Centrosome amplification promotes cell invasion via cell-cell contact disruption and Rap-1 activation.

Centrosome amplification (CA) is a prominent feature of human cancers linked to tumorigenesis in vivo. Here, we report mechanistic contributions of CA induction alone to tumour architecture and extracellular matrix (ECM) remodelling. CA induction in non-tumorigenic breast cells MCF10A causes cell migration and invasion, with underlying disruption of epithelial cell-cell junction integrity and dysregulation of expression and subcellular localisation of cell junction proteins. CA also elevates expression of integrin ß-3, its binding partner fibronectin-1 and matrix metalloproteinase enzymes, promoting cell-ECM attachment, ECM degradation, and a migratory and invasive cell phenotype. Using a chicken embryo xenograft model for in vivo validation, we show that CA-induced (+CA) MCF10A cells invade into the chick mesodermal layer, with inflammatory cell infiltration and marked focal reactions between chorioallantoic membrane and cell graft. We also demonstrate a key role of small GTPase Rap-1 signalling through inhibition using GGTI-298, which blocked various CA-induced effects. These insights reveal that in normal cells, CA induction alone (without additional oncogenic alterations) is sufficient to confer early pro-tumorigenic changes within days, acting through Rap-1-dependent signalling to alter cell-cell contacts and ECM disruption.

A method of characterising the complex anatomy of vascular occlusions and 3D printing biomimetic analogues.

Chronic total occlusions (CTOs) occur in approximately 40% of individuals with symptomatic peripheral arterial disease and are indicative of critical limb ischaemia. Currently, few medical devices can effectively treat CTOs long-term, with amputation often required. This is due to a lack of knowledge of CTO anatomy, making device design and testing difficult. This study is a proof-of-concept study, which aimed to develop a workflow for further characterising the complex multi-material anatomy of CTOs and creating 3D models of CTO components, which may be useful in producing a vascular CTO biomimetic for device testing. Here, we establish such a workflow using samples of atheromatous plaques. We focus on a high-resolution, non-destructive microcomputed tomography (µCT) technique which enables visualisation of occlusion anatomy at a greater resolution than computed tomography angiography (CTA), which is the typical modality used for CTO clinical visualisation. Four arteries (n = 2 superficial femoral; n = 2 popliteal) with evidence of atheromatous plaques were cut into 8 cm segments, which were then stained with iodine and scanned at low resolution, with calcified regions rescanned at high resolution. Resulting files were manually segmented to generate 3D models, which were then 3D printed in resin using a stereolithography printer to produce parts suitable for creating a biomimetic. In total, µCT files from three arterial segments (n = 2 high resolution, n = 1 low resolution) were deemed suitably calcified for segmentation, and thus were segmented to produce 3D models. 3D models of the arterial wall, intima and atheromatous calcium deposits from a high-resolution popliteal artery scan were successfully 3D printed at several scales. While this research is at an early stage, it holds great promise. The workflow for segmentation and 3D printing various components of an atheromatous plaque established here is replicable and uses software and equipment which are accessible to research laboratories in both academia and industry. The ability to print detailed models on a desktop 3D printer is unprecedented and can be improved further, which is promising for future development of biomimetics with multi-material detail of both soft tissue and calcified components of a vascular occlusion. Indeed, this workflow provides a solid foundation for future studies of CTO anatomy and the creation of true, multi-material CTO biomimetics. Such biomimetics may enable the development of improved interventional devices, as they would mimic the general in vivo CTO environment. As this method cannot be applied in vivo, we cannot yet produce patient-specific biomimetics, however, these analogues would still be important in device development, which would improve patient outcomes in critical limb ischaemia.

In Vitro Evolution of Listeria monocytogenes Reveals Selective Pressure for Loss of SigB and AgrA Function at Different Incubation Temperatures.

