Sequential changes in cellular properties accompanying amniote somite formation.

Somites are transient structures derived from the pre-somitic mesoderm (PSM), involving mesenchyme-to-epithelial transition (MET) where the cells change their shape and polarize. Using Scanning electron microscopy (SEM), immunocytochemistry and confocal microscopy, we study the progression of these events along the tail-to-head axis of the embryo, which mirrors the progression of somitogenesis (younger cells located more caudally). SEM revealed that PSM epithelialization is a gradual process, which begins much earlier than previously thought, starting with the dorsalmost cells, then the medial ones, and then, simultaneously, the ventral and lateral cells, before a somite fully separates from the PSM. The core (internal) cells of the PSM and somites never epithelialize, which suggests that the core cells could be 'trapped' within the somitocoele after cells at the surfaces of the PSM undergo MET. Three-dimensional imaging of the distribution of the cell polarity markers PKCζ, PAR3, ZO1, the Golgi marker GM130 and the apical marker N-cadherin reveal that the pattern of polarization is distinctive for each marker and for each surface of the PSM, but the order of these events is not the same as the progression of cell elongation. These observations challenge some assumptions underlying existing models of somite formation.

Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene dose-sensitive AD suppressor in human brain.

A population of more than six million people worldwide at high risk of Alzheimer's disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of ß-amyloid-(Aß)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aß deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome 21 gene BACE2, but prevented by combined chemical ß and γ-secretase inhibition. We found that T21 organoids secrete increased proportions of Aß-preventing (Aß1-19) and Aß-degradation products (Aß1-20 and Aß1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1 inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.

Development of a porcine acellular bladder matrix for tissue-engineered bladder reconstruction.

PURPOSE: Enterocystoplasty is adopted for patients requiring bladder augmentation, but significant long-term complications highlight need for alternatives. We established a protocol for creating a natural-derived bladder extracellular matrix (BEM) for developing tissue-engineered bladder, and investigated its structural and functional characteristics. METHODS: Porcine bladders were de-cellularised with a dynamic detergent-enzymatic treatment using peristaltic infusion. Samples and fresh controls were evaluated using histological staining, ultrastructure (electron microscopy), collagen, glycosaminoglycans and DNA quantification and biomechanical testing. Compliance and angiogenic properties (Chicken chorioallantoic membrane [CAM] assay) were evaluated. T test compared stiffness and glycosaminoglycans, collagen and DNA quantity. p value of < 0.05 was regarded as significant. RESULTS: Histological evaluation demonstrated absence of cells with preservation of tissue matrix architecture (collagen and elastin). DNA was 0.01 µg/mg, significantly reduced compared to fresh tissue 0.13 µg/mg (p < 0.01). BEM had increased tensile strength (0.259 ± 0.022 vs 0.116 ± 0.006, respectively, p < 0.0001) and stiffness (0.00075 ± 0.00016 vs 0.00726 ± 0.00216, p = 0.011). CAM assay showed significantly increased number of convergent allantoic vessels after 6 days compared to day 1 (p < 0.01). Urodynamic studies showed that BEM maintains or increases capacity and compliance. CONCLUSION: Dynamic detergent-enzymatic treatment produces a BEM which retains structural characteristics, increases strength and stiffness and is more compliant than native tissue. Furthermore, BEM shows angiogenic potential. These data suggest the use of BEM for development of tissue-engineered bladder for patients requiring bladder augmentation.

Multiple roles of integrin-α3 at the neuromuscular junction.

The neuromuscular junction (NMJ) is the synapse between motoneurons and skeletal muscle, and is responsible for eliciting muscle contraction. Neurotransmission at synapses depends on the release of synaptic vesicles at sites called active zones (AZs). Various proteins of the extracellular matrix are crucial for NMJ development; however, little is known about the identity and functions of the receptors that mediate their effects. Using genetically modified mice, we find that integrin-α3 (encoded by Itga3), an adhesion receptor at the presynaptic membrane, is involved in the localisation of AZ components and efficient synaptic vesicle release. Integrin-α3 also regulates integrity of the synapse - mutant NMJs present with progressive structural changes and upregulated autophagy, features commonly observed during ageing and in models of neurodegeneration. Unexpectedly, we find instances of nerve terminal detachment from the muscle fibre; to our knowledge, this is the first report of a receptor that is required for the physical anchorage of pre- and postsynaptic elements at the NMJ. These results demonstrate multiple roles of integrin-α3 at the NMJ, and suggest that alterations in its function could underlie defects that occur in neurodegeneration or ageing.

