Synaptophysin sustains presynaptic performance by preserving vesicular synaptobrevin-II levels.

The two most abundant molecules on synaptic vesicles (SVs) are synaptophysin and synaptobrevin-II (sybII). SybII is essential for SV fusion, whereas synaptophysin is proposed to control the trafficking of sybII after SV fusion and its retrieval during endocytosis. Despite controlling key aspects of sybII packaging into SVs, the absence of synaptophysin results in negligible effects on neurotransmission. We hypothesised that this apparent absence of effect may be because of the abundance of sybII on SVs, with the impact of inefficient sybII retrieval only revealed during periods of repeated SV turnover. To test this hypothesis, we subjected primary cultures of synaptophysin knockout neurons to repeated trains of neuronal activity, while monitoring SV fusion events and levels of vesicular sybII. We identified a significant decrease in both the number of SV fusion events (monitored using the genetically encoded reporter vesicular glutamate transporter-pHluorin) and vesicular sybII levels (via both immunofluorescence and Western blotting) using this protocol. This revealed that synaptophysin is essential to sustain both parameters during periods of repetitive SV turnover. This was confirmed by the rescue of presynaptic performance by the expression of exogenous synaptophysin. Importantly, the expression of exogenous sybII also fully restored SV fusion events in synaptophysin knockout neurons. The ability of additional copies of sybII to fully rescue presynaptic performance in these knockout neurons suggests that the principal role of synaptophysin is to mediate the efficient retrieval of sybII to sustain neurotransmitter release.

In vivo validation of a miniaturized electrochemical oxygen sensor for measuring intestinal oxygen tension.

Recent advances in the fields of electronics and microfabrication techniques have led to the development of implantable medical devices for use within the field of precision medicine. Monitoring visceral surface tissue O2 tension (PTo2) by means of an implantable sensor is potentially useful in many clinical situations, including the perioperative management of patients undergoing intestinal resection and anastomosis. This concept could provide a means by which treatment could be tailored to individual patients. This study describes the in vivo validation of a novel, miniaturized electrochemical O2 sensor to provide real-time data on intestinal PTo2. A single O2 sensor was placed onto the serosal surface of the small intestine of anesthetized rats that were exposed to ischemic (superior mesenteric artery occlusion) and hypoxemic (alterations in inspired fractional O2 concentrations) insults. Control experiments demonstrated that the sensors can function and remain stable in an in vivo environment. Intestinal PTo2 decreased following superior mesenteric artery occlusion and with reductions in inspired O2 concentrations. These results were reversible after reinstating blood flow or by increasing inspired O2 concentrations. We have successfully developed an anesthetized rat intestinal ischemic and hypoxic model for validation of a miniaturized O2 sensor to provide real-time measurement of intestinal PTo2. Our results support further validation of the sensors in physiological conditions using a large animal model to provide evidence of their use in clinical applications where monitoring visceral surface tissue O2 tension is important.NEW & NOTEWORTHY This is the first report of real-time continuous measurements of intestinal oxygen tension made using a microfabricated O2 sensor. Using a developed rodent model, we have validated this sensor's ability to accurately measure dynamic and reversible changes in intestinal oxygenation that occur through ischemic and hypoxemic insults. Continuous monitoring of local intestinal oxygenation could have value in the postoperative monitoring of patients having undergone intestinal surgery.

Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity-dependent bulk endocytosis.

Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. By definition this mode is triggered by neuronal activity; however, key questions regarding its mechanism of activation remain unaddressed. To determine the basic requirements for ADBE triggering in central nerve terminals, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. ADBE was monitored both optically and morphologically by observing uptake of the fluid phase markers tetramethylrhodamine-dextran and horse radish peroxidase respectively. Ablation of SV fusion with tetanus toxin resulted in the arrest of ADBE, but had no effect on other calcium-dependent events such as activity-dependent dynamin I dephosphorylation, indicating that SV exocytosis is necessary for triggering. Furthermore, the calcium chelator EGTA abolished ADBE while leaving SV exocytosis intact, demonstrating that ADBE is triggered by intracellular free calcium increases outside the active zone. Activity-dependent dynamin I dephosphorylation was also arrested in EGTA-treated neurons, consistent with its proposed role in triggering ADBE. Thus, SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient individually to trigger ADBE. Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. To determine the minimal requirements for ADBE triggering, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. We found that SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient to trigger ADBE.

ARF6 directs axon transport and traffic of integrins and regulates axon growth in adult DRG neurons.

Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of α9 integrin promotes axon regeneration, so we have investigated α9ß1 trafficking and transport in axons and at the growth cone. We have previously found that α9 and ß1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied α9 and ß1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of ß1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.

Rac GTPase-activating protein (Rac GAP) α1-Chimaerin undergoes proteasomal degradation and is stabilized by diacylglycerol signaling in neurons.