The alternative sigma factor B (σB) contributes to the stress tolerance of the foodborne pathogen Listeria monocytogenes by upregulating the general stress response. We previously showed that σB loss-of-function mutations arise frequently in strains of L. monocytogenes and suggested that mild stresses might favor the selection of such mutations. In this study, we performed in vitro evolution experiments (IVEE) where L. monocytogenes was allowed to evolve over 30 days at elevated (42°C) or lower (30°C) incubation temperatures. Isolates purified throughout the IVEE revealed the emergence of sigB operon mutations at 42°C. However, at 30°C, independent alleles in the agr locus arose, resulting in the inactivation of Agr quorum sensing. Colonies of both sigB mutants and agr mutants exhibited a greyer coloration on 7-days-old agar plates than those of the parental strain. Scanning electron microscopy revealed a more complex colony architecture in the wild type than in the mutant strains. sigB mutant strains outcompeted the parental strain at 42°C but not at 30°C, while agr mutant strains showed a small increase in competitive fitness at 30°C. Analysis of 40,080 L. monocytogenes publicly available genome sequences revealed a high occurrence rate of premature stop codons in both the sigB and agrCA loci. An analysis of a local L. monocytogenes strain collection revealed 5 out of 168 strains carrying agrCA alleles. Our results suggest that the loss of σB or Agr confer an increased competitive fitness in some specific conditions and this likely contributes to the emergence of these alleles in strains of L. monocytogenes. IMPORTANCE To withstand environmental aggressions, L. monocytogenes upregulates a large regulon through the action of the alternative sigma factor B (σB). However, σB becomes detrimental for L. monocytogenes growth under mild stresses, which confer a competitive advantage to σB loss-of-function alleles. Temperatures of 42°C, a mild stress, are often employed in mutagenesis protocols of L. monocytogenes and promote the emergence of σB loss-of-function alleles in the sigB operon. In contrast, lower temperatures of 30°C promote the emergence of Agr loss-of-function alleles, a cell-cell communication mechanism in L. monocytogenes. Our findings demonstrate that loss-of-function alleles emerge spontaneously in laboratory-grown strains. These alleles rise in the population as a consequence of the trade-off between growth and survival imposed by the activation of σB in L. monocytogenes. Additionally, our results demonstrate the importance of identifying unwanted hitchhiker mutations in newly constructed mutant strains.

Anatomy of the mesentery: Current understanding and mechanisms of attachment.

An understanding of the anatomy of the mesentery is necessary to undertake any appraisal of the literature on its development. The mesentery is the collection of tissues that maintain all abdominal digestive organs in position and connection with the rest of the body. Therefore, it is also necessary to detail the exact mechanisms that maintain the mesentery in position. We explore these mechanisms, including the supportive functions of structures such as Toldt's fascia, the peritoneal reflection, and vascular connections, in this article.

NRXN1α+/- is associated with increased excitability in ASD iPSC-derived neurons.

BACKGROUND: NRXN1 deletions are identified as one of major rare risk factors for autism spectrum disorder (ASD) and other neurodevelopmental disorders. ASD has 30% co-morbidity with epilepsy, and the latter is associated with excessive neuronal firing. NRXN1 encodes hundreds of presynaptic neuro-adhesion proteins categorized as NRXN1α/ß/γ. Previous studies on cultured cells show that the short NRXN1ß primarily exerts excitation effect, whereas the long NRXN1α which is more commonly deleted in patients involves in both excitation and inhibition. However, patient-derived models are essential for understanding functional consequences of NRXN1α deletions in human neurons. We recently derived induced pluripotent stem cells (iPSCs) from five controls and three ASD patients carrying NRXN1α+/- and showed increased calcium transients in patient neurons. METHODS: In this study we investigated the electrophysiological properties of iPSC-derived cortical neurons in control and ASD patients carrying NRXN1α+/- using patch clamping. Whole genome RNA sequencing was carried out to further understand the potential underlying molecular mechanism. RESULTS: NRXN1α+/- cortical neurons were shown to display larger sodium currents, higher AP amplitude and accelerated depolarization time. RNASeq analyses revealed transcriptomic changes with significant upregulation glutamatergic synapse and ion channels/transporter activity including voltage-gated potassium channels (GRIN1, GRIN3B, SLC17A6, CACNG3, CACNA1A, SHANK1), which are likely to couple with the increased excitability in NRXN1α+/- cortical neurons. CONCLUSIONS: Together with recent evidence of increased calcium transients, our results showed that human NRXN1α+/- isoform deletions altered neuronal excitability and non-synaptic function, and NRXN1α+/- patient iPSCs may be used as an ASD model for therapeutic development with calcium transients and excitability as readouts.