Salmonella exploits HLA-B27 and host unfolded protein responses to promote intracellular replication.

OBJECTIVE: Salmonella enterica infections can lead to Reactive Arthritis (ReA), which can exhibit an association with human leucocyte antigen (HLA)-B*27:05, a molecule prone to misfolding and initiation of the unfolded protein response (UPR). This study examined how HLA-B*27:05 expression and the UPR affect the Salmonella life-cycle within epithelial cells. METHODS: Isogenic epithelial cell lines expressing two copies of either HLA-B*27:05 and a control HLA-B*35:01 heavy chain (HC) were generated to determine the effect on the Salmonella infection life-cycle. A cell line expressing HLA-B*27:05.HC physically linked to the light chain beta-2-microglobulin and a specific peptide (referred to as a single chain trimer, SCT) was also generated to determine the effects of HLA-B27 folding status on S.enterica life-cycle. XBP-1 venus and AMP dependent Transcription Factor (ATF6)-FLAG reporters were used to monitor UPR activation in infected cells. Triacin C was used to inhibit de novo lipid synthesis during UPR, and confocal imaging of ER tracker stained membrane allowed quantification of glibenclamide-associated membrane. RESULTS: S.enterica demonstrated enhanced replication with an altered cellular localisation in the presence of HLA-B*27:05.HC but not in the presence of HLA-B*27:05.SCT or HLA-B*35:01. HLA-B*27:05.HC altered the threshold for UPR induction. Salmonella activated the UPR and required XBP-1 for replication, which was associated with endoreticular membrane expansion and lipid metabolism. CONCLUSIONS: HLA-B27 misfolding and a UPR cellular environment are associated with enhanced Salmonella replication, while Salmonella itself can activate XBP-1 and ATF6. These data provide a potential mechanism linking the life-cycle of Salmonella with the physicochemical properties of HLA-B27 and cellular events that may contribute to ReA pathogenesis. Our observations suggest that the UPR pathway maybe targeted for future therapeutic intervention.

Wnt signaling regulates cytosolic translocation of connexin 43.

The availability of intracellular, stabilized ß-catenin, a transcription factor coactivator, is tightly regulated; ß-catenin is translocated into the nucleus in response to Wnt ligand binding to its cell membrane receptors. Here we show that Wnt signal activation in mammalian cells activates intracellular mobilization of connexin 43 (Cx43), which belongs to a gap junction protein family, a new target protein in response to extracellular Wnt signal activation. Transmission electron microscopy showed that the nuclear localization of Cx43 was increased by 8- to 10-fold in Wnt5A- and 9B-treated cells compared with controls; this Wnt-induced increase was negated in the cells where Cx43 and ß-catenin were knocked down using shRNA. There was a significant (P < 0.001) and concomitant depletion of the cell membrane and cytosolic signal of Cx43 in Wnt-treated cells with an increase in the nuclear signal for Cx43; this was more obvious in cells where ß-catenin was knocked down using shRNA. Conversely, Cx43 knockdown resulted in increased ß-catenin in the nucleus in the absence of Wnt activation. Coimmunoprecipitation of Cx43 and ß-catenin proteins with a casein kinase (CKIδ) antibody showed that Cx43 interacts with ß-catenin and may form part of the so-called destruction complex. Functionally, Wnt activation increased the rate of wound reepithelization in rat skin in vivo.

Impairments in contractility and cytoskeletal organisation cause nuclear defects in nemaline myopathy.

Nemaline myopathy (NM) is a skeletal muscle disorder caused by mutations in genes that are generally involved in muscle contraction, in particular those related to the structure and/or regulation of the thin filament. Many pathogenic aspects of this disease remain largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mouse models and patients with NM: irregular spacing and morphology of nuclei; disrupted nuclear envelope; altered chromatin arrangement; and disorganisation of the cortical cytoskeleton. Impairments in contractility are the primary cause of these nuclear defects. We also establish the role of microtubule organisation in determining nuclear morphology, a phenomenon which is likely to contribute to nuclear alterations in this disease. Our results overlap with findings in diseases caused directly by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to explain some of the hallmarks of NM, including contractile filament disarray, altered mechanical properties and broad transcriptional alterations.

