphones, tablets, and computers and know how to search the internet for almost any information. However, very few of them know how to select proper information from reliable sources. This can become a problem when health issues are concerned, where it is vital to identify incorrect or misleading information. The competence to critically evaluate digital information on health issues is of increasing importance for adolescents. OBJECTIVE: The aim of this study was to assess how children and adolescents rate their internet-based health literacy and how their actual literacy differs from their ratings. In addition, there was a question on how their search performance is related to their self-efficacy. To evaluate these questions, a criteria-based analysis of the quality of the websites they visited was performed. Finally, the possibility to increase their internet-based health literacy in a 3-day workshop was explored. METHODS: A workshop with a focus on health literacy was attended by 14 children and adolescents in an Austrian secondary school. After prior assessments (Culture Fair Intelligence Test, revised German version; Reading Speed and Reading Comprehension Test for Grades 6 to 12, German; electronic health literacy scale [eHEALS]; and General Self-Efficacy Scale, Reversed Version, German), the students were asked to perform an internet-based search on a health-related issue. Browser histories and screenshots of all internet searches were gathered, clustered, and analyzed. After the workshop, the health literacy of the students was assessed again by using the eHEALS. RESULTS: The 14 students opened a total of 85 homepages, but only eight of these homepages were rated as good or fair by two experts (independent rating) based on specific criteria. The analysis showed that the students judged their own internet-based health literacy much higher than the actual value, and students who had rated themselves better did not visit websites of high quality. Internet-based health literacy correlated significantly with the self-efficacy of the students (rs=0.794, P=.002). CONCLUSIONS: Our study showed that it is possible to draw the attention of students to critical aspects of internet search and to slightly improve their search competence in a workshop. Targeted improvement of health literacy is urgently required, and students need special instruction for this purpose. Further investigations in this area with larger sets of data, which could be feasible with the help of a computer program, are urgently needed.

Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine.

BACKGROUND: Bacterial nanocellulose (BNC) is considered a promising carrier for various substances and novel approaches using BNC in combination with antiseptics are well documented. However, the difference in the molecular weight of these molecules influences their uptake by and release from BNC. Analysing the diffusion of standard molecules with different weight, e.g. dextrans, offers the possibility to investigate the mobility of various molecules. We aimed to test the use of BNC regarding uptake and release of different standard molecules as well as two commercially available antiseptics for possible applications in future wound dressings. MATERIAL AND METHODS: Diffusion profiles, uptake and release of three FITC-dextran molecules differing in weight as well as octenidine (Octenisept®) and povidone-iodine (Betaisodona®)-based antiseptics were tested with BNC-based wound dressings. Furthermore, the antiseptic efficacy of BNC in combination with antiseptics against Staphylococcus aureus was tested. RESULTS: Uptake and release capacity for FITC-dextran molecules showed a molecular weight-dependent mobility from BNC into an agarose gel. The loading capacity of BNC was also inversely proportional to the molecular weight of the antiseptics. The release test for octenidine showed a sustained and prolonged delivery into a solid matrix, whereas povidone-iodine was released faster. Both antiseptic solutions combined with BNC showed a good dose-dependent efficacy against S. aureus. CONCLUSION: Results obtained from the mobility of FITC-dextran molecules in the BNC matrix could open possible applications for the combination of BNC with other molecules for medical applications. Combination of both tested antiseptics with BNC showed to be an efficient approach to control bacterial infections.

Topical application of haemoglobin: a safety study.

OBJECTIVE: Ischaemia is one of the biggest problems in wound healing. It causes chronic wounds and also prevents normal wound healing because the tissue is oxygen deprived. Most oxygen-supplying therapies are only feasible in a clinical setting, but topical haemoglobin applications, such as Granulox, can be used in a non-clinical setting. For home application, the haemoglobin solution is sprayed topically onto the wound using a pressurised ready-to-use device with a bag-on-valve system. Although this system does not mix product and propellant, the risk of product inhalation by the patient, user or bystanders has to be minimised. This safety study aimed to determine particle size and product concentration in the surroundings after application to determine if there is a risk that product particles enter the respiratory tract. METHODS: Measurements were performed using a laser scattered light photometer and a Scanning Mobility Particle Sizer (SMPS)-Spectrometer at different distances from the measuring devices to determine the inhalation risk for a possible user, patient and bystander. At all measuring points the amount of particles, their size and the formation of dust were measured. RESULTS: No nanoparticles or dust were created during the application of the haemoglobin spray. The concentrations of the measured particles are below the allowed limits defined by Austrian law. CONCLUSION: There is no risk of inhaling nanoparticles or being exposed to harmful concentrations of larger particles of the tested product. All the product's ingredients can be degraded and excreted by the human body through natural pathways.

Uptake of PHMB in a bacterial nanocellulose-based wound dressing: A feasible clinical procedure.

BACKGROUND: With the increase of antimicrobial resistance in recent decades, other methods of preventing and fighting infections must be considered. Burn patients, whose wound areas are often extensive, are especially prone to wound infections. The loading of bacterial nanocellulose (BNC) with antiseptics has already been successfully performed but unfortunately, the described procedure is time-consuming and thus not applicable in a clinical emergency setting. Therefore, a clinically feasible approach was established. MATERIAL AND METHODS: Sheets of BNC-based wound dressings were placed into antiseptic solutions containing PHMB (Prontosan® and LAVANID® 2) and were left to soak for up to two hours. At different time points, samples were analysed for their concentration of PHMB and antiseptic efficacy. RESULTS: Within 30min, clinically relevant concentrations of PHMB were achieved in the BNC-based wound dressing. The 30-min PHMB uptake for Prontosan® and LAVANID® 2 resulted in concentrations of 0.05% and 0.019%, respectively. Samples from the PHMB loaded dressing showed a dose dependent antiseptic efficacy for Staphylococcus aureus. CONCLUSION: This experiment showed that the loading of BNC-based wound dressings with PHMB-containing antiseptics was achieved by a simple and quick procedure. According to studies a PHMB concentration of 0.001% can already inhibits all bacterial growth, indicating that the concentrations of PHMB in the BNC-based wound dressings after 30min are higher than the minimal inhibitory concentration and the antiseptic efficacy after 120min loading analysed by an standardized bacterial disk diffusion assay was shown to be comparable to the clinically used Suprasorb® X+PHMB wound dressing.

An Untargeted Metabolomics Approach to Characterize Short-Term and Long-Term Metabolic Changes after Bariatric Surgery.

Bariatric surgery is currently one of the most effective treatments for obesity and leads to significant weight reduction, improved cardiovascular risk factors and overall survival in treated patients. To date, most studies focused on short-term effects of bariatric surgery on the metabolic profile and found high variation in the individual responses to surgery. The aim of this study was to identify relevant metabolic changes not only shortly after bariatric surgery (Roux-en-Y gastric bypass) but also up to one year after the intervention by using untargeted metabolomics. 132 serum samples taken from 44 patients before surgery, after hospital discharge (1-3 weeks after surgery) and at a 1-year follow-up during a prospective study (NCT01271062) performed at two study centers (Austria and Switzerland). The samples included 24 patients with type 2 diabetes at baseline, thereof 9 with diabetes remission after one year. The samples were analyzed by using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS, HILIC-QExactive). Raw data was processed with XCMS and drift-corrected through quantile regression based on quality controls. 177 relevant metabolic features were selected through Random Forests and univariate testing and 36 metabolites were identified. Identified metabolites included trimethylamine-N-oxide, alanine, phenylalanine and indoxyl-sulfate which are known markers for cardiovascular risk. In addition we found a significant decrease in alanine after one year in the group of patients with diabetes remission relative to non-remission. Our analysis highlights the importance of assessing multiple points in time in subjects undergoing bariatric surgery to enable the identification of biomarkers for treatment response, cardiovascular benefit and diabetes remission. Key-findings include different trend pattern over time for various metabolites and demonstrated that short term changes should not necessarily be used to identify important long term effects of bariatric surgery.

Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary between regions of the human gastrointestinal tract.

PURPOSE: Vitamin D is well known for its effects on bone mineralisation but has also been attributed immunomodulatory properties. It positively influences human health, but in vivo data describing vitamin D effects on the human gut microbiome are missing. We aimed to investigate the effects of oral vitamin D3 supplementation on the human mucosa-associated and stool microbiome as well as CD8(+) T cells in healthy volunteers. METHODS: This was an interventional, open-label, pilot study. Sixteen healthy volunteers (7 females, 9 males) were endoscopically examined to access a total of 7 sites. We sampled stomach, small bowel, colon, and stools before and after 8 weeks of vitamin D3 supplementation. Bacterial composition was assessed by pyrosequencing the 16S rRNA gene (V1-2), and CD8(+) T cell counts were determined by flow cytometry. RESULTS: Vitamin D3 supplementation changed the gut microbiome in the upper GI tract (gastric corpus, antrum, and duodenum). We found a decreased relative abundance of Gammaproteobacteria including Pseudomonas spp. and Escherichia/Shigella spp. and increased bacterial richness. No major changes occurred in the terminal ileum, appendiceal orifice, ascending colon, and sigmoid colon or in stools, but the CD8(+) T cell fraction was significantly increased in the terminal ileum. CONCLUSION: Vitamin D3 modulates the gut microbiome of the upper GI tract which might explain its positive influence on gastrointestinal diseases, such as inflammatory bowel disease or bacterial infections. The local effects of vitamin D demonstrate pronounced regional differences in the response of the GI microbiome to external factors, which should be considered in future studies investigating the human microbiome.

Recirculation--a novel approach to quantify interstitial analytes in living tissue by combining a sensor with open-flow microperfusion.

We report a novel approach to quantify interstitial analytes in living tissue by combining open-flow microperfusion (OFM) with a sensor and the re-circulation method. OFM is based on the unrestricted exchange of molecules between the interstitial fluid (ISF) and a perfusion medium through macroscopic perforations that enables direct access to the ISF. By re-circulating the perfusate and monitoring the changes of the analytes' concentration with a sensor, the absolute analyte concentration in the ISF can be calculated. In order to validate the new concept, the absolute electrical conductivity of the ISF was identified in six subjects to be 1.33 ± 0.08 S/m (coefficient of variation CV = 6 %), showing the robustness of this approach. The most striking feature of this procedure is the possibility to monitor several compounds simultaneously by applying different sensors which will allow not only the determination of the concentration of a single substance in vivo but also the simultaneous dynamics of different analytes. This will open new fields in analytical chemistry, pharmacology, as well as clinical experimental research.

Failure to control hyperglycemia in noncritically ill diabetes patients despite standard glycemic management in a hospital setting.

BACKGROUND: Successful control of hyperglycemia has been shown to improve outcomes for diabetes patients in a clinical setting. We assessed the quality of physician-based glycemic management in two general wards, considering the most recent recommendations for glycemic control for noncritically ill patients (<140 mg/dl for premeal glucose). METHODS: Quality of glycemic management of 50 patients in two wards (endocrinology, cardiology) was assessed retrospectively by analyzing blood glucose (BG) levels, the glycemic management effort, and the online questionnaire. RESULTS: Glycemic control was clearly above the recommended target (mean BG levels: endocrinology: 175 ± 62 mg/dl; cardiology: 186 ± 68 mg/dl). When comparing the first half with the second half of the hospital stay, we found no difference in glycemic control (endocrinology: 168 ± 32 vs 164 ± 42 mg/dl, P = .67; cardiology: 174 ± 36 mg/dl vs 170 ± 42 mg/dl, P =.51) and in insulin dose (endocrinology: 15 ± 14 IU vs 15 ± 13 IU per day, P = .87; cardiology: 27 ± 17 IU vs 27 ± 18 IU per day, P = .92), despite frequent BG measurements (endocrinology: 2.7 per day; cardiology: 3.2 per day). A lack of clearly defined BG targets was indicated in the questionnaire. CONCLUSION: The recommended BG target range was not achieved in both wards. Analysis of routine glycemic management demonstrated considerable glycemic management effort, but also a lack of translation into adequate insulin therapy. Implementation of corrective measures, such as structured treatment protocols, is essential.

Enhanced absorption of insulin aspart as the result of a dispersed injection strategy tested in a randomized trial in type 1 diabetic patients.

OBJECTIVE: We investigated the impact of two different injection strategies on the pharmacokinetics and pharmacodynamics of insulin aspart in vivo in an open-label, two-period crossover study and verified changes in the surface-to-volume ratio ex vivo. RESEARCH DESIGN AND METHODS: Before the clinical trial, insulin aspart was injected ex vivo into explanted human abdominal skin flaps. The surface-to-volume ratio of the subcutaneous insulin depot was assessed by microfocus computed tomography that compared 1 bolus of 18 IU with 9 dispersed boluses of 2 IU. These two injection strategies were then tested in vivo, in 12 C-peptide-negative type 1 diabetic patients in a euglycemic glucose clamp (glucose target 5.5 ± 1.1 mmol/L) for 8 h after the first insulin administration. RESULTS: The ex vivo experiment showed a 1.8-fold higher mean surface-to-volume ratio for the dispersed injection strategy. The maximum glucose infusion rates (GIR) were similar for the two strategies (10 ± 4 vs. 9 ± 4; P = 0.5); however, times to reach maximum GIR and 50% and 10% of the maximum GIR were significantly reduced by using the 9 × 2 IU strategy (68 ± 33 vs. 127 ± 93 min; P = 0.01; 38 ± 9 vs. 49 ± 16 min; P < 0.01; 23 ± 6 vs. 30 ± 10 min; P < 0.05). For 9 × 2 IU, the area under the GIR curve was greater during the first 60 min (219 ± 89 vs. 137 ± 75; P < 0.01) and halved until maximum GIR (242 ± 183 vs. 501 ± 396; P < 0.01); however, it was similar across the whole study period (1,361 ± 469 vs. 1,565 ± 527; P = 0.08). CONCLUSIONS: A dispersed insulin injection strategy enhanced the effect of a fast-acting insulin analog. The increased surface-to-volume ratio of the subcutaneous insulin depot can facilitate insulin absorption into the vascular system.

Population dynamics with a mixed type of sexual and asexual reproduction in a fluctuating environment.