Neutralisation of SARS-CoV-2 by anatomical embalming solutions.

Teaching and learning anatomy by using human cadaveric specimens has been a foundation of medical and biomedical teaching for hundreds of years. Therefore, the majority of institutions that teach topographical anatomy rely on body donation programmes to provide specimens for both undergraduate and postgraduate teaching of gross anatomy. The COVID-19 pandemic has posed an unprecedented challenge to anatomy teaching because of the suspension of donor acceptance at most institutions. This was largely due to concerns about the potential transmissibility of the SARS-CoV-2 virus and the absence of data about the ability of embalming solutions to neutralise the virus. Twenty embalming solutions commonly used in institutions in the United Kingdom and Ireland were tested for their ability to neutralise SARS-CoV-2, using an established cytotoxicity assay. All embalming solutions tested neutralised SARS-CoV-2, with the majority of solutions being effective at high-working dilutions. These results suggest that successful embalming with the tested solutions can neutralise the SARS-CoV-2 virus, thereby facilitating the safe resumption of body donation programmes and cadaveric anatomy teaching.

A stereological study of developmental changes in hepatocyte ultrastructure of zebrafish (Danio rerio).

Histopathology can reveal toxicant-induced changes in the structure of a tissue or organ. A prerequisite for histopathological studies is a sound knowledge of the morphology of the anatomical structure in the normal or healthy state. Zebrafish larvae can provide a tool for studies focused on hepatotoxicity at early stages of development; therefore, the fine structure of the organ should be well characterised. To date, liver structure at 72 and 120 hr post-fertilisation (hpf) has not been reported in detail and this study aimed to fill this scientific gap. A stereological approach allowed for quantitative description of the liver and revealed ultrastructural alterations occurring with time of development. These included a significant increase in the absolute volume of hepatocytes, mitochondria and rough endoplasmic reticulum (rER) during the period of study. The surface area of rER, and of outer and inner mitochondrial membranes also increased. There was no change in the absolute volume of the nuclei. This study provides a quantitative spatial and temporal framework for future research aiming to detect early developmental changes in the liver.

COVID-19 and anatomy: Stimulus and initial response.

The outbreak of COVID-19, resulting from widespread transmission of the SARS-CoV-2 virus, represents one of the foremost current challenges to societies across the globe, with few areas of life remaining untouched. Here, we detail the immediate impact that COVID-19 has had on the teaching and practice of anatomy, providing specific examples of the varied responses from several UK, Irish and German universities and medical schools. Alongside significant issues for, and suspension of, body donation programmes, the widespread closure of university campuses has led to challenges in delivering anatomy education via online methods, a particular problem for a practical, experience-based subject such as anatomy. We discuss the short-term consequences of COVID-19 for body donation programmes and anatomical education, and highlight issues and challenges that will need to be addressed in the medium to long term in order to restore anatomy education and practice throughout the world.

Novel Pt(IV) Prodrugs Displaying Antimitochondrial Effects.

