Genetic diversity of Dinaric brown bears (Ursus arctos) in Croatia with implications for bear conservation in Europe

Brown bears have lost most of their range on the European continent. The remaining western populations are small, isolated and highly endangered. The Dinaric-Pindos brown bear population is the western-most stable population and the fourth largest in Europe. It has been recognized as a potential source for recolonization of populations whose survival is at risk. Indeed, several translocations of Dinaric bears to Italy, Austria and France have recently been made. Despite the importance of the Dinaric bear population, its genetic status remains poorly understood. Using tissue samples from 156 hunted or accidentally killed Dinaric bears in Croatia, this study analysed genetic diversity at 12 microsatellite loci, as well as population structure and past reductions in size. In addition, a subset of 59 samples was used to assess diversity of the mitochondrial DNA control region. The results indicate that Dinaric bears have high nuclear genetic diversity, as compared to other extant brown bear populations, despite genetic evidence of a bottleneck caused by past persecutions. However, haplotype diversity was low, probably as a result of male-biased dispersal and female philopatry. Not surprisingly, no evidence of population sub-structure was found using nuclear markers, as the bear habitat has remained continuous and the highway network has been built only recently. Management should focus on maintaining habitat connectivity and keeping the effective population size as large as possible. In addition, when removing individuals, care should be taken not to further deplete the population of rare haplotypes. A coordinated transboundary management of the entire Dinaric-Pindos brown bear population should be a priority for its long-term conservation.     

Temporomandibular joint disorder in a patient with multiple sclerosis--review of literature with a clinical report

Temporomandibular disorders are a form of musculoskeletal disorders, which reduce the function of stomatognathic system and they are related to some other diseases causing painful conditions and disorders of oral function. The aim of this paper is to describe a one year follow up clinical case of a female patient with comorbid multiple sclerosis and a relatively rare form of articular disc disorder. Primary clinical diagnostics encompassed manual methods of TMJ examination. Definite diagnosis included radiologic examination. Clinical hyperextensive condyle position was palpated bilaterally and subsequently confirmed by a functional panoramic radiograph of TMJ. The anterior displacement of disc with reduction was diagnosed by magnetic resonance and in the right joint there was a disc displacement upon excursive movement. From relevant literature, the relationship of a number of diseases that can be related to functional disorder of the orofacial system, such as multiple sclerosis, has been described from many aspects. Also, apart from the standard classification of one form of anterior displacement of the disc, made primarily by magnetic resonance, cases of disc displacement upon excursive mandibular movement can rarely be found in literature.     

Genetic diversity, structure, and size of an endangered brown bear population threatened by highway construction in the Pindos Mountains, Greece

One of the major negative effects of roads is the creation of barriers to the movement of wildlife, ultimately disconnecting populations and increasing extinction risk. We collected genetic data from a threatened brown bear population in the central part of the Pindos mountain range in northwestern Greece to provide information about this, as yet genetically undescribed, population and to evaluate its status prior to the construction of a major highway. We used noninvasive genetic sampling methods and microsatellite analysis to investigate nuclear genetic diversity, population genetic structure, demographic history, relatedness within the population and estimated effective and total population size. Brown bears in the study area were found to possess a relatively high level of nuclear genetic diversity and low levels of inbreeding; the population did not show any signs of substructuring but seems to have gone through a genetic bottleneck in the recent past. The estimated effective population size was 29, and the total population size estimate obtained by two different methods was 33 and 51 individuals, respectively. Our results indicate a good conservation status of this bear population and provide baseline genetic data for the future evaluation of the effects on bears from the construction of a major highway, for monitoring the genetic status of this and other bear populations in Greece and for assessing gene flow in bear populations in southern Europe.     

Detection of human herpesvirus 6 and 7 DNA in saliva from healthy adults from Rio de Janeiro, Brazil

In this study, we aimed to evaluate virus shedding in the saliva of healthy adults from the metropolitan region of the city of Rio de Janeiro, Brazil, in order to verify the prevalence of both human herpesviruses 6 and 7 (HHV-6, HHV-7). The studied group comprised 182 healthy individuals at Pedro Ernesto University Hospital, who were being seen for annual odontologic revisions. Saliva specimens were subjected to a multiplex polymerase chain reaction (PCR) to detect the presence of HHV-6A, HHV-6B and HHV-7. The total Roseolovirus DNA prevalence was 22.4%. The PCR detected a HHV-6 prevalence of 9.8%, with HHV-6A detected in 7.1% of the samples and HHV-6B in 2.7%. HHV-7 DNA was revealed in 12.6% of the studied cases. Multiple infections caused by HHV-6A and 7 were found in 2.1% of the samples. No statistical differences were observed regarding age, but for HHV-7 infection, an upward trend was observed in female patients. Compared to studies from other countries, low prevalence rates of herpesvirus DNA were detected in saliva from the healthy individuals in our sample. PCR methodology thus proved to be a useful tool for Roseolovirus detection and it is important to consider possible geographic and populations differences that could explain the comparatively low prevalence rates described here.     

