Skeletal muscle regeneration after extensive cryoinjury of caudal myomeres in adult zebrafish.

Skeletal muscles can regenerate after minor injuries, but severe structural damage often leads to fibrosis in mammals. Whether adult zebrafish possess the capacity to reproduce profoundly destroyed musculature remains unknown. Here, a new cryoinjury model revealed that several myomeres efficiently regenerated within one month after wounding the zebrafish caudal peduncle. Wound clearance involved accumulation of the selective autophagy receptor p62, an immune response and Collagen XII deposition. New muscle formation was associated with proliferation of Pax7 expressing muscle stem cells, which gave rise to MyoD1 positive myogenic precursors, followed by myofiber differentiation. Monitoring of slow and fast muscles revealed their coordinated replacement in the superficial and profound compartments of the myomere. However, the final boundary between the muscular components was imperfectly recapitulated, allowing myofibers of different identities to intermingle. The replacement of connective with sarcomeric tissues required TOR signaling, as rapamycin treatment impaired new muscle formation, leading to persistent fibrosis. The model of zebrafish myomere restoration may provide new medical perspectives for treatment of traumatic injuries.

A Cryoinjury Model for Studying Skeletal Muscle Regeneration of the Caudal Peduncle in Adult Zebrafish.

Skeletal muscle undergoes renewal and restoration after minor injury through the activation of satellite-like stem cells. Severe injuries of the musculature often lead to fibrosis in humans. In comparison to mammals, zebrafish possess a higher innate capacity for organ regeneration, providing a powerful model for studying tissue restoration after extensive damage to the organ. Here, a cryoinjury model is described to induce profound damage to four myomeres of the caudal peduncle in adult zebrafish. A custom-made cryoprobe was designed to fit the body shape and reproducibly injure the lateral musculature from the skin to the midline. Importantly, the body integrity remained intact, and the fish continued their swimming activity. Changes to the skeletal muscle were assessed by histological staining and fluorescence staining of sarcomeric proteins on tissue sections. This method will open up new avenues of research aiming to understand how the degeneration of the skeletal muscle induces reparative responses and, thus, the reactivation of the myogenic program in adult zebrafish.

Alveolar macrophages in early stage COPD show functional deviations with properties of impaired immune activation.

Despite its high prevalence, the cellular and molecular mechanisms of chronic obstructive pulmonary disease (COPD) are far from being understood. Here, we determine disease-related changes in cellular and molecular compositions within the alveolar space and peripheral blood of a cohort of COPD patients and controls. Myeloid cells were the largest cellular compartment in the alveolar space with invading monocytes and proliferating macrophages elevated in COPD. Modeling cell-to-cell communication, signaling pathway usage, and transcription factor binding predicts TGF-ß1 to be a major upstream regulator of transcriptional changes in alveolar macrophages of COPD patients. Functionally, macrophages in COPD showed reduced antigen presentation capacity, accumulation of cholesteryl ester, reduced cellular chemotaxis, and mitochondrial dysfunction, reminiscent of impaired immune activation.

The Degradation of Automotive Radar Sensor Signals Caused by Vehicle Vibrations and other Nonlinear Movements.

As the demands on modern radar systems with respect to accuracy, reliability, and availability increase, a detailed assessment of the influence of nonlinear movements has become necessary. In particular, from the point of view of radar, different types of movements, such as any kind of acceleration, braking situation, or vehicle vibration, are essential parts of any traffic scenario. These unavoidable motions, in which the relative velocity changes within one measurement cycle, are called nonlinear movements. These nonlinearities contribute to intermediate frequencies, which are comparable to the extensively described nonlinearities of a frequency ramp. This additional contribution to the intermediate signal has a direct effect on the signal-to-noise ratio and thus on the accuracy and probability of target detection. This paper presents a study of various types of nonlinear motion and a detailed definition of the resulting parameters based on a variety of vehicle-based measurements. An advanced signal model of frequency-modulated continuous wave (FMCW) radar is introduced and verified in addition to a detailed mathematical description of spectral signal behaviour in sinusoidal motions and linear acceleration. The theoretical and experimental results in idealised point targets are transferred to real complex road users. Furthermore, by applying established automotive signal processing steps in the form of an ordered statistical constant false alarm rate (OS CFAR), the consequences of determining the noise level are also shown. In combination with the already introduced signal behaviour, these results enabled general description of the signal-to-noise ratio of nonlinear movements in complex traffic scenarios.