The design, synthesis, characterization, and biological activity of a series of platinum(IV) prodrugs containing the axial ligand 3-(4-phenylquinazoline-2-carboxamido)propanoate (L3) are reported. L3 is a derivative of the quinazolinecarboxamide class of ligands that binds to the translocator protein (TSPO) at the outer mitochondrial membrane. The cytotoxicities of cis,cis,trans-[Pt(NH3)2Cl2(L3)(OH)] (C-Pt1), cis,cis,trans-[Pt(NH3)2Cl2(L3)(BZ)] (C-Pt2), trans-[Pt(DACH)(OX)(L3)(OH)] (C-Pt3), and trans-[Pt(DACH)(OX)(L3)(BZ)] (C-Pt4) (DACH: R,R-diaminocyclohexane, BZ: benzoate, OX: oxalate) in MCF-7 breast cancer and noncancerous MCF-10A epithelial cells were assessed and compared with those of cisplatin, oxaliplatin, and the free ligand L3. Moreover, the cellular uptake, ROS generation, DNA damage, and the effect on the mitochondrial function, mitochondrial membrane potential, and morphology were investigated. Molecular interactions of L3 in the TSPO binding site were studied using molecular docking. The results showed that complex C-Pt1 is the most effective Pt(IV) complex and exerts a multimodal mechanism involving DNA damage, potent ROS production, loss of the mitochondrial membrane potential, and mitochondrial damage.

Cell viability in three ex vivo rat models of spinal cord injury.

Spinal cord injury (SCI) is a devastating disorder that has a poor prognosis of recovery. Animal models of SCI are useful to understand the pathophysiology of SCI and the potential use of therapeutic strategies for human SCI. Ex vivo models of central nervous system (CNS) trauma, particularly mechanical trauma, have become important tools to complement in vivo models of injury in order to reproduce the sequelae of human CNS injury. Ex vivo organotypic slice cultures (OSCs) provide a reliable model platform for the study of cell dynamics and therapeutic intervention following SCI. In addition, these ex vivo models support the 3R concept of animal use in SCI research - replacement, reduction and refinement. Ex vivo models cannot be used to monitor functional recovery, nor do they have the intact blood supply of the in vivo model systems. However, the ex vivo models appear to reproduce many of the post traumatic events including acute and secondary injury mechanisms. Several well-established OSC models have been developed over the past few years for experimental spinal injuries ex vivo in order to understand the biological response to injury. In this study, we investigated cell viability in three ex vivo OSC models of SCI: stab injury, transection injury and contusion injury. Injury was inflicted in postnatal day 4 rat spinal cord slices. Stab injury was performed using a needle on transverse slices of spinal cord. Transection injury was performed on longitudinal slices of spinal cord using a double blade technique. Contusion injury was performed on longitudinal slices of spinal cord using an Infinite Horizon impactor device. At days 3 and 10 post-injury, viability was measured using dual staining for propidium iodide and fluorescein diacetate. In all ex vivo SCI models, the slices showed more live cells than dead cells over 10 days in culture, with higher cell viability in control slices compared with injured slices. Although no change in cell viability was observed between time-points in stab- and contusion-injured OSCs, a reduction in cell viability was observed over time in transection-injured OSCs. Taken together, ex vivo SCI models are a useful and reliable research tool that reduces the cost and time involved in carrying out animal studies. The use of OSC models provides a simple way to study the cellular consequences following SCI, and they can also be used to investigate potential therapeutics regimes for the treatment of SCI.

Ulk4 deficiency leads to hypomyelination in mice.