STAT3 Controls the Long-Term Survival and Phenotype of Repair Schwann Cells during Nerve Regeneration.

After nerve injury, Schwann cells convert to a phenotype specialized to promote repair. But during the slow process of axonal regrowth, these repair Schwann cells gradually lose their regeneration-supportive features and eventually die. Although this is a key reason for the frequent regeneration failures in humans, the transcriptional mechanisms that control long-term survival and phenotype of repair cells have not been studied, and the molecular signaling underlying their decline is obscure. We show, in mice, that Schwann cell STAT3 has a dual role. It supports the long-term survival of repair Schwann cells and is required for the maintenance of repair Schwann cell properties. In contrast, STAT3 is less important for the initial generation of repair Schwann cells after injury. In repair Schwann cells, we find that Schwann cell STAT3 activation by Tyr705 phosphorylation is sustained during long-term denervation. STAT3 is required for maintaining autocrine Schwann cell survival signaling, and inactivation of Schwann cell STAT3 results in a striking loss of repair cells from chronically denervated distal stumps. STAT3 inactivation also results in abnormal morphology of repair cells and regeneration tracks, and failure to sustain expression of repair cell markers, including Shh, GDNF, and BDNF. Because Schwann cell development proceeds normally without STAT3, the function of this factor appears restricted to Schwann cells after injury. This identification of transcriptional mechanisms that support long-term survival and differentiation of repair cells will help identify, and eventually correct, the failures that lead to the deterioration of this important cell population.SIGNIFICANCE STATEMENT Although injured peripheral nerves contain repair Schwann cells that provide signals and spatial clues for promoting regeneration, the clinical outcome after nerve damage is frequently poor. A key reason for this is that, during the slow growth of axons through the proximal parts of injured nerves repair, Schwann cells gradually lose regeneration-supporting features and eventually die. Identification of signals that sustain repair cells is therefore an important goal. We have found that in mice the transcription factor STAT3 protects these cells from death and contributes to maintaining the molecular and morphological repair phenotype that promotes axonal regeneration. Defining the molecular mechanisms that maintain repair Schwann cells is an essential step toward developing therapeutic strategies that improve nerve regeneration and functional recovery.

Photoreceptor phagosome processing defects and disturbed autophagy in retinal pigment epithelium of Cln3Δex1-6 mice modelling juvenile neuronal ceroid lipofuscinosis (Batten disease).

Retinal degeneration and visual impairment are the first signs of juvenile neuronal ceroid lipofuscinosis caused by CLN3 mutations, followed by inevitable progression to blindness. We investigated retinal degeneration in Cln3(Δex1-6) null mice, revealing classic 'fingerprint' lysosomal storage in the retinal pigment epithelium (RPE), replicating the human disease. The lysosomes contain mitochondrial F0-ATP synthase subunit c along with undigested membranes, indicating a reduced degradative capacity. Mature autophagosomes and basal phagolysosomes, the terminal degradative compartments of autophagy and phagocytosis, are also increased in Cln3(Δex1) (-6) RPE, reflecting disruption to these key pathways that underpin the daily phagocytic turnover of photoreceptor outer segments (POS) required for maintenance of vision. The accumulated autophagosomes have post-lysosome fusion morphology, with undigested internal contents visible, while accumulated phagosomes are frequently docked to cathepsin D-positive lysosomes, without mixing of phagosomal and lysosomal contents. This suggests lysosome-processing defects affect both autophagy and phagocytosis, supported by evidence that phagosomes induced in Cln3(Δex1) (-) (6)-derived mouse embryonic fibroblasts have visibly disorganized membranes, unprocessed internal vesicles and membrane contents, in addition to reduced LAMP1 membrane recruitment. We propose that defective lysosomes in Cln3(Δex1) (-) (6) RPE have a reduced degradative capacity that impairs the final steps of the intimately connected autophagic and phagocytic pathways that are responsible for degradation of POS. A build-up of degradative organellar by-products and decreased recycling of cellular materials is likely to disrupt processes vital to maintenance of vision by the RPE.

Immunomodulatory effect of a decellularized skeletal muscle scaffold in a discordant xenotransplantation model.

Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.

c-Jun activation in Schwann cells protects against loss of sensory axons in inherited neuropathy.