Individual face- and house-related eye movement patterns distinctively activate FFA and PPA.

We investigated if the fusiform face area (FFA) and the parahippocampal place area (PPA) contain a representation of fixation sequences that are typically used when looking at faces or houses. Here, we instructed observers to follow a dot presented on a uniform background. The dot's movements represented gaze paths acquired separately from observers looking at face or house pictures. Even when gaze dispersion differences were controlled, face- and house-associated gaze patterns could be discriminated by fMRI multivariate pattern analysis in FFA and PPA, more so for the current observer's own gazes than for another observer's gaze. The discrimination of the observer's own gaze patterns was not observed in early visual areas (V1 - V4) or superior parietal lobule and frontal eye fields. These findings indicate a link between perception and action-the complex gaze patterns that are used to explore faces and houses-in the FFA and PPA.

Production of human milk oligosaccharides by enzymatic and whole-cell microbial biotransformations.

Human milk oligosaccharides (HMO) are almost unique constituents of breast milk and are not found in appreciable amounts in cow milk. Due to several positive aspects of HMO for the development, health, and wellbeing of infants, production of HMO would be desirable. As a result, scientists from different disciplines have developed methods for the preparation of single HMO compounds. Here, we review approaches to HMO preparation by (chemo-)enzymatic syntheses or by whole-cell biotransformation with recombinant bacterial cells. With lactose as acceptor (in vitro or in vivo), fucosyltransferases can be used for the production of 2'-fucosyllactose, 3-fucosyllactose, or more complex fucosylated core structures. Sialylated HMO can be produced by sialyltransferases and trans-sialidases. Core structures as lacto-N-tetraose can be obtained by glycosyltransferases from chemical donor compounds or by multi-enzyme cascades; recent publications also show production of lacto-N-tetraose by recombinant Escherichia coli bacteria and approaches to obtain fucosylated core structures. In view of an industrial production of HMOs, the whole cell biotransformation is at this stage the most promising option to provide human milk oligosaccharides as food additive.

A studyforrest extension, simultaneous fMRI and eye gaze recordings during prolonged natural stimulation.

Here we present an update of the studyforrest (http://studyforrest.org) dataset that complements the previously released functional magnetic resonance imaging (fMRI) data for natural language processing with a new two-hour 3 Tesla fMRI acquisition while 15 of the original participants were shown an audio-visual version of the stimulus motion picture. We demonstrate with two validation analyses that these new data support modeling specific properties of the complex natural stimulus, as well as a substantial within-subject BOLD response congruency in brain areas related to the processing of auditory inputs, speech, and narrative when compared to the existing fMRI data for audio-only stimulation. In addition, we provide participants' eye gaze location as recorded simultaneously with fMRI, and an additional sample of 15 control participants whose eye gaze trajectories for the entire movie were recorded in a lab setting-to enable studies on attentional processes and comparative investigations on the potential impact of the stimulation setting on these processes.

Synthesis of fucosylated lacto-N-tetraose using whole-cell biotransformation.