Brain nerve fibers are insulated by myelin which is produced by oligodendrocytes. Defects in myelination are increasingly recognized as a common pathology underlying neuropsychiatric and neurodevelopmental disorders, which are associated with deletions of the Unc-51-like kinase 4 (ULK4) gene. Key transcription factors have been identified for oligodendrogenesis, but little is known about their associated regulators. Here we report that Ulk4 acts as a key regulator of myelination. Myelination is reduced by half in the Ulk4tm1a/tm1a hypomorph brain, whereas expression of axonal marker genes Tubb3, Nefh, Nefl and Nefm remains unaltered. Transcriptome analyses reveal that 8 (Gfap, Mbp, Mobp, Plp1, Slc1a2, Ttr, Cnp, Scd2) of the 10 most significantly altered genes in the Ulk4tm1a/tm1a brain are myelination-related. Ulk4 is co-expressed in Olig2+ (pan-oligodendrocyte marker) and CC1+ (mature myelinated oligodendrocyte marker) cells during postnatal development. Major oligodendrogeneic transcription factors, including Olig2, Olig1, Myrf, Sox10, Sox8, Sox6, Sox17, Nkx2-2, Nkx6-2 and Carhsp1, are significantly downregulated in the mutants. mRNA transcripts enriched in oligodendrocyte progenitor cells (OPCs), the newly formed oligodendrocytes (NFOs) and myelinating oligodendrocytes (MOs), are significantly attenuated. Expression of stage-specific oligodendrocyte factors including Cspg4, Sox17, Nfasc, Enpp6, Sirt2, Cnp, Plp1, Mbp, Ugt8, Mag and Mog are markedly decreased. Indirect effects of axon caliber and neuroinflammation may also contribute to the hypomyelination, as Ulk4 mutants display smaller axons and increased neuroinflammation. This is the first evidence demonstrating that ULK4 is a crucial regulator of myelination, and ULK4 may therefore become a novel therapeutic target for hypomyelination diseases.

CEP164-null cells generated by genome editing show a ciliation defect with intact DNA repair capacity.

Primary cilia are microtubule structures that extend from the distal end of the mature, mother centriole. CEP164 is a component of the distal appendages carried by the mother centriole that is required for primary cilium formation. Recent data have implicated CEP164 as a ciliopathy gene and suggest that CEP164 plays some roles in the DNA damage response (DDR). We used reverse genetics to test the role of CEP164 in the DDR. We found that conditional depletion of CEP164 in chicken DT40 cells using an auxin-inducible degron led to no increase in sensitivity to DNA damage induced by ionising or ultraviolet irradiation. Disruption of CEP164 in human retinal pigmented epithelial cells blocked primary cilium formation but did not affect cellular proliferation or cellular responses to ionising or ultraviolet irradiation. Furthermore, we observed no localisation of CEP164 to the nucleus using immunofluorescence microscopy and analysis of multiple tagged forms of CEP164. Our data suggest that CEP164 is not required in the DDR.

Ulk4 Is Essential for Ciliogenesis and CSF Flow.

UNLABELLED: Ciliopathies are an emerging class of devastating disorders with pleiotropic symptoms affecting both the central and peripheral systems and commonly associated with hydrocephalus. Even though ciliary components and three master transcriptional regulators have been identified, little is known about the signaling molecules involved. We previously identified a novel gene, Unc51-like-kinase 4 (ULK4), as a risk factor of neurodevelopmental disorders. Here we took multidisciplinary approaches and uncovered essential roles of Ulk4 in ciliogenesis. We show that Ulk4 is predominantly expressed in the ventricular system, and Ulk4(tm1a/tm1a) ependymal cells display reduced/disorganized cilia with abnormal axonemes. Ulk4(tm1a/tm1a) mice exhibit dysfunctional subcommissural organs, obstructive aqueducts, and impaired CSF flow. Mechanistically, we performed whole-genome RNA sequencing and discovered that Ulk4 regulates the Foxj1 pathway specifically and an array of other ciliogenesis molecules. This is the first evidence demonstrating that ULK4 plays a vital role in ciliogenesis and that deficiency of ULK4 can cause hydrocephalus and ciliopathy-related disorders. SIGNIFICANCE STATEMENT: Ciliopathies are an emerging class of devastating disorders with pleiotropic symptoms affecting both the central and peripheral systems. Ciliopathies are commonly associated with hydrocephalus, and Unc51-like-kinase 4 (Ulk4) has been identified as one of 12 genes causing hydrocephalus in mutants. Here we uncover an essential role of Ulk4 in ciliogenesis. Ulk4 is predominantly expressed in the ventricles, and mutant ependymal cells display reduced/disorganized/nonfunctional motile cilia with abnormal axonemes and impaired CSF flow. Ulk4 modulates expression of the master regulator of ciliogenesis, Foxj1, and other ciliogenesis molecules. This is the first report demonstrating a vital role of Ulk4 in ciliogenesis. ULK4 deficiency may be implicated in human hydrocephalus and other ciliopathy-related disorders.

