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•The neural underpinning of separate BOLD phases is unclear.•A reproducible multi-phase BOLD responses were obtained.•MUA power exhibited the same multiphasic dynamics as the BOLD signal.•Post-stimulus BOLD phases can be better explained by high-frequency neuronal signal. Visual stimulation-evoked blood-oxygen-level dependent (BOLD) responses can exhibit more complex temporal dynamics than a simple monophasic response. For instance, BOLD responses sometimes include a phase of positive response followed by a phase of post-stimulus undershoot. Whether the BOLD response during these phases reflects the underlying neuronal signal fluctuations or is contributed by non-neuronal physiological factors remains elusive. When presenting blocks of sustained (i.e. DC) light ON-OFF stimulations to unanesthetized rats, we observed that the response following a decrease in illumination (i.e. OFF stimulation-evoked BOLD response) in the visual cortices displayed reproducible multiple phases, including an initial positive BOLD response, followed by an undershoot and then an overshoot before the next ON trial. This multi-phase BOLD response did not result from the entrainment of the periodic stimulation structure. When we measured the neural correlates of these responses, we found that the high-frequency band from the LFP power (300 – 3000 Hz, multi-unit activity (MUA)), but not the power in the gamma band (30 – 100 Hz) exhibited the same multiphasic dynamics as the BOLD signal. This study suggests that the post-stimulus phases of the BOLD response can be better explained by the high-frequency neuronal signal.

Germination, the process whereby a dry, quiescent seed springs to life, has been a focus of plant biologist for many years, yet the early events following water uptake, during which metabolism of the embryo is restarted, remain enigmatic. Here, the nature of the cues required for this restarting in oilseed rape (Brassica napus) seed has been investigated. A holistic in vivo approach was designed to display the link between the entry and allocation of water, metabolic events and structural changes occurring during germination. For this, we combined functional magnetic resonance imaging with Fourier transform infrared microscopy, fluorescence-based respiration mapping, computer-aided seed modeling and biochemical tools. We uncovered an endospermal lipid gap, which channels water to the radicle tip, from whence it is distributed via embryonic vasculature toward cotyledon tissues. The resumption of respiration is initiated first in the endosperm, only later spreading to the embryo. Sugar metabolism and lipid utilization are linked to the spatiotemporal sequence of tissue rehydration. Together, this imaging study provides insights into the spatial aspects of key events in oilseed rape seeds leading to germination. It demonstrates how seed architecture predetermines the pattern of water intake, which sets the stage for the orchestrated restart of life.

Constrained to develop within the seed, the plant embryo must adapt its shape and size to fit the space available. Here, we demonstrate how this adjustment shapes metabolism of photosynthetic embryo. Noninvasive NMR-based imaging of the developing oilseed rape (Brassica napus) seed illustrates that, following embryo bending, gradients in lipid concentration became established. These were correlated with the local photosynthetic electron transport rate and the accumulation of storage products. Experimentally induced changes in embryo morphology and/or light supply altered these gradients and were accompanied by alterations in both proteome and metabolome. Tissue-specific metabolic models predicted that the outer cotyledon and hypocotyl/radicle generate the bulk of plastidic reductant/ATP via photosynthesis, while the inner cotyledon, being enclosed by the outer cotyledon, is forced to grow essentially heterotrophically. Under field-relevant highlight conditions, major contribution of the ribulose-1,5-bisphosphate carboxylase/oxygenase—bypass to seed storage metabolism is predicted for the outer cotyledon and the hypocotyl/radicle only. Differences between in vitro— versus in planta—grown embryos suggest that metabolic heterogeneity of embryo is not observable by in vitro approaches. We conclude that in vivo metabolic fluxes are locally regulated and connected to seed architecture, driving the embryo toward an efficient use of available light and space.

Background Studying dynamic processes in living organisms with MRI is one of the most promising research areas. The use of paramagnetic compounds as contrast agents (CA), has proven key to such studies, but so far, the lack of appropriate techniques limits the application of CA-technologies in experimental plant biology. The presented proof-of-principle aims to support method and knowledge transfer from medical research to plant science. Results In this study, we designed and tested a new approach for plant Dynamic Contrast Enhanced Magnetic Resonance Imaging (pDCE-MRI). The new approach has been applied in situ to a cereal crop ( Hordeum vulgare ). The pDCE-MRI allows non-invasive investigation of CA allocation within plant tissues. In our experiments, gadolinium-DTPA, the most commonly used contrast agent in medical MRI, was employed. By acquiring dynamic T 1 -maps, a new approach visualizes an alteration of a tissue-specific MRI parameter T 1 (longitudinal relaxation time) in response to the CA. Both, the measurement of local CA concentration and the monitoring of translocation in low velocity ranges (cm/h) was possible using this CA-enhanced method. Conclusions A novel pDCE-MRI method is presented for non-invasive investigation of paramagnetic CA allocation in living plants. The temporal resolution of the T 1 -mapping has been significantly improved to enable the dynamic in vivo analysis of transport processes at low-velocity ranges, which are common in plants. The newly developed procedure allows to identify vascular regions and to estimate their involvement in CA allocation. Therefore, the presented technique opens a perspective for further development of CA-aided MRI experiments in plant biology.