Fucosylated oligosaccharides present a predominant group of free oligosaccharides found in human milk. Here, a microbial conversion of lactose, D-glucose and L-fucose to fucosylated lacto-N-tetraose by growing Escherichia coli cultures is presented. The recombinant expression of genes encoding for the ß1,3-N-acetylglucosaminyltransferase (LgtA) and the ß1,3-galactosyltransferase (WbgO) enables the whole-cell biotransformation of lactose to lacto-N-tetraose. By the additional expression of a recombinant GDP-L-fucose salvage pathway together with a bacterial fucosyltransferase, lacto-N-tetraose is further converted into the respective fucosylated compounds. The expression of a gene encoding the α1,2-fucosyltransferase (FutC) in the lacto-N-tetraose producing E. coli strain led to the formation of lacto-N-fucopentaose I, whereas the expression of a gene encoding the α1,4-fucosyltransferase (FucT14) mainly led to the conversion of lacto-N-tetraose to lacto-N-difucohexaose II.

Galactose-limited fed-batch cultivation of Escherichia coli for the production of lacto-N-tetraose.

Lacto-N-tetraose (Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc) is one of the most abundant oligosaccharide structures in human milk. We recently described the synthesis of lacto-N-tetraose by a whole-cell biotransformation with recombinant Escherichia coli cells. However, only about 5% of the lactose was converted into lacto-N-tetraose by this approach. The major product obtained was the intermediate lacto-N-triose II (GlcNAc(ß1-3)Gal(ß1-4)Glc). In order to improve the bioconversion of lactose to lacto-N-tetraose, we have investigated the influence of the carbon source on the formation of lacto-N-tetraose and on the intracellular availability of the glycosyltransferase substrates, UDP-N-acetylglucosamine and UDP-galactose. By growth of the recombinant E. coli cells on D-galactose, the yield of lacto-N-tetraose (810.8 mg L(-1) culture) was 3.6-times higher compared to cultivation on D-glucose. Using fed-batch cultivation with galactose as sole energy and carbon source, a large-scale synthesis of lacto-N-tetraose was demonstrated. During the 26 h feeding phase the growth rate (µ = 0.05) was maintained by an exponential galactose feed. In total, 16 g L(-1) lactose were fed and resulted in final yields of 12.72 ± 0.21 g L(-1) lacto-N-tetraose and 13.70 ± 0.10 g L(-1) lacto-N-triose II. In total, 173 g of lacto-N-tetraose were produced with a space-time yield of 0.37 g L(-1) h(-1).

Investigating the brain basis of facial expression perception using multi-voxel pattern analysis.

Humans can readily decode emotion expressions from faces and perceive them in a categorical manner. The model by Haxby and colleagues proposes a number of different brain regions with each taking over specific roles in face processing. One key question is how these regions directly compare to one another in successfully discriminating between various emotional facial expressions. To address this issue, we compared the predictive accuracy of all key regions from the Haxby model using multi-voxel pattern analysis (MVPA) of functional magnetic resonance imaging (fMRI) data. Regions of interest were extracted using independent meta-analytical data. Participants viewed four classes of facial expressions (happy, angry, fearful and neutral) in an event-related fMRI design, while performing an orthogonal gender recognition task. Activity in all regions allowed for robust above-chance predictions. When directly comparing the regions to one another, fusiform gyrus and superior temporal sulcus (STS) showed highest accuracies. These results underscore the role of the fusiform gyrus as a key region in perception of facial expressions, alongside STS. The study suggests the need for further specification of the relative role of the various brain areas involved in the perception of facial expression. Face processing appears to rely on more interactive and functionally overlapping neural mechanisms than previously conceptualised.

Synthesis of the human milk oligosaccharide lacto-N-tetraose in metabolically engineered, plasmid-free E. coli.