Quantifying phenotype-environment matching in the protected Kerry spotted slug (Mollusca: Gastropoda) using digital photography: exposure to UV radiation determines cryptic colour morphs.

BACKGROUND: Animal colours and patterns commonly play a role in reducing detection by predators, social signalling or increasing survival in response to some other environmental pressure. Different colour morphs can evolve within populations exposed to different levels of predation or environmental stress and in some cases can arise within the lifetime of an individual as the result of phenotypic plasticity. Skin pigmentation is variable for many terrestrial slugs (Mollusca: Gastropoda), both between and within species. The Kerry spotted slug Geomalacus maculosus Allman, an EU protected species, exhibits two distinct phenotypes: brown individuals occur in forested habitats whereas black animals live in open habitats such as blanket bog. Both colour forms are spotted and each type strongly resembles the substrate of their habitat, suggesting that G. maculosus possesses camouflage. RESULTS: Analysis of digital images of wild slugs demonstrated that each colour morph is strongly and positively correlated with the colour properties of the background in each habitat but not with the substrate of the alternative habitats, suggesting habitat-specific crypsis. Experiments were undertaken on laboratory-reared juvenile slugs to investigate whether ultraviolet (UV) radiation or diet could induce colour change. Exposure to UV radiation induced the black (bog) phenotype whereas slugs reared in darkness did not change colour. Diet had no effect on juvenile colouration. Examination of skin tissue from specimens exposed to either UV or dark treatments demonstrated that UV-exposed slugs had significantly higher concentrations of black pigment in their epithelium. CONCLUSIONS: These results suggest that colour dimorphism in G. maculosus is an example of phenotypic plasticity which is explained by differential exposure to UV radiation. Each resulting colour morph provides incidental camouflage against the different coloured substrate of each habitat. This, to our knowledge, is the first documented example of colour change in response to UV radiation in a terrestrial mollusc. Pigmentation appears to be correlated with a number of behavioural traits in G. maculosus, and we suggest that understanding melanisation in other terrestrial molluscs may be useful in the study of pestiferous and invasive species. The implications of colour change for G. maculosus conservation are also discussed.

An appraisal of the computed axial tomographic appearance of the human mesentery based on mesenteric contiguity from the duodenojejunal flexure to the mesorectal level.

OBJECTIVE: The human mesentery is now regarded as contiguous from the duodenojejunal (DJ) to anorectal level. This interpretation prompts re-appraisal of computed tomography (CT) images of the mesentery. METHODS: A digital model and reference atlas of the mesentery were generated using the full-colour data set of the Visible Human Project (VHP). Seventy one normal abdominal CT images were examined to identify mesenteric regions. CT appearances were correlated with cadaveric and histological appearances at corresponding levels. RESULTS: Ascending, descending and sigmoid mesocolons were identifiable in 75%, 86% and 88% of the CTs, respectively. Flexural contiguity was evident in 66%, 68%, 71% and 80% for the ileocaecal, hepatic, splenic and rectosigmoid flexures, respectively. A posterior mesocolic boundary corresponding to the anterior renal fascia was evident in 40% and 54% of cases on the right and left, respectively. The anterior pararenal space (in front of the boundary) corresponded to the mesocolon. CONCLUSIONS: Using the VHP, a mesenteric digital model and reference atlas were developed. This enabled re-appraisal of CT images of the mesentery, in which contiguous flexural and non-flexural mesenteric regions were repeatedly identifiable. The anterior pararenal space corresponded to the mesocolon. KEY POINTS: The Visible Human Project (VHP) allows direct identification of mesenteric structures. Correlating CT and VHP allows identification of flexural and non-flexural mesenteric components. Radiologic appearance of intraperitoneal structures is assessed, starting from a mesenteric platform.