Craniomaxillofacial reconstruction is challenging due to the requirement for diverse manual surgical interventions, which significantly increase as the defect volume enlarges. To address these concerns, we utilized intraoperative bioprinting (IOB) to reconstruct cranial bone defects in surgical settings. We formulated an innovative collagen‐based bioink supplemented with human adipose‐derived stem cells (hADSCs) or bone morphogenetic protein‐2 (BMP‐2). The concentration and dispersion state of collagen along with hADSCs were precisely adjusted to enhance cytocompatibility, bioprintability, and osteogenic activities. IOB was first performed via a 3‐axis bioprinter on a rat model having a critical‐sized calvarial defect (39.3 mm3), which was infilled within ≈30 s and resulted in ≈90% bone coverage area in 8 weeks. Secondly, IOB was conducted on sheep calvarial defects (1,209 mm3, ≈31‐fold larger compared to the rat defects) using a 6‐axis robotic arm, where IOB took ≈5 min per defect. On Week 12, sheep defects treated with IOB revealed accelerated bone repair (≈80% bone coverage area) and mechanical enhancement with 240%, 235%, and 358% increments in Young's modulus, peak force, and energy compared to the non‐treated group. The successful execution of IOB in small and large animal models validates the translation potential of IOB for automated surgical interventions. In rat and sheep calvarial models, a highly concentrated collagen‐based bioink (HC‐ink) is bioprinted and compared in combination with recombinant human bone morphogenetic protein‐2 (rhBMP‐2) or human adipose‐derived stem cells. Collectively, HC‐ink with the highest cell density and the addition of rhBMP‐2 contributes to improved new bone formation in rat and sheep models, respectively.

The brain and skull represent a complex arrangement of integrated anatomical structures composed of various cell and tissue types that maintain structural and functional association throughout development. Morphological integration, a concept developed in vertebrate morphology and evolutionary biology, describes the coordinated variation of functionally and developmentally related traits of organisms. Syndromic craniosynostosis is characterized by distinctive changes in skull morphology and perceptible, though less well studied, changes in brain structure and morphology. Using mouse models for craniosynostosis conditions, our group has precisely defined how unique craniosynostosis causing mutations in affect brain and skull morphology and dysgenesis involving coordinated tissue-specific effects of these mutations. Here we examine integration of brain and skull in two mouse models for craniosynostosis: one carrying the FGFR2c C342Y mutation associated with Pfeiffer and Crouzon syndromes and a mouse model carrying the FGFR2 S252W mutation, one of two mutations responsible for two-thirds of Apert syndrome cases. Using linear distances estimated from three-dimensional coordinates of landmarks acquired from dual modality imaging of skull (high resolution micro-computed tomography and magnetic resonance microscopy) of mice at embryonic day 17.5, we confirm variation in brain and skull morphology in mice, mice, and their unaffected littermates. Mutation-specific variation in neural and cranial tissue notwithstanding, patterns of integration of brain and skull differed only subtly between mice carrying either the FGFR2c C342Y or the FGFR2 S252W mutation and their unaffected littermates. However, statistically significant and substantial differences in morphological integration of brain and skull were revealed between the two mutant mouse models, each maintained on a different strain. Relative to the effects of disease-associated mutations, our results reveal a stronger influence of the background genome on patterns of brain-skull integration and suggest robust genetic, developmental, and evolutionary relationships between neural and skeletal tissues of the head.