BACKGROUND: Carassius gibelio, a cyprinid fish from Eurasia, has the ability to reproduce both sexually and asexually. This fish is also known as an invasive species which colonized almost all continental Europe, most likely originating from Asia and Eastern Europe. Populations of both sexually and asexually reproducing individuals exist in sympatry. In this study we try to elucidate the advantages of such a mixed type of reproduction. We investigate the dynamics of two sympatric populations with sexual and asexual reproduction in a periodically fluctuating environment. We define an individual-based computational model in which genotypes are represented by L loci, and the environment is composed of L resources for which the two populations compete. RESULTS: Our model demonstrates advantageous population dynamics where the optimal percentage of asexual reproduction depends on selection strength, on the number of selected loci and on the timescale of environmental fluctuations. We show that the sexual reproduction is necessary for "generating" fit genotypes, while the asexual reproduction is suitable for "amplifying" them. The simulations show that the optimal percentage of asexual reproduction increases with the length of the environment stability period and decrease with the strength of the selection and the number of loci. CONCLUSIONS: In this paper we addressed the advantages of a mixed type of sexual and asexual reproduction in a changing environment and explored the idea that a species that is able to adapt itself to environmental fluctuation can easily colonize a new habitat. Our results could provide a possible explanation for the rapid and efficient invasion of species with a variable ratio of sexual and asexual reproduction such as Carassius gibelio.

Genetic and morphological population differentiation in the rock-dwelling and specialized shrimp-feeding cichlid fish species Altolamprologus compressiceps from Lake Tanganyika, East Africa.

With about 250 endemic species, Lake Tanganyika contains an extraordinarily diverse cichlid fish fauna, and thus represents an ideal model system for the study of pathways and processes of speciation. The Lamprologini form the most species-rich tribe in Lake Tanganyika comprising about 100 species in seven genera, most of which are endemic to the lake. They are territorial substrate-breeders and represent a monophyletic tribe. By combined analysis of population genetics and geometric morphometric markers, we assessed gene flow among three populations of the highly specialized shrimp-feeding rock-dweller Altolamprologus compressiceps, separated by geographic distance and ecological barriers. Five highly polymorphic microsatellite markers were analyzed in conjunction with 17 landmarks in order to compare genetic differences to body shape differences among populations. Both genetic and morphological analyses revealed significant differentiation among the three studied populations. A significant, but overall relatively low degree of genetic differentiation supports a very recent divergence. Phenotypic differentiation was primarily found in the head region of A. compressiceps. In agreement with findings in other cichlid species, similar adaptations to specialized feeding mechanisms can consequently lead to marginal shape changes in the trophic apparatus.

Introgression of Alectoris chukar genes into a Spanish wild Alectoris rufa population.

In order to detect introgression of other Alectoris genus species into wild populations of Spanish Alectoris rufa, we studied a sample of 93 red-legged partridges (supposed to be A. rufa) captured in the island of Majorca. A set of 31 chukar partridges (Alectoris chukar) from Cyprus and 33 red-legged partridges (A. rufa) from one Spanish farm were also studied to provide suitable populations for comparison. Factorial correspondence analysis on microsatellite genotypes supported a clear distinction of birds from Cyprus, whereas partridges from Majorca and the Spanish farm overlapped in a wide area. The existence of A. chukar mitochondrial DNA in 16 individuals from Majorca indicated introgression into their maternal lineage even if their phenotypes were not different from A. rufa. Bayesian inference based on microsatellite analysis indicated a noticeable degree of genetic proximity to A. chukar only for one of these hybrids.

Determining the environmental fate of a filamentous fungus, Trichoderma reesei, in laboratory-contained intact soil-core microcosms using competitive PCR and viability plating.

Trichoderma spp. are used extensively in industry and are routinely disposed of in landfill sites as spent biomass from fermentation plants. However, little is known regarding the environmental fate of this biomass. We tracked the survival of T. reesei strain QM6A#4 (a derivative of strain QM6A marked with a recombinant construct) over a 6-month period in laboratory-contained, intact soil-core microcosms incubated in a growth chamber. Survival was tested in 3 different soils and the effect of a plant rhizosphere (bush lima beans, Phaseolus limensis) was investigated. Levels and viability of the fungus were determined, respectively, by quantitative competitive polymerase chain reaction analysis of total soil DNA extracts and dilution-plating of soil on a semiselective growth medium. Whereas chemically killed QM6A#4 became undetectable within 3 d, QM6A#4 added as a live inoculum decreased approximately 4- to approximately 160-fold over the first 1-3 months and then reached a steady state. After 4 months, soil cores were subjected to a 1.5-month simulated winter period, which did not significantly affect QM6A#4 levels. Throughout the experiment, QM6A#4 remained viable. These results indicate that, following release into the environment, live T. reesei will persist in soil for at least 2 seasons.