Charcot-Marie-Tooth disease type 1A is the most frequent inherited peripheral neuropathy. It is generally due to heterozygous inheritance of a partial chromosomal duplication resulting in over-expression of PMP22. A key feature of Charcot-Marie-Tooth disease type 1A is secondary death of axons. Prevention of axonal loss is therefore an important target of clinical intervention. We have previously identified a signalling mechanism that promotes axon survival and prevents neuron death in mechanically injured peripheral nerves. This work suggested that Schwann cells respond to injury by activating/enhancing trophic support for axons through a mechanism that depends on upregulation of the transcription factor c-Jun in Schwann cells, resulting in the sparing of axons that would otherwise die. As c-Jun orchestrates Schwann cell support for distressed neurons after mechanical injury, we have now asked: do Schwann cells also activate a c-Jun dependent neuron-supportive programme in inherited demyelinating disease? We tested this by using the C3 mouse model of Charcot-Marie-Tooth disease type 1A. In line with our previous findings in humans with Charcot-Marie-Tooth disease type 1A, we found that Schwann cell c-Jun was elevated in (uninjured) nerves of C3 mice. We determined the impact of this c-Jun activation by comparing C3 mice with double mutant mice, namely C3 mice in which c-Jun had been conditionally inactivated in Schwann cells (C3/Schwann cell-c-Jun(-/-) mice), using sensory-motor tests and electrophysiological measurements, and by counting axons in proximal and distal nerves. The results indicate that c-Jun elevation in the Schwann cells of C3 nerves serves to prevent loss of myelinated sensory axons, particularly in distal nerves, improve behavioural symptoms, and preserve F-wave persistence. This suggests that Schwann cells have two contrasting functions in Charcot-Marie-Tooth disease type 1A: on the one hand they are the genetic source of the disease, on the other, they respond to it by mounting a c-Jun-dependent response that significantly reduces its impact. Because axonal death is a central feature of much nerve pathology it will be important to establish whether an axon-supportive Schwann cell response also takes place in other conditions. Amplification of this axon-supportive mechanism constitutes a novel target for clinical intervention that might be useful in Charcot-Marie-Tooth disease type 1A and other neuropathies that involve axon loss.

CD63 is an essential cofactor to leukocyte recruitment by endothelial P-selectin.

The activation of endothelial cells is critical to initiating an inflammatory response. Activation induces the fusion of Weibel-Palade Bodies (WPB) with the plasma membrane, thus transferring P-selectin and VWF to the cell surface, where they act in the recruitment of leukocytes and platelets, respectively. CD63 has long been an established component of WPB, but the functional significance of its presence within an organelle that acts in inflammation and hemostasis was unknown. We find that ablating CD63 expression leads to a loss of P-selectin-dependent function: CD63-deficient HUVECs fail to recruit leukocytes, CD63-deficient mice exhibit a significant reduction in both leukocyte rolling and recruitment and we show a failure of leukocyte extravasation in a peritonitis model. Loss of CD63 has a similar phenotype to loss of P-selectin itself, thus CD63 is an essential cofactor to P-selectin.

Detergent enzymatic treatment for the development of a natural acellular matrix for oesophageal regeneration.

PURPOSE: Tissue engineering of the oesophagus has been proposed as a therapeutic alternative to organ transplantation. We previously demonstrated that a detergent enzymatic treatment (DET) is a valid method to obtain an acellular matrix with preservation of the native architecture. In this study, we aimed to develop a natural acellular matrix from pig oesophagus, as a valid framework for oesophageal replacement. METHODS: Pig oesophagi (n = 4) were decellularized with continuous luminal infusion of DET. To evaluate the efficiency of the decellularization, samples were assessed by histology and DNA quantification. Moreover, the ultra-structural characteristics of the acellular matrix were investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: Decellularization of the oesophagus was achieved after three cycles of DET. Histological analysis showed the maintenance of tissue matrix architecture with absence of cellular elements, verified by measurement of DNA. SEM and TEM analysis confirmed preservation of the ultra-structural characteristics of the native tissue. CONCLUSIONS: Oesophageal acellular matrix can be successfully obtained by decellularization of pig oesophagus using a gentle DET via the oesophageal lumen. This decellularization method preserves the ultrastructure of the native tissue and could represent the basis for a tissue-engineered oesophagus.

SARM1 detection in myelinating glia: sarm1/Sarm1 is dispensable for PNS and CNS myelination in zebrafish and mice.

