Genetic testing of canine degenerative myelopathy in the South African Boxer dog population

Canine degenerative myelopathy (DM) is a progressive disease process that is diagnosed late in life and mainly affects the pelvic limbs. Factors that make an ante-mortem definitive diagnosis of DM include: an insidious onset and clinical manifestation that mimics other disease processes of the pelvic limbs (hip dysplasia, cranial cruciate ligament rupture, etc.) or there may even be concurrent disease processes, old-age onset and lack of reliable diagnostic methods. Until recently, South African dog owners had to submit samples to laboratories overseas for genetic testing in order to confirm an affected dog (homozygous A/A) and to aid in the ante-mortem diagnosis of DM. Only affected dogs have been confirmed to manifest the clinical signs of DM. This study aimed to verify whether genetic testing by a local genetic laboratory was possible in order to detect a missense mutation of the superoxide dismutase gene (SOD1) that is implicated in causing the clinical signs of DM. The study also aimed to detect and map the inheritance of this disease process in a local Boxer dog population where the pedigree of the sampled population was known. Venous blood collected from Boxer dogs using a simple random sampling technique. The samples were genotyped for the SOD1:c.118G>A polymorphism. Carrier and affected Boxer dogs were detected. A pedigree that demonstrated the significance of inheriting a carrier or affected state in the population was mapped. The present study concludes that genotyping of the missense mutation in Boxer dogs is possible in South Africa. There are carrier and affected Boxer dogs in the local population, making DM a plausible diagnosis in aged dogs presenting with pelvic limb pathology

Sélection de clones résistants appartenant aux genres Kiebsiella, Serratia et Pseudomonas afin de suivre leur implantation dans un biofiltre

Des souches appartenant aux espèces : Klebsiella oxytoca, Serratia marcescens et Pseudomonas putida, isolées d'un biofiltre utilisé pour le traitement d'effluents urbains ont été choisies parmi une centaine d'autres pour être réimplantées dans un réacteur du même type. Dans le but de suivre leur fixation en réacteur ouvert, une méthode spécifique de sélection a été développée. Des clones de ces souches résistant naturellement à des antibiotiques (rifampicine, streptomycine, acide nalidixique) et à des substrats suicides (chlorate, bromoacétate, fluorouracile) ont été recherchés. Cette sélection a permis d'obtenir des clones de Klebsiella et de Serratia résistants à 2 g/l de streptomycine, 1 g/l de rifampicine et à 2 g/l de chlorate ainsi que des clones de Pseudomonas résistants à 0,5 g/l d'acide nalidixique et à 2 g/l de bromoacétate ou à 40 mgll de fluorouracile.Les clones résistants dont les caractéristiques de croissance et les activités enzymatiques sont identiques à celles de la souche sauvage et dont la stabilité génétique a été maintenue après de nombreux repiquages ont été retenus. Afin de valider notre méthode de reconnaissance, une numération de la flore indigène d'un effluent urbain a été réalisée sur les milieux spécifiques des clones résistants : seule une faible proportion de cette flore, à savoir 0,02 % est capable de s'y développer. Des essais préliminaires d'ensemencement du biofiltre avec les souches sélectionnées ont été réalisés, ils montrent que celles-ci s'implantent puisqu'elles sont retrouvées sur les grains de matériau de garnissage et que chacune d'elle représente 1 % de la flore totale.Comparison with free tell system, fixed process applied for biological wastewater treatment have been shown to offer numerous advantages. The Biocarbone process, an aerobic down flow immersed bed reactor (ODA patent n° 78-30246), has been selected for many industrial and municipal wastewater treatment facilities.From this type of aerobic fixed-bed reactor, made of expanded schist as a granular support and fed with clarified domestic wastewater, eigthy-eigth strains were isolated (ZINEBI et al., 1992). Three of the bacterial strains were chosen for their abilities to express high levels of glucidolytic, proteolytic or lipolytic activities and to grow on the granular support as microcolonies which developed into a film of organisms over the whole surface.Our objective was to initiate biofilm formation by feeding the clean support with thon selected strains named : Klebsiella oxytoca, 501; Serratia marcescens, 532 and Pseudomonas putida, 601. In order to follow attachment kinetics of these selected strains of this biofilter, and to verify their perenity within the biofilm in non sterile conditions (mixed with indigeneous flora from the influent), a specific labelling method was required.As antibiotic-resistant mutants are easily isolated and the resistances can often serve as convenient genetic markers for use in characterizing bacterial strains, a direct selection of tells acquiring resistance to various antibiotics (ampicillin, streptomycin, nalidixic acid and rifampicin) bas been performed. Selected antibiotic-resistant strains were further incubated in presence of growth inhibitors or suicide substrates in order to select again spontaneous arising mutants well characterized by two distinct markers. From the two bacteria belonging to the Enterobacteriaceae family, mutants having lost the nitrate reductase have been isolated under anaerobic growth conditions in the presence of chlorate. In the case of Pseudomonas strain, mutants resistant towards substrate halogen analogues were obtained.Colonies resistant to antibiotics and resistant to lethal substrates were isolated : thus, colonies of Klebsiella resistant to streptomycin at 2 g/l, to rifampicin al 1 g/l and chlorate al 2 g/l ; colonies of Serratia resistant to streptomycin at 2 g/l or to rifampicine at 1 g/l and chlorate at 2 g/l and colonies of Pseudomonas resistant to nalidixic acid at 0.5 g/l and to bromoacetate at 2 g/l or to fluorouracil at 40 mg/l, were obtained. We have selected : trains showing the same doubling time as well as the same final population titan the parental strains when growths were performed with or without the markers. The three strains retained were : Klebsiellaoxytoca, 501 R1S2Cl2 which grew on the Mac Conkey medium added with 1 g/l of rifampicin, 2 g/l at streptomycin and 2 g/l of chlorate; Serratia marcescens, 532 S2Cl2 (on Mac Conkey plus 2 g/l of streptomycin and 2 g/l of chlorate) and Pseudomonas putida, 601 NB2 (on King plus 0,5 g/l of nalidixic acid and 2 g/l of bromoacetate). These specific media for the detection of selectionned clones were selective toward a fixed indigenous flora since only 0,02 % of total heterotrophic population can grow.A column filled with grains of « Biodagen » either colonized by natural, microbial populations or with clean grains of « Biodagen » was fed with a population of the : train Klebsiella 501 R1S2Cl2. The strain colonized virgin « Biodagen » and maintained population of 4.106 CFU per grain for 9 days with new material and 105 CFU for 7 days with precolonized material.Experiment with a mixed population resulting from the three identified microbial species have been conducted with clean grains of « Biodagen », a whole population of 107 CFU per grain was obtained after two days and each identified strain corresponded to 1 % of the entire bacterial population. The relative concentrations of the three : trains did not decrease feeding the column with a mixture of the three : trains and of wastewater but slightly decreased when the column was fed with wastewater only

