Noninvasive metabolic profiling using microfluidics for analysis of single preimplantation embryos

Noninvasive analysis of metabolism at the single cell level will have many applications in evaluating cellular physiology. One clinically relevant application would be to determine the metabolic activities of embryos produced through assisted reproduction. There is increasing evidence that embryos with greater developmental capacity have distinct metabolic profiles. One of the standard techniques for evaluating embryonic metabolism has been to evaluate consumption and production of several key energetic substrates (glucose, pyruvate, and lactate) using microfluorometric enzymatic assays. These assays are performed manually using constriction pipets, which greatly limits the utility of this system. Through multilayer soft-lithography, we have designed a microfluidic device that can perform these assays in an automated fashion. Following manual loading of samples and enzyme cocktail reagents, this system performs sample and enzyme cocktail aliquotting, mixing of reagents, data acquisition, and data analysis without operator intervention. Optimization of design and operating regimens has resulted in the ability to perform serial measurements of glucose, pyruvate, and lactate in triplicate with submicroliter sample volumes within 5 min. The current architecture allows for automated analysis of 10 samples and intermittent calibration over a 3 h period. Standard curves generated for each metabolite have correlation coefficients that routinely exceed 0.99. With the use of a standard epifluorescent microscope and CCD camera, linearity is obtained with metabolite concentrations in the low micromolar range (low femtomoles of total analyte). This system is inherently flexible, being easily adapted for any NAD(P)H-based assay and scaled up in terms of sample ports. Open source JAVA-based software allows for simple alterations in routine algorithms. Furthermore, this device can be used as a standalone device in which media samples are loaded or be integrated into microfluidic culture systems for in line, real time metabolic evaluation. With the improved throughput and flexibility of this system, many barriers to evaluating metabolism of embryos and single cells are eliminated. As a proof of principle, metabolic activities of single murine embryos were evaluated using this device.     

Encapsulation of a proteolytically active novel bioproduct; controlling the release of proteinous components

Low trophic level organisms like zooplankton not only represent a valuable and unutilized source for marine proteins and lipids, but are also challenging with respect to high post-mortem protease activity. As an example, Ca–alginate encapsulated homogenized fresh zooplankton (Calanus finmarchicus) exhibited a high release of protein components when immersed in water due to diffusion of proteolytically degraded proteins. Initial diffusion rates at pH 7 increased with temperature up to 60 °C. Above 50 °C, the release was reduced because of protease instability after 12 h. The release of protein also increased with increasing ionic strength, most likely due to decreased electrostatic interaction between the alginate matrix and protein. As function of pH, the release of both amino groups as well as larger protein entities was apparently highest under alkaline conditions. Encapsulated heat-treated or alkali treated zooplankton had a high degree of release, the first 2 h reflecting the presence of osmoregulating amino acids as well as pre-digested proteins. After 2 h, no further release of protein was observed, which can be attributed to protease inactivation caused by these treatments. The present data show that all studied parameters (temperature, pH and ionic strength) have a profound impact on protein loss from the encapsulated model feed particles. Therefore, possible conservation methods to control the observed protein loss from marine raw materials in, e.g. marine feed formulations are suggested.     

Combined electrical and mechanical model of electric submersiblepumps

The electric submersible pump unit consists of a pump powered by a medium-voltage three-phase induction motor. The power transmission system is integrated with the riser pipes. Starting the pump causes heavy dynamic stresses on the motor shaft and the mechanical connection between pump (impellers) and shaft. The motor and its load will generate transient torque pulsations that may be damaging to shaft and coupling, particularly to the key grooves. System models are developed to predict the electrical and mechanical conditions on starting. Different torsional models with certain types of nonlinearities, combined with different motor models have been examined to find combinations that give the best results. The motor models applied include saturation in the main flux path and the leakage flux paths, as well as rotor deep bar effect. It has been shown how the pump parameters, material coefficients, design dimensions, and number of impellers affect the dynamic stresses. The aim has been to optimize the design with respect to the transient stresses. The simulations reveal that the resulting shaft torque, caused by excitation from resonant frequencies during the acceleration period, amount to high values that may result in excessive overloading of shafts, couplings, and key grooves. Maximum torque is, as expected, strongly dependent on the shaft dimensions. Certain shaft diameters may cause resonance and, thereby, heavy torsional amplitudes. It has been shown how the model can be a tool in the struggle to find the optimum shaft diameter     

The impact of monetary costs on commuting flows*

In Western Norway, fjords cause disconnections in the road network, necessitating the use of ferries. In several cases, ferries have been replaced by roads, often part‐financed by tolls. We use data on commuting from a region with a high number of ferries, tunnels and bridges. Using a doubly‐constrained gravity‐based model specification, we focus on how commuting responds to varying tolls and ferry prices. Focus is placed on the role played by tolls on infrastructure in inhibiting spatial interaction. We show there is considerable latent demand, and suggest that these tolls contradict the aim of greater territorial cohesion.     

Bulk tank raw milk microbiota differs within and between farms: A moving goalpost challenging quality control

Microbial contamination of bovine raw milk often occurs at the farm. To acquire a deeper knowledge of the microbiota of farm tank milk, we studied milk from 45 farms situated in 2 geographical areas in Norway. Each farm was visited on 3 different occasions, with at least 2 wk between visits. We combined both bacterial cell counts and a sequence variant inference method of amplicon-based high-throughput sequencing to achieve a high-resolution overview of the microbiota in each sample. Compositional variation of the farm milk microbiota was shown in relation to the 2 areas, between the farms and between the sampling times. Despite the near constant level of bacteria enumerated in milk from each individual farm, the dominant microbiota differed significantly between the samplings. The predominant microbiota was dominated by spoilage genera, such as Pseudomonas and Bacillus, as well as the dairy fermentation genus Lactococcus and mastitis-causing organisms (Streptococcus). Analysis of the identified sequence variants within these genera showed that the populations of Pseudomonas and Lactococcus in milk had similar composition between the farms, but that Bacillus and, in particular, Streptococcus populations changed between collection days from the same farm and between farms and geographical areas. Furthermore, the levels and composition of Bacillus and Paenibacillus were different between the 2 geographical areas. The results presented here provide new insight into the farm milk microbiota and show that this microbiota is a dynamic community highly subject to variation.     

Detection of Clostridium perfringens type A enterotoxin in faecal and food samples using immunomagnetic separation (IMS)-ELISA

A simple, rapid and sensitive immunoassay, based on immunomagnetic particles (Dynabeads M-280) was developed for detection and quantitation of Clostridium perfringens type A enterotoxin from faecal and food extracts. The assay had a detection limit of 2.5 ng/ml enterotoxin in homogenates of faeces and inoculated meat extracts. The specificity was confirmed by both crossed immunoelectrophoresis and Western immunoblotting techniques, using a purified enterotoxin as standard.     

Microorganisms associated with Maari, a Baobab seed fermented product

A microbiological study was carried out on Baobab fermented seeds (Maari) obtained from 4 different production sites in Burkina Faso (Mansila, Toulfé, Ouagadougou and Gorgadji).A total of 390 representative isolates comprising 251 aerobic mesophilic bacteria (AMB) and 139 lactic acid bacteria (LAB) were isolated and identified to species level using a combination of pheno- and genotypic methods including conventional morphological analysis, carbohydrate fermentation profiling, rep-PCR ((GTG)₅-fingerprinting) and 16S rRNA gene sequencing.The fermentation of Baobab seeds was initiated by the AMB identified as Bacillus subtilis (82% of AMB isolates) and Staphylococcus sciuri (18% of AMB isolates). No lactic acid bacteria were isolated at the beginning of the process. After 24 h fermentation time, Enterococcus faecium appeared in the fermenting seeds and remained until the end of the fermentation, as the predominant LAB.In Maari collected from retail outlets the AMB count ranged from 6.7 log10 CFU/g to 10 log10 CFU/g while the LAB load ranged from 4.4 log10 CFU/g to 9.9 log10 CFU/g. The AMB were identified as belonging to genus Bacillus (12 species), Staphylococcus (3 species) and one species of Aerococcus, Macrococcus, Leifsonia, Kurthia, Proteus, Acinetobacter and Globicatella, respectively. A putatively novel, previously undescribed Corynebacterium sp. was also found. E. faecium was the dominant LAB in all investigated retail samples except one sample dominated by Pediococcus acidilactici.     

Gain tilt of erbium-doped fiber amplifiers due to signal-inducedinversion locking

A theoretical analysis describing an interpretation of the fundamental processes, responsible for the second-order distortion of AM signals in erbium-doped fiber amplifiers (EDFAs) is presented. It is shown that the locked-inversion gain tilt rather than the widely used continuous wave gain tilt is responsible for the distortion. The theoretical analysis is confirmed by experiments showing that the two types of gain tilt may differ by factors of up to 60. In particular, it is demonstrated that the locked-inversion gain-tilt may be significant even at the CW gain peak. Experimental results indicate that the relevant gain tilt can be reduced to acceptable levels by proper design of the EDFA     

Liquid waveguide-based evanescent wave sensor that uses two light sources with different wavelengths

We demonstrate a prototypic optofluidic evanescent wave sensor made of poly(dimethylsiloxane) (PDMS) elastomer in which two light sources with different wavelengths are coupled into an optofluidic liquid-core/liquid-cladding (L(2)) waveguide. The exponentially decaying evanescent wave interacts with analyte molecules dissolved in the cladding fluids or products formed by in situ reactions at the core-cladding interface. The analyte molecules exhibit distinctly different light absorbance at the two wavelengths during the light-analyte interaction. Therefore, by using the normalized absorbance calculated from the intensity ratio of the two wavelengths instead of the absolute magnitude of either signal, unwanted effects from omnipresent external noise sources can be reduced. In addition, the differential absorption of the two beams by the analyte solutions can be used to enhance the resolution of sample analysis. The evanescent wave sensor based on a liquid waveguide can also be used for real-time monitoring of chemical reactions, because the core and cladding fluids in the L(2) waveguide are slightly miscible at the core-cladding interface due to the diffusional mixing.     

Enterotoxins and emetic toxins production by Bacillus cereus and other species of Bacillus isolated from Soumbala and Bikalga, African alkaline fermented food condiments

The ability of various species of Bacillus from fermented seeds of Parkia biglobosa known as African locust bean (Soumbala) and fermented seeds of Hibiscus sabdariffa (Bikalga) was investigated. The study included screening of the isolates by haemolysis on blood agar, detection of toxins in broth and during the fermentation of African locust bean using the Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (BDEVIA). Detection of genes encoding cytotoxin K (CytK), haemolysin BL (Hbl A, Hbl C, Hbl D), non-hemolytic enterotoxin (NheA, NheB, NheC) and EM1 specific of emetic toxin producers was also investigated using PCR with single pair and multiplex primers. Of 41 isolates, 29 Bacillus belonging to the species of B. cereus, Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus showed haemolysis on blood agar. Using RPLA, enterotoxin production was detected for three isolates of B. cereus in broth and all B. cereus (9) in fermented seeds. Using BDEVIA, enterotoxin production was detected in broth as well as in fermented seeds for all B. cereus isolates. None of the isolates belonging to the other Bacillus species was able to produce enterotoxins either by RPLA or BDEVIA. Nhe genes were detected in all B. cereus while Hbl and CytK genes were detected respectively in five and six B. cereus strains. A weak presence of Hbl (A, D) and CytK genes was detected in two isolates of B. subtilis and one of B. licheniformis but results were inconsistent, especially for Hbl genes. The emetic specific gene fragment EM1 was not detected in any of the isolates studied.