Non-invasive and label-free detection of oral squamous cell carcinoma using saliva surface-enhanced Raman spectroscopy and multivariate analysis.

Reported here is the application of silver nanoparticle-based surface-enhanced Raman spectroscopy (SERS) as a label-free, non-invasive technique for detection of oral squamous cell cancer (OSCC) using saliva and desquamated oral cells. A total of 180 SERS spectra were acquired from saliva and 120 SERS spectra from oral cells collected from normal healthy individuals and from confirmed oropharyngeal cancer patients. Notable biochemical peaks in the SERS spectra were tentatively assigned to various components. Data were subjected to multivariate statistical techniques including principal component analysis, linear discriminate analysis (PCA-LDA) and logistic regression (LR) revealing a sensitivity of 89% and 68% and a diagnostic accuracy of 73% and 60% for saliva and oral cells, respectively. The results from this study demonstrate the potential of saliva and oral cell SERS combined with PCA-LDA or PCA-LR diagnostic algorithms as a promising clinical adjunct for the non-invasive detection of oral cancer.

Three-Dimensional Microgel Platform for the Production of Cell Factories Tailored for the Nucleus Pulposus.

Intradiscal injection of growth factors or cells has been shown to attenuate symptoms of intervertebral disc degeneration. However, different approaches are needed to overcome limitations such as short-term efficacy and leakage of the injected solutions. The current study aims at creating a platform for the realization of functional cell factories by using in parallel cell delivery and gene therapy approaches. Superfect, a transfecting agent, was used as nonviral gene vector because of its ability to form complexes with plasmid DNA (polyplexes). Polyplexes were loaded into collagen hollow microsphere reservoirs, and their ability to transfect cells was ascertained in vitro. Adipose-derived stem cells were then embedded in three-dimensional (3D) microgels composed of type II collagen/hyaluronan, which mimics the environmental cues typical of the healthy nucleus pulposus. These were functionalized with polyplex-loaded collagen hollow spheres and the secretion of the target protein was assessed quantitatively. Delivery of polyplexes from a reservoir system lowered their toxicity significantly while maintaining high levels of transfection in a monolayer culture. In 3D microgels, lower levels of transfection were observed, however; increasing levels of luciferase were secreted from the microgels over 7 days of culture. These results indicate that 3D microgels, functionalized with polyplex-loaded reservoirs offer a reliable platform for the production of cell factories that are able to manufacture targeted therapeutic proteins for regenerative therapies that have applications in nucleus pulposus repair.

Hyaluronic Acid Based Hydrogels Attenuate Inflammatory Receptors and Neurotrophins in Interleukin-1ß Induced Inflammation Model of Nucleus Pulposus Cells.

Inflammation plays an important role in symptomatic intervertebral disc degeneration and is associated with the production of neurotrophins in sensitizing innervation into the disc. The use of high molecular weight (HMw) hyaluronic acid (HA) hydrogels offers a potential therapeutic biomaterial for nucleus pulposus (NP) regeneration as it exerts an anti-inflammatory effect and provides a microenvironment that is more suitable for NP. Therefore, it was hypothesized that cross-linked HMw HA hydrogels modulate the inflammatory receptor of IL-1R1, MyD88 and neurotrophin expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in an in vitro inflammation model of NP. HA cross-linking was optimized using various concentrations of 4-arm PEG-amine by determination of free carboxyl groups of HA and unreacted free amine groups of PEG-amine. The optimally cross-linked HA hydrogels were characterized for hydrolytic stability, enzymatic degradation and cytotoxicity on NP cells. The therapeutic effect of HA hydrogels was further investigated in IL-1ß induced inflammation on NP cell cultures and the mechanism of HA by examining the expression of cell surface receptor of CD44. Hydrogel was optimally cross-linked at 75 mM PEG, stable in phosphate buffered saline, and showed greater than 40% resistance to enzymatic degradation. No cytotoxic effect of NP cells was observed in the presence of hydrogels for 1, 3, and 7 days. IL-1R1 and MyD88 were significantly suppressed. Additionally, NGF and BDNF mRNA were down-regulated after treatment with cross-linked HA hydrogel. Possible protective mechanism of HA is shown by high expression of CD44 receptor of NP cells after HA treatment in which suggest the binding of HA to CD44 receptor and prevent NP cells from further undergoing inflammation. These results indicate that optimally stabilized cross-linked HMw HA hydrogel has a therapeutic effect in response to inflammation-associated pain and becomes an ideal matrices hydrogel for NP regeneration.

Mesenteric-Based Surgery Exploits Gastrointestinal, Peritoneal, Mesenteric and Fascial Continuity from Duodenojejunal Flexure to the Anorectal Junction--A Review.

INTRODUCTION: It is now well established that mesenteric-based colorectal surgery is associated with superior outcomes. Recent anatomic observations have demonstrated that the mesenteric organ is contiguous from the duodenojejunal to the anorectal junction. This led to similar observations in relation to associated peritoneum and fascia. The aim of this review was to demonstrate the relevance of the contiguity principle to resectional colorectal surgery. METHODS: All literature in relation to mesenteric anatomy was reviewed from 1873 to the present, without language restriction. RESULTS: Mesenteric-based surgery (i.e. complete mesocolic excision, total mesocolic and mesorectal excision) requires division of the peritoneal reflection (i.e. peritonotomy), and mesenteric mobilisation in the mesofascial plane. These are the fundamental technical elements of mesenterectomy. Mesenteric, peritoneal and fascial contiguity mean that in resectional surgery, these technical elements can be reproducibly applied at all levels from the origin at the superior mesenteric root, to the anorectal junction. CONCLUSIONS: The goals of complete mesocolic, total mesocolic and mesorectal excision can be universally achieved at any level from duodenojejunal flexure to anorectal junction, by adopting technical elements based on mesenteric, peritoneal and fascial contiguity.

The mesocolon: a histological and electron microscopic characterization of the mesenteric attachment of the colon prior to and after surgical mobilization.

BACKGROUND: Colonic mobilization requires separation of mesocolon from underlying fascia. Despite the surgical importance of planes formed by these structures, no study has formally characterized their microscopic features. The aim of this study was to determine the histological and electron microscopic appearance of mesocolon, fascia, and retroperitoneum, prior to and after colonic mobilization. METHODS: In 24 cadavers, samples were taken from right, transverse, descending, and sigmoid mesocolon. In 12 cadavers, specimens were stained with hematoxylin and eosin (3 sections) or Masson trichrome (3 sections). In the second 12 cadavers, lymphatic channels were identified by staining immunohistochemically for podoplanin. The ascending mesocolon was assessed with scanning electron microscopy. The above process was first conducted with the mesocolon in situ. The mesocolon was then surgically mobilized, and the process was repeated on remaining structures. RESULTS: The microscopic structure of mesocolon and associated fascia was consistent from ileocecal to mesorectal level. A surface mesothelium and underlying connective tissue were evident throughout. Fibrous septae separated adipocyte lobules. Where apposed to retroperitoneum, 2 mesothelial layers separated mesocolon and underlying retroperitoneum. A connective tissue layer occurred between these (ie, Toldt's fascia). Lymphatic channels were evident both in mesocolic connective tissue and Toldt's fascia. After surgical separation of mesocolon and fascia both remained contiguous, the fascia remained in situ and the retroperitoneum undisturbed. CONCLUSIONS: The findings demonstrate that the contiguous mesocolon and retroperitoneum are separated by mesothelial and connective tissue layers. These properties generate the surgical planes (ie, meso- and retrofascial planes) exploited in colonic and mesocolic mobilization.