α1-Chimaerin is a neuron-specific member of the Rho GTPase-activating protein family that selectively inactivates the small GTPase Rac. It is known to regulate the structure of dendrites and dendritic spines. We describe here that under basal conditions α1-chimaerin becomes polyubiquitinated and undergoes rapid proteasomal degradation. This degradation is partly dependent on the N-terminal region that is unique to this isoform. Mimicking diacylglycerol (DAG) signaling with a phorbol ester stabilizes endogenous α1-chimaerin against degradation and causes accumulation of the protein. The stabilization requires phorbol ester binding via the C1 domain of the protein and is independent of PKC activity. In addition, overexpression of a constitutively active Rac1 mutant is sufficient to cause an accumulation of α1-chimaerin through a phospholipase C-dependent mechanism, showing that endogenous DAG signaling can also stabilize the protein. These results suggest that signaling via DAG may regulate the abundance of α1-chimaerin under physiological conditions, providing a new model for understanding how its activity could be controlled.

An Impedance Sensor for Pathologically Relevant Detection of In-Stent Restenosis In Vitro.

Cardiovascular disease (CVD) is the biggest cause of death globally. CVD is caused by atherosclerosis which is the accumulation of fatty deposits, often within the fine arteries of the heart or brain. These blockages reduce blood flow and lead to oxygen starvation (ischemia) which can lead to heart attacks and strokes. To treat blocked arteries an implantable device called a stent re-opens the artery to reinstate blood flow to the organ. The stent itself can become blocked over time by cell growth (intimal hyperplasia) which is characterised by excessive smooth muscle cell proliferation. Sensors based on electrical impedance spectroscopy (EIS) embedded in a stent could detect this re-blocking to allow for early intervention. Using platinum interdigitated electrodes on silicon sensor wafers we were able to co-culture different ratios of mouse smooth muscle cells and mouse endothelial cells on these sensors. This mimics the complex, multicellular environment which a stent is found in vivo when undergoing neo-intimal hyperplasia. Trends in the cell impedances were then characterised using the detection frequency and the gradient of change between populations over time which we termed 'Peak Cumulative Gradients (PCG). PCGs were calculated to successfully discriminate each cell type. This work moves towards a sensor that may help guide clinician's decision-making in a disease that is historically silent and difficult to detect. Clinical Relevance-This moves towards an early warning system for the detection of neo intimal hyperplasia ultimately leading to a reduction in stent complications.

Predicting Cardiovascular Stent Complications Using Self-Reporting Biosensors for Noninvasive Detection of Disease.

Self-reporting implantable medical devices are the future of cardiovascular healthcare. Cardiovascular complications such as blocked arteries that lead to the majority of heart attacks and strokes are frequently treated with inert metal stents that reopen affected vessels. Stents frequently re-block after deployment due to a wound response called in-stent restenosis (ISR). Herein, an implantable miniaturized sensor and telemetry system are developed that can detect this process, discern the different cell types associated with ISR, distinguish sub plaque components as demonstrated with ex vivo samples, and differentiate blood from blood clot, all on a silicon substrate making it suitable for integration onto a vascular stent. This work shows that microfabricated sensors can provide clinically relevant information in settings closer to physiological conditions than previous work with cultured cells.

In vivo application of an implantable tri-anchored methylene blue-based electrochemical pH sensor.

The development of robust implantable sensors is important in the successful advancement of personalised medicine as they have the potential to provide in situ real-time data regarding the status of health and disease and the effectiveness of treatment. Tissue pH is a key physiological parameter and herein, we report the design, fabrication, functionalisation, encapsulation and protection of a miniaturised, self-contained, electrochemical pH sensor system and characterisation of sensor performance. Notably for the first time in this environment the pH sensor was based on a methylene blue redox reporter which showed remarkable robustness, accuracy and sensitivity. This was achieved by encapsulation of a self-assembled monolayer containing methylene blue entrapped within a Nafion layer. Another powerful feature was the incorporation, within the same implanted device, of a fabricated on-chip Ag/AgCl reference electrode - vital in any electrochemical sensor, but often ignored. When utilised in vivo, the sensor allowed accurate tracking of externally induced pH changes within a naturally occurring ovine lung cancer model, and correlated well with single point laboratory measurements made on extracted arterial blood, whilst enabling in vivo time-dependent measurements. The sensors functioned robustly whilst implanted, and maintained in vitro function once extracted and together, these results demonstrate proof-of-concept of the ability to sense real-time intratumoral tissue pH changes in vivo.

Predictive and Diagnostic Biomarkers of Anastomotic Leakage: A Precision Medicine Approach for Colorectal Cancer Patients.

Development of an anastomotic leak (AL) following intestinal surgery for the treatment of colorectal cancers is a life-threatening complication. Failure of the anastomosis to heal correctly can lead to contamination of the abdomen with intestinal contents and the development of peritonitis. The additional care that these patients require is associated with longer hospitalisation stays and increased economic costs. Patients also have higher morbidity and mortality rates and poorer oncological prognosis. Unfortunately, current practices for AL diