LacSwitchTM Inducible Mammalian Expression System in mouse Swiss 3T3 fibroblasts

Swiss 3T3 fibroblasts were transfected with the provided plasmids of LacSwitch Inducible Mammalian Expression System (Stratagene). Stable transfectants were selected, expanded and characterised. At first, the production of CAT in these cell lines could be induced by IPTG treatment, but the inducibility was lost after a few months in culture in a reproducible manner. Further analysis revealed that the transfectants did not lose the cat gene nor the lac repressor protein. As a result, we conclude that LacSwitch Inducible Mammalian Expression System needs further modification for use in Swiss 3T3 fibroblasts.     

Dynamic Localization of Tat Protein Transport Machinery Components in Streptomyces coelicolor

The Tat pathway transports folded proteins across the bacterial cytoplasmic membrane and is a major route of protein export in the Streptomyces genus of bacteria. In this study, we have examined the localization of Tat components in the model organism Streptomyces coelicolor by constructing enhanced green fluorescent protein (eGFP) and mCherry fusions with the TatA, TatB, and TatC proteins. All three components colocalized dynamically in the vegetative hyphae, with foci of each tagged protein being prominent at the tips of emerging germ tubes and of the vegetative hyphae, suggesting that this may be a primary site of Tat secretion. Time-lapse imaging revealed that localization of the Tat components was highly dynamic during tip growth and again demonstrated a strong preference for apical sites in growing hyphae. During aerial hypha formation, TatA-eGFP and TatB-eGFP fusions relocalized to prespore compartments, indicating repositioning of Tat components during the Streptomyces life cycle.     

Protein tyrosine phosphatase gene expression analysis in Swiss 3T3 fibroblasts

The aim of this study was to identify protein tyrosine phosphatases (PTPs) expressed in Swiss 3T3 fibroblasts and to examine their expression levels as well as to characterize quantitative aspects of RT-PCR based on degenerate deoxyoligonucleotides. By using an RT-PCR assay based on degenerate deoxyoligonucleotide primers, expression of mRNAs for two cytoplasmic- and six transmembrane-type PTPs in Swiss 3T3 cells was detected. The sequences of two of them are new. Among nine analyzed PTPs expressed to widely varied extends, only three have mRNA levels high enough to be seen on Northern blots with 10 µg of total RNA per lane. The frequencies with which the examined PTPs are represented among the PCR amplification products, correlate stronger with the primer fidelity, defined as the number of mismatches between the primer- and the cDNA target-sequences, rather than with the PTP expression levels. In conclusion, an RT-PCR assay based on degenerate primers can be successfully used to sample the expressed PTPs and to identify new members of this gene family. However, reliable quantification of their mRNA levels can only be achieved using the classical approaches, like Northern, RNase protection assay or non-degenerate quantitative RT-PCR.     

An APD-based iterative reconstruction method for simultaneous technetium-99m/iodine-123 SPECT imaging

Dual-isotope SPECT (DI-SPECT) studies offer significant advantages over sequential scans, foremost among them faster acquisition and perfect image registration. However, reconstructed images may be affected by substantial cross-talk contamination rendering them inadequate for diagnosis. This effect is especially strong for isotopes with close photopeak energies, such as ^99mTc (140 keV) and ¹²³I (159 keV). In this paper we present an iterative DI-SPECT reconstruction method which includes accurate, analytically computed scatter corrections provided by the APD (analytical photon distribution) algorithm. This algorithm calculates first and second order Compton scatter (based on the Klein–Nishina formula) and first order Rayleigh scatter. Both self-scatter and cross-talk between the two isotopes are evaluated using patient specific attenuation maps and an initial activity distribution estimate. To validate our method we performed experiments using the Data Spectrum, Inc. thorax phantom and a SPECT/CT camera system. Reconstructed images demonstrate significant improvement in data quantitation. Their quantitative accuracy increases up to a factor of two, even for activity ratios which strongly enhance cross-talk effects and seriously degrade projections.     

Unsupervised machine-learning method for improving the performance of ambulatory fall-detection systems

Background  

Falls can cause trauma, disability and death among older people. Ambulatory accelerometer devices are currently capable of detecting falls in a controlled environment. However, research suggests that most current approaches can tend to have insufficient sensitivity and specificity in non-laboratory environments, in part because impacts can be experienced as part of ordinary daily living activities.     

Structured morphological modeling as a framework for rational strain design of Streptomyces species

Successful application of a computational model for rational design of industrial Streptomyces exploitation requires a better understanding of the relationship between morphology—dictated by microbial growth, branching, fragmentation and adhesion—and product formation. Here we review the state-of-the-art in modeling of growth and product formation by filamentous microorganisms and expand on existing models by combining a morphological and structural approach to realistically model and visualize a three-dimensional pellet. The objective is to provide a framework to study the effect of morphology and structure on natural product and enzyme formation and yield. Growth and development of the pellet occur via the processes of apical extension, branching and cross-wall formation. Oxygen is taken to be the limiting component, with the oxygen concentration at the tips regulating growth kinetics and the oxygen profile within the pellet affecting the probability of branching. Biological information regarding the processes of differentiation and branching in liquid cultures of the model organism Streptomyces coelicolor has been implemented. The model can be extended based on information gained in fermentation trials for different production strains, with the aim to provide a test drive for the fermentation process and to pre-assess the effect of different variables on productivity. This should aid in improving Streptomyces as a production platform in industrial biotechnology.     

Single particle tracking of dynamically localizing TatA complexes in Streptomyces coelicolor

The Tat (twin-arginine translocation) pathway transports folded proteins across the bacterial cytoplasmic membrane and is a major route of protein export in the mycelial soil-dwelling bacterium Streptomyces. We recently examined the localization of Tat components (TatABC) in time-lapse imaging and demonstrated that all three components colocalize dynamically with a preference for apical sites. Here we apply an in-house single particle tracking package to quantitatively analyze the movement of the TatA subunit, the most abundant of the Tat components. Segmentation and analysis of trajectories revealed that TatA transitions from free to confined movement and then to fixed localization. The sequence starts with a mixed punctate and dispersed localization of TatA oligomers, which then develop into a few larger still foci, and finally colocalize with TatBC to form a functional translocation system. It takes 15–30 min for the Tat export complex to assemble and most likely become active. With this study we provide the first example of quantitative analysis of dynamic protein localization in Streptomyces, which is applicable to the study of many other dynamically localizing proteins identified in these complex bacteria.     

A gradient model of cardiac pacemaker myocytes

We have formulated a spatial-gradient model of action potential heterogeneity within the rabbit sinoatrial node (SAN), based on cell-specific ionic models of electrical activity from its central and peripheral regions. The ionic models are derived from a generic cell model, incorporating five background and exchange currents, and seven time-dependent currents based on three- or four-state Markov schemes. State transition rates are given by non-linear sigmoid functions of membrane potential.

By appropriate selection of parameters, the generic model is able to accurately reproduce a wide range of action potential waveforms observed experimentally. Specifically, the model can fit recordings from central and peripheral regions of the SAN with RMS errors of 0.3987 and 0.7628 mV, respectively. Using a custom least squares parameter optimisation routine, we have constructed a spatially-varying gradient model that exhibits a smooth transition in action potential characteristics from the central to the peripheral region, whilst ensuring individual membrane currents remain physiologically accurate. Smooth transition action potential characteristics include maximum diastolic potential, overshoot potential, upstroke velocity, action potential duration and cycle length. The gradient model is suitable for developing higher dimensional models of the right atrium, in which action potential heterogeneity within nodal tissue may be readily incorporated.