Since SARM1 mutations have been identified in human neurological disease, SARM1 inhibition has become an attractive therapeutic strategy to preserve axons in a variety of disorders of the peripheral (PNS) and central nervous system (CNS). While SARM1 has been extensively studied in neurons, it remains unknown whether SARM1 is present and functional in myelinating glia? This is an important question to address. Firstly, to identify whether SARM1 dysfunction in other cell types in the nervous system may contribute to neuropathology in SARM1 dependent diseases? Secondly, to ascertain whether therapies altering SARM1 function may have unintended deleterious impacts on PNS or CNS myelination? Surprisingly, we find that oligodendrocytes express sarm1 mRNA in the zebrafish spinal cord and that SARM1 protein is readily detectable in rodent oligodendrocytes in vitro and in vivo. Furthermore, activation of endogenous SARM1 in cultured oligodendrocytes induces rapid cell death. In contrast, in peripheral glia, SARM1 protein is not detectable in Schwann cells and satellite glia in vivo and sarm1/Sarm1 mRNA is detected at very low levels in Schwann cells, in vivo, in zebrafish and mouse. Application of specific SARM1 activators to cultured mouse Schwann cells does not induce cell death and nicotinamide adenine dinucleotide (NAD) levels remain unaltered suggesting Schwann cells likely contain no functionally relevant levels of SARM1. Finally, we address the question of whether SARM1 is required for myelination or myelin maintenance. In the zebrafish and mouse PNS and CNS, we show that SARM1 is not required for initiation of myelination and myelin sheath maintenance is unaffected in the adult mouse nervous system. Thus, strategies to inhibit SARM1 function to treat neurological disease are unlikely to perturb myelination in humans.

Generation of mice with a conditional null Fraser syndrome 1 (Fras1) allele.

Fraser syndrome (FS) is an autosomal recessive disease characterized by skin lesions and kidney and upper airway malformations. Fraser syndrome 1 (FRAS1) is an extracellular matrix protein, and FRAS1 homozygous mutations occur in some FS individuals. FRAS1 is expressed at the epithelial-mesenchymal interface in embryonic skin and kidney. blebbed mice have a null Fras1 mutation and phenocopy human FS. Like humans with FS, they exhibit a high fetal and neonatal mortality, precluding studies of FRAS1 functions in later life. We generated conditional Fras1 null allele mice. Cre-mediated generalized deletion of this allele generated embryonic skin blisters and renal agenesis characteristic of blebbed mice and human FS. Targeted deletion of Fras1 in kidney podocytes circumvented skin blistering, renal agenesis, and early death. FRAS1 expression was downregulated in maturing glomeruli which then became sclerotic. The data are consistent with the hypothesis that locally produced FRAS1 has roles in glomerular maturation and integrity. This conditional allele will facilitate study of possible role for FRAS1 in other tissues such as the skin.

Hypoxia mimetics restore bone biomineralisation in hyperglycaemic environments.

Diabetic patients have an increased risk of fracture and an increased occurrence of impaired fracture healing. Diabetic and hyperglycaemic conditions have been shown to impair the cellular response to hypoxia, via an inhibited hypoxia inducible factor (HIF)-1α pathway. We investigated, using an in vitro hyperglycaemia bone tissue engineering model (and a multidisciplinary bone characterisation approach), the differing effects of glucose levels, hypoxia and chemicals known to stabilise HIF-1α (CoCl2 and DMOG) on bone formation. Hypoxia (1% O2) inhibited bone nodule formation and resulted in discrete biomineralisation as opposed to the mineralised extracellular collagen fibres found in normoxia (20% O2). Unlike hypoxia, the use of hypoxia mimetics did not prevent nodule formation in normal glucose level. Hyperglycaemic conditions (25 mM and 50 mM glucose) inhibited biomineralisation. Interestingly, both hypoxia mimetics (CoCl2 and DMOG) partly restored hyperglycaemia inhibited bone nodule formation. These results highlight the difference in osteoblast responses between hypoxia mimetics and actual hypoxia and suggests a role of HIF-1α stabilisation in bone biomineralisation that extends that of promoting neovascularisation, or other system effects associated with hypoxia and bone regeneration in vivo. This study demonstrates that targeting the HIF pathway may represent a promising strategy for bone regeneration in diabetic patients.

Monitoring tissue engineered constructs and protocols with laboratory-based x-ray phase contrast tomography.