While often thought of as a smoking drug, tobacco (Nicotiana spp.) is now considered as a plant of choice for molecular farming and biofuel production. Here, we describe a noninvasive means of deriving both the distribution of lipid and the microtopology of the submillimeter tobacco seed, founded on nuclear magnetic resonance (NMR) technology. Our platform enables counting of seeds inside the intact tobacco capsule to measure seed sizes, to model the seed interior in three dimensions, to quantify the lipid content, and to visualize lipid gradients. Hundreds of seeds can be simultaneously imaged at an isotropic resolution of 25 µm, sufficient to assess each individual seed. The relative contributions of the embryo and the endosperm to both seed size and total lipid content could be assessed. The extension of the platform to a range of wild and cultivated Nicotiana species demonstrated certain evolutionary trends in both seed topology and pattern of lipid storage. The NMR analysis of transgenic tobacco plants with seed-specific ectopic expression of the plastidial phosphoenolpyruvate/phosphate translocator, displayed a trade off between seed size and oil concentration. The NMR-based assay of seed lipid content and topology has a number of potential applications, in particular providing a means to test and optimize transgenic strategies aimed at the manipulation of seed size, seed number, and lipid content in tobacco and other species with submillimeter seeds.

Ketogenic diets (KDs) are very low-carbohydrate, very high-fat diets which promote nutritional ketosis and impact energetic metabolism. Aquaporins (AQPs) are transmembrane channels that facilitate water and glycerol transport across cell membranes and are critical players in energy homeostasis. Altered AQP expression or function impacts fat accumulation and related comorbidities, such as the metabolic syndrome. Here, we sought to determine whether nutritional ketosis impacts AQPs expression in the context of an atherogenic model. To do this, we fed ApoE−/− (apolipoprotein E-deficient) mice, a model of human atherosclerosis, a KD (Kcal%: 1/81/18, carbohydrate/fat/protein) or a control diet (Kcal%: 70/11/18, carbohydrate/fat/protein) for 12 weeks. Plasma was collected for biochemical analysis. Upon euthanasia, livers, white adipose tissue (WAT), and brown adipose tissue (BAT) were used for gene expression studies. Mice fed the KD and control diets exhibited similar body weights, despite the profoundly different fat contents in the two diets. Moreover, KD-fed mice developed nutritional ketosis and showed increased expression of thermogenic genes in BAT. Additionally, these mice presented an increase in Aqp9 transcripts in BAT, but not in WAT, which suggests the participation of Aqp9 in the influx of excess plasma glycerol to fuel thermogenesis, while the up-regulation of Aqp7 in the liver suggests the involvement of this aquaporin in glycerol influx into hepatocytes. The relationship between nutritional ketosis, energy homeostasis, and the AQP network demands further investigation.

The dysfunction of vascular endothelial cells is profoundly implicated in the pathogenesis of atherosclerosis and cardiovascular disease, the global leading cause of death. Aquaporins (AQPs) are membrane channels that facilitate water and glycerol transport across cellular membranes recently implicated in the homeostasis of the cardiovascular system. Apolipoprotein-E deficient (apoE−/−) mice are a common model to study the progression of atherosclerosis. Nevertheless, the pattern of expression of AQPs in this atheroprone model is poorly characterized. In this study, apoE−/− mice were fed an atherogenic high-fat (HF) or a control diet. Plasma was collected at multiple time points to assess metabolic disturbances. At the endpoint, the aortic atherosclerotic burden was quantified using high field magnetic resonance imaging. Moreover, the transcriptional levels of several AQP isoforms were evaluated in the liver, white adipocyte tissue (WAT), and brown adipocyte tissue (BAT). The results revealed that HF-fed mice, when compared to controls, presented an exacerbated systemic inflammation and atherosclerotic phenotype, with no major differences in systemic methylation status, circulating amino acids, or plasma total glutathione. Moreover, an overexpression of the isoform AQP5 was detected in all studied tissues from HF-fed mice when compared to controls. These results suggest a novel role for AQP5 on diet-induced atherosclerosis that warrants further investigation.

Stem cell transplantation is a promising therapeutic approach for several neurological disorders. However, it has yet to fulfill its high expectations, partially due to the lack of a reliable noninvasive method for monitoring the biodistribution of the grafted stem cells in vivo. We have used high-resolution magnetic resonance imaging (MRI) at 17.6 T, combined with efficient magnetic labeling of the stem cells with iron oxide nanoparticles, in order to assess the in vivo detection limit in small animal models. Injection of different concentrations of magnetically labeled stem cells in gel phantoms led to significant reductions in image intensity from small cellular clusters of less than 10 cells. To determine the detection limit in vivo, various numbers of both labeled and unlabeled cells were injected stereotactically into the striatum of rats. Significant hypointense signal changes were observed for 100 labeled cells. After injection of approximately 20 labeled cells, signal reduction at the injection site was observed but could not be assigned unambiguously to the cells. Our results show that high-field MRI allows tracking of a minimal number of cells in vivo, well below the number used in previous studies, opening the possibility of gaining new insights into cell migration and differentiation.