Empirical Geographic Modeling of Switchgrass Yields in the United States

Switchgrass (Panicum virgatum L.) is a perennial grass native to the United States that has been studied as a sustainable source of biomass fuel. Although many field‐scale studies have examined the potential of this grass as a bioenergy crop, these studies have not been integrated. In this study, we present an empirical model for switchgrass yield and use this model to predict yield for the conterminous United States. We added environmental covariates to assembled yield data from field trials based on geographic location. We developed empirical models based on these data. The resulting empirical models, which account for spatial autocorrelation in the field data, provide the ability to estimate yield from factors associated with climate, soils, and management for both lowland and upland varieties of switchgrass. Yields of both ecotypes showed quadratic responses to temperature, increased with precipitation and minimum winter temperature, and decreased with stand age. Only the upland ecotype showed a positive response to our index of soil wetness and only the lowland ecotype showed a positive response to fertilizer. We view this empirical modeling effort, not as an alternative to mechanistic plant‐growth modeling, but rather as a first step in the process of functional validation that will compare patterns produced by the models with those found in data. For the upland variety, the correlation between measured yields and yields predicted by empirical models was 0.62 for the training subset and 0.58 for the test subset. For the lowland variety, the correlation was 0.46 for the training subset and 0.19 for the test subset. Because considerable variation in yield remains unexplained, it will be important in the future to characterize spatial and local sources of uncertainty associated with empirical yield estimates

Structure of a robust bacterial protein cage and its application as a versatile biocatalytic platform through enzyme encapsulation

Using a newly discovered encapsulin from Mycolicibacterium hassiacum, several biocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produce as recombinant protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of 60 protomers (diameter of 23 nm) with narrow pores. By developing an effective coexpression and isolation procedure, the effect of packaging a variety of biocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stability of the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in the encapsulin. Due to the restricted diameters of the encapsulin pores (5–9 Å), the protein cage protects the encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuable tools to tune the properties of biocatalysts such as stability and substrate specificity

Functional MRI Readouts From BOLD and Diffusion Measurements Differentially Respond to Optogenetic Activation and Tissue Heating

Functional blood-oxygenation-level-dependent (BOLD) MRI provides a brain-wide readout that depends on the hemodynamic response to neuronal activity. Diffusion fMRI has been proposed as an alternative to BOLD fMRI and has been postulated to directly rely on neuronal activity. These complementary functional readouts are versatile tools to be combined with optogenetic stimulation to investigate networks of the brain. The cell-specificity and temporal precision of optogenetic manipulations promise to enable further investigation of the origin of fMRI signals. The signal characteristics of the diffusion fMRI readout vice versa may better resolve network effects of optogenetic stimulation. However, the light application needed for optogenetic stimulation is accompanied by heat deposition within the tissue. As both diffusion and BOLD are sensitive to temperature changes, light application can lead to apparent activations confounding the interpretation of fMRI data. The degree of tissue heating, the appearance of apparent activation in different fMRI sequences and the origin of these phenomena are not well understood. Here, we disentangled apparent activations in BOLD and diffusion measurements in rats from physiological activation upon sensory or optogenetic stimulation. Both, BOLD and diffusion fMRI revealed similar signal shapes upon sensory stimulation that differed clearly from those upon heating. Apparent activations induced by high-intensity light application were dominated by T2∗-effects and resulted in mainly negative signal changes. We estimated that even low-intensity light application used for optogenetic stimulation reduces the BOLD response close to the fiber by up to 0.4%. The diffusion fMRI signal contained T2, T2∗ and diffusion components. The apparent diffusion coefficient, which reflects the isolated diffusion component, showed negative changes upon both optogenetic and electric forepaw stimulation. In contrast, positive changes were detected upon high-intensity light application and thus ruled out heating as a major contributor to the diffusion fMRI signal