Tissue engineering (TE) aims to generate bioengineered constructs which can offer a surgical treatment for many conditions involving tissue or organ loss. Construct generation must be guided by suitable assessment tools. However, most current tools (e.g. histology) are destructive, which restricts evaluation to a single-2D anatomical plane, and has no potential for assessing constructs prior to or following their implantation. An alternative can be provided by laboratory-based x-ray phase contrast computed tomography (PC-CT), which enables the extraction of 3D density maps of an organ's anatomy. In this work, we developed a semi-automated image processing pipeline dedicated to the analysis of PC-CT slices of oesophageal constructs. Visual and quantitative (density and morphological) information is extracted on a volumetric basis, enabling a comprehensive evaluation of the regenerated constructs. We believe the presented tools can enable the successful regeneration of patient-specific oesophagus, and bring comparable benefit to a wide range of TE applications. STATEMENT OF SIGNIFICANCE: Phase contrast computed tomography (PC-CT) is an imaging modality which generates high resolution volumetric density maps of biological tissue. In this work, we demonstrate the use of PC-CT as a new tool for guiding the progression of an oesophageal tissue engineering (TE) protocol. Specifically, we developed a semi-automated image-processing pipeline which analyses the oesophageal PC-CT slices, extracting visual and quantitative (density and morphological) information. This information was proven key for performing a comprehensive evaluation of the regenerated constructs, and cannot be obtained through existing assessment tools primarily due to their destructive nature (e.g. histology). This work paves the way for using PC-CT in a wide range of TE applications which can be pivotal for unlocking the potential of this field.

Connexin 43 mediates the tangential to radial migratory switch in ventrally derived cortical interneurons.

The adult cerebral cortex is composed of excitatory and inhibitory neurons that arise from progenitor cells in disparate proliferative regions in the developing brain and follow different migratory paths. Excitatory pyramidal neurons originate near the ventricle and migrate radially to their position in the cortical plate along radial glial fibers. On the other hand, inhibitory interneurons arise in the ventral telencephalon and migrate tangentially to enter the developing cortex before migrating radially to reach their correct laminar position. Gap junction adhesion has been shown to play an important mechanistic role in the radial migration of excitatory neurons. We asked whether a similar mechanism governs the tangential or radial migration of inhibitory interneurons. Using short hairpin RNA knockdown of Connexin 43 (Cx43) and Cx26 together with rescue experiments, we found that gap junctions are dispensable for the tangential migration of interneurons, but that Cx43 plays a role in the switch from tangential to radial migration that allows interneurons to enter the cortical plate and find their correct laminar position. Moreover this action is dependent on the adhesive properties and the C terminus of Cx43 but not the Cx43 channel. Thus, the radial phase of interneuron migration resembles that of excitatory neuron migration in terms of dependence on Cx43 adhesion. Furthermore, gap junctions between migrating interneurons and radial processes were observed by electron microscopy. These findings provide mechanistic and structural support for a gap junction-mediated interaction between migrating interneurons and radial glia during the switch from tangential to radial migration.

Cellular internalization of silver nanoparticles in gut epithelia of the estuarine polychaete Nereis diversicolor.

Silver nanoparticles (AgNPs) are widely used which may result in environmental impacts, notably within aquatic ecosystems. As estuarine sediments are sinks for numerous pollutants, but also habitat and food for deposit feeders such as Nereis diversicolor, ingested sediments must be investigated as an important route of uptake for NPs. N. diversicolor were fed sediment spiked with either citrate capped AgNPs (30 ± 5 nm) or aqueous Ag for 10 days. Postexposure AgNPs were observed in the lumen of exposed animals, and three lines of evidence indicated direct internalization of AgNPs into the gut epithelium. With TEM, electron-dense particles resembling AgNPs were observed associated with the apical plasma membrane, in endocytotic pits and in endosomes. Energy dispersive X-ray analysis (EDX) confirmed the presence of Ag in these particles, which were absent in controls. Subcellular fractionation revealed that Ag accumulated from AgNPs was predominantly associated with inorganic granules, organelles, and the heat denatured proteins; whereas dissolved Ag was localized to the metallothionein fraction. Collectively, these results indicate separate routes of cellular internalization and differing in vivo fates of Ag delivered in dissolved and NP form. For AgNPs an endocytotic pathway appears to be a key route of cellular uptake.