Mathematical modelling and process optimization of a continuous 5-stage bioreactor cascade for production of poly[-(R)-3-hydroxybutyrate] by Cupriavidus necator

A multistage system for poly(hydroxyalkanoate) (PHA) production consisting of five continuous stirred tank reactors in series (5-CSTR) with Cupriavidus necator DSM 545 as production strain was modelled using formal kinetic relations. Partially growth-associated production of PHA under nitrogen limited growth was chosen as modelling strategy, thus the Luedeking-Piret’s model of partial growth-associated product synthesis was applied as working hypothesis. Specific growth rate relations adjusted for double substrate (C and N source) limited growth according to Megee et al. and Mankad-Bungay relation were tested. The first stage of the reactor cascade was modelled according to the principle of nutrient balanced continuous biomass production system, the second one as two substrate controlled process, while the three subsequent reactors were adjusted to produce PHB under continuous C source fed and nitrogen deficiency. Simulated results of production obtained by the applied mathematical models and computational optimization indicate that PHB productivity of the whole system could be significantly increased (from experimentally achieved 2.14 g L^?1 h^?1 to simulated 9.95 g L^?1 h^?1) if certain experimental conditions would have been applied (overall dilution rate, C and N source feed concentration). Additionally, supplemental feeding strategy for switching from batch to continuous mode of cultivation was proposed to avoid substrate inhibition.     

In silico optimization and low structured kinetic model of poly[(R)-3-hydroxybutyrate] synthesis by Cupriavidus necator DSM 545 by fed-batch cultivation on glycerol

Glycerol was utilized by Cupriavidus necator DSM 545 for production of poly-3-hydroxybutyrate (PHB) in fed-batch fermentation. Maximal specific growth rates (0.12 and 0.3 h⁻¹) and maximal specific non-growth PHB production rate (0.16 g g⁻¹ h⁻¹) were determined from two experiments (inocula from exponential and stationary phase). Saturation constants for nitrogen (0.107 and 0.016 g L⁻¹), glycerol (0.05 g L⁻¹), non-growth related PHB synthesis (0.011 g L⁻¹) and nitrogen/PHB related inhibition constant (0.405 g L⁻¹), were estimated. Five relations for specific growth rate were tested using mathematical models. In silico performed optimization procedures (varied glycerol/nitrogen ratio and feeding) has resulted in a PHB content of 70.9%, shorter cultivation time (23 h) and better PHB yield (0.347 g g⁻¹). Initial concentration of biomass 16.8 g L⁻¹ and glycerol concentration in broth between 3 and 5 g L⁻¹ were decisive factors for increasing of productivity.     

Study of metabolic network of Cupriavidus necator DSM 545 growing on glycerol by applying elementary flux modes and yield space analysis

A metabolic network consisting of 48 reactions was established to describe intracellular processes during growth and poly-3-hydroxybutyrate (PHB) production for Cupriavidus necator DSM 545. Glycerol acted as the sole carbon source during exponential, steady-state cultivation conditions. Elementary flux modes were obtained by the program Metatool and analyzed by using yield space analysis. Four sets of elementary modes were obtained, depending on whether the pair NAD/NADH or FAD/FADH₂ contributes to the reaction of glycerol-3-phosphate dehydrogenase (GLY-3-P DH), and whether 6-phosphogluconate dehydrogenase (6-PG DH) is present or not. Established metabolic network and the related system of equations provide multiple solutions for the simultaneous synthesis of PHB and biomass; this number of solutions can be further increased if NAD/NADH or FAD/FADH₂ were assumed to contribute in the reaction of GLY-3-P DH. As a major outcome, it was demonstrated that experimentally determined yields for biomass and PHB with respect to glycerol fit well to the values obtained in silico when the Entner–Doudoroff pathway (ED) dominates over the glycolytic pathway; this is also the case if the Embden–Meyerhof–Parnas pathway dominates over the ED.     

Micromorphological and chemotaxonomical traits of Micromeria croatica (Pers.) Schott

A study on the types and distribution of trichomes, pollen morphology, chemical composition of essential oil (analyzed by GC-FID and GC/MS), and the content of macroelements (Na, K, Ca, and Mg) and trace elements (B, Fe, Cu, Mn, Zn, Al, Pb, Cr, Cd, Ni, Hg, and As) analyzed by ICP-AES (=inductively coupled plasma atomic emission spectroscopy) was conducted on Micromeria croatica (Pers.) Schott. Non-glandular trichomes, peltate trichomes (typical hairs of Lamiaceae), small capitate trichomes (composed of one basal epidermal cell and one head cell), and larger capitate trichomes (composed of one basal epidermal cell, two to three stalk cells, and one head cell with subcuticular space) were observed on leaves, stems, the calyx, and corolla. Pollen of M. croatica had six apertures (hexacolpate) which were set in the equatorial pollen belt (zonocolpate) and showed medium reticulate ornamentation (hetrobrochate type). The essential oil contained from 27 to 39?constituents, the most representative of which were caryophyllene oxide and E-caryophyllene. Among the investigated macroelements, the content of K was highest (8730-10080?mg/kg). The content of trace elements ranged from 0.12?mg/kg (Cr) to 78.00?mg/kg (Fe), while the content of Cd, Ni, Hg, and As were lower than the limit of quantification.     

Fast analysis of pravastatin in production media

High throughput methods (high performance liquid chromatography and capillary electrophoresis) were developed to determine pravastatin in production media. The analyses were performed on particle column, monolithic column and silica capillary filled with borate buffer pH 9.3 containing 20 mM SDS. All three methods successfully separate pravastatin from interfering compounds (matrix, mevastatin and 6-epi pravastatin) and runtimes are shorter than 1 min. Solvent consumptions for methods using small particle column, monolith column and MECK were 132, 510 and 1.5 mL h(-1). The most sensitive was the method using particle column (LOD was about 10(-5) mg mL(-1)), followed by the system using monolith column (LOD was 2 x 10(-4) mg mL(-1)) and the MECK method (LOD was about 0.02 mg mL(-1)).