Human milk oligosaccharides (HMOs) constitute the third most abundant solid component of human milk. HMOs have been demonstrated to show positive effects on the infant's well-being. Despite numerous studies, more physiological analyses of single compounds are needed to fully elucidate these effects. Although being one of the most abundant core structures in human milk, the HMO lacto-N-tetraose (LNT) is not available at reasonable prices. In this study, we demonstrate the construction of the first E. coli strain capable of producing LNT in vivo. The strain was constructed by chromosomally integrating the genes lgtA and wbgO, encoding ß-1,3-N-acetylglucosaminyltransferase and ß-1,3-galactosyltransferase. In shake-flask cultivations, the strain yielded a total concentration of 219.1±3.5 mg L(-1) LNT (LNT in culture broth and the cell pellet). After recovery of LNT, structural analysis by NMR spectroscopy confirmed the molecule structure.

The right temporo-parietal junction contributes to visual feature binding.

We investigated the neural basis of conjoined processing of color and spatial frequency with functional magnetic resonance imaging (fMRI). A multivariate classification algorithm was trained to differentiate between either isolated color or spatial frequency differences, or between conjoint differences in both feature dimensions. All displays were presented in a singleton search task, avoiding confounds between conjunctive feature processing and search difficulty that arose in previous studies contrasting single feature and conjunction search tasks. Based on patient studies, we expected the right temporo-parietal junction (TPJ) to be involved in conjunctive feature processing. This hypothesis was confirmed in that only conjoined color and spatial frequency differences, but not isolated feature differences could be classified above chance level in this area. Furthermore, we could show that the accuracy of a classification of differences in both feature dimensions was superadditive compared to the classification accuracies of isolated color or spatial frequency differences within the right TPJ. These data provide evidence for the processing of feature conjunctions, here color and spatial frequency, in the right TPJ.

Functional asymmetry and effective connectivity of the auditory system during speech perception is modulated by the place of articulation of the consonant- A 7T fMRI study.

To differentiate between stop-consonants, the auditory system has to detect subtle place of articulation (PoA) and voice-onset time (VOT) differences between stop-consonants. How this differential processing is represented on the cortical level remains unclear. The present functional magnetic resonance (fMRI) study takes advantage of the superior spatial resolution and high sensitivity of ultra-high-field 7 T MRI. Subjects were attentively listening to consonant-vowel (CV) syllables with an alveolar or bilabial stop-consonant and either a short or long VOT. The results showed an overall bilateral activation pattern in the posterior temporal lobe during the processing of the CV syllables. This was however modulated strongest by PoA such that syllables with an alveolar stop-consonant showed stronger left lateralized activation. In addition, analysis of underlying functional and effective connectivity revealed an inhibitory effect of the left planum temporale (PT) onto the right auditory cortex (AC) during the processing of alveolar CV syllables. Furthermore, the connectivity result indicated also a directed information flow from the right to the left AC, and further to the left PT for all syllables. These results indicate that auditory speech perception relies on an interplay between the left and right ACs, with the left PT as modulator. Furthermore, the degree of functional asymmetry is determined by the acoustic properties of the CV syllables.

A high-resolution 7-Tesla fMRI dataset from complex natural stimulation with an audio movie.

Here we present a high-resolution functional magnetic resonance (fMRI) dataset - 20 participants recorded at high field strength (7 Tesla) during prolonged stimulation with an auditory feature film ("Forrest Gump"). In addition, a comprehensive set of auxiliary data (T1w, T2w, DTI, susceptibility-weighted image, angiography) as well as measurements to assess technical and physiological noise components have been acquired. An initial analysis confirms that these data can be used to study common and idiosyncratic brain response patterns to complex auditory stimulation. Among the potential uses of this dataset are the study of auditory attention and cognition, language and music perception, and social perception. The auxiliary measurements enable a large variety of additional analysis strategies that relate functional response patterns to structural properties of the brain. Alongside the acquired data, we provide source code and detailed information on all employed procedures - from stimulus creation to data analysis. In order to facilitate replicative and derived works, only free and open-source software was utilized.

Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2'-fucosyllactose.

BACKGROUND: The trisaccharide 2'-fucosyllactose (2'-FL) is one of the most abundant oligosaccharides found in human milk. Due to its prebiotic and anti-infective properties, 2'-FL is discussed as nutritional additive for infant formula. Besides chemical synthesis and extraction from human milk, 2'-FL can be produced enzymatically in vitro and in vivo. The most promising approach for a large-scale formation of 2'-FL is the whole cell biosynthesis in Escherichia coli by intracellular synthesis of GDP-L-fucose and subsequent fucosylation of lactose with an appropriate α1,2-fucosyltransferase. Even though whole cell approaches have been demonstrated for the synthesis of 2'-FL, further improvements of the engineered E. coli host are required to increase product yields. Furthermore, an antibiotic-free method of whole cell synthesis of 2'-FL is desirable to simplify product purification and to avoid traces of antibiotics in a product with nutritional purpose. RESULTS: Here we report the construction of the first selection marker-free E. coli strain that produces 2'-FL from lactose and glycerol. To construct this strain, recombinant genes of the de novo synthesis pathway for GDP-L-fucose as well as the gene for the H. pylori fucosyltransferase futC were integrated into the chromosome of E. coli JM109 by using the λ-Red recombineering technique. Strains carrying additional copies of the futC gene and/or the gene fkp (from Bacteroides fragilis) for an additional salvage pathway for GDP-L-fucose production were used and shown to further improve production of 2'-FL in shake flask experiments. An increase of the intracellular GDP-L-fucose concentration by expression of fkp gene as well as an additional copy of the futC gene lead to an enhanced formation of 2'-FL. Using an improved production strain, feasibility of large scale 2'-FL production was demonstrated in an antibiotic-free fed-batch fermentation (13 l) with a final 2'-FL concentration of 20.28 ± 0.83 g l(-1) and a space-time-yield of 0.57 g l(-1) h(-1). CONCLUSIONS: By chromosomal integration of recombinant genes, altering the copy number of these genes and analysis of 2'-FL and intracellular GDP-L-fucose levels, we were able to construct and improve the first selection marker-free E. coli strain which is capable to produce 2'-FL without the use of expression plasmids. Analysis of intracellular GDP-L-fucose levels identified the de novo synthesis pathway of GDP-L-fucose as one bottleneck in 2'-FL production. In antibiotic-free fed-batch fermentation with an improved strain, scale-up of 2'-FL could be demonstrated.

Possible pitfalls investigating cell death responses in genetically engineered mouse models and derived cell lines.

Genetically engineered mouse models are frequently used to identify pathophysiological consequences of deregulated cell death. Targeting pro-apoptotic or anti-apoptotic proteins of the extrinsic or intrinsic apoptotic signalling cascade is state of the art since more than two decades. Such animal models have been increasingly made use of over the past years to study loss- or gain-of-function consequences of one or more components of the molecular machinery leading to cell death. These studies have helped to separate redundant from non-redundant functions of apoptosis-related proteins in normal physiology and sometimes unravelled unexpected phenotypes. However, correct interpretation of data derived from knockout mice or derived cells and cell lines is often flawed by the comparison of cells originating from different inbred or mixed genetic backgrounds. Here we want to highlight some basic problems associated with genetic background-based modulation of cell death sensitivity and describe some methods that we use to investigate cell death responses in hematopoietic and non-hematopoietic cells. Thereby, we show that hematopoietic cells derived from wild type mice on a C57BL/6:129/SvJ recombinant mixed genetic background are significantly more resistant to spontaneous cell death or DNA-damage induced apoptosis in vitro than cells derived from inbred C57BL/6 mice. Furthermore, we show as an example that C57BL/6 mice are more susceptible to γ-irradiation induced cell death after whole body irradiation in vivo and subsequent T cell lymphomagenesis.

A behavioral task for the validation of a gaze-contingent simulated scotoma.

Gaze-contingent displays provide a valuable method in visual research for controlling visual input and investigating its visual and cognitive processing. Although the body of research using gaze-contingent retinal stabilization techniques has grown considerably during the last decade, only few studies have been concerned with the reliability of the specific real-time simulations applied. Using a Landolt ring discrimination task, we present a behavioral validation of gaze-contingent central scotoma simulation in healthy observers. Importantly, behavioral testing is necessary to show whether the simulation impairs foveal processing of visual information. This test becomes even more crucial when researchers are faced with null results in a task performed with the scotoma, as compared with a control condition. It must be ruled out that the lack of behavioral effects results from a type II error caused by improper implementation before conclusions about foveal contributions to the given task may be drawn. In our experiment, the scotoma effectively prevented foveal processing of the visual stimuli, leading to significantly reduced response accuracies, as compared with unimpaired vision. Moreover, the final fixation at the time of the participants' responses was placed close to the target position in the unimpaired condition, whereas the distance to the target was enhanced with the scotoma, indicating that the observers were not able to discriminate visual target stimuli from distractors, due to the scotoma. The present work presents a validated behavioral testing method for the efficiency of gaze-contingent scotoma simulations, including code for implementation. In addition, solutions for common methodological problems are discussed.

Evidence for feature binding in the superior parietal lobule.

The neural substrates of feature binding are an old, yet still not completely resolved problem. While patient studies suggest that posterior parietal cortex is necessary for feature binding, imaging evidence has been inconclusive in the past. These studies compared visual feature and conjunction search to investigate the neural substrate of feature conjunctions. However, a common problem of these comparisons was a confound with search difficulty. To circumvent this confound, we directly investigated the localized representation of features (color and spatial frequency) and feature conjunctions in a single search task by using multivariate pattern analysis at high field strength (7T). In right superior parietal lobule, we found evidence for the representation of feature conjunctions that could not be explained by the summation of individual feature representations and thus indicates conjoined processing of color and spatial frequency.

Dorsal and ventral working memory-related brain areas support distinct processes in contextual cueing.

Behavioral evidence suggests that the use of implicitly learned spatial contexts for improved visual search may depend on visual working memory resources. Working memory may be involved in contextual cueing in different ways: (1) for keeping implicitly learned working memory contents available during search or (2) for the capture of attention by contexts retrieved from memory. We mapped brain areas that were modulated by working memory capacity. Within these areas, activation was modulated by contextual cueing along the descending segment of the intraparietal sulcus, an area that has previously been related to maintenance of explicit memories. Increased activation for learned displays, but not modulated by the size of contextual cueing, was observed in the temporo-parietal junction area, previously associated with the capture of attention by explicitly retrieved memory items, and in the ventral visual cortex. This pattern of activation extends previous research on dorsal versus ventral stream functions in memory guidance of attention to the realm of attentional guidance by implicit memory.

Medial temporal lobe-dependent repetition suppression and enhancement due to implicit vs. explicit processing of individual repeated search displays.

Using visual search, functional magnetic resonance imaging (fMRI) and patient studies have demonstrated that medial temporal lobe (MTL) structures differentiate repeated from novel displays-even when observers are unaware of display repetitions. This suggests a role for MTL in both explicit and, importantly, implicit learning of repeated sensory information (Greene et al., 2007). However, recent behavioral studies suggest, by examining visual search and recognition performance concurrently, that observers have explicit knowledge of at least some of the repeated displays (Geyer et al., 2010). The aim of the present fMRI study was thus to contribute new evidence regarding the contribution of MTL structures to explicit vs. implicit learning in visual search. It was found that MTL activation was increased for explicit and, respectively, decreased for implicit relative to baseline displays. These activation differences were most pronounced in left anterior parahippocampal cortex (aPHC), especially when observers were highly trained on the repeated displays. The data are taken to suggest that explicit and implicit memory processes are linked within MTL structures, but expressed via functionally separable mechanisms (repetition-enhancement vs. -suppression). They further show that repetition effects in visual search would have to be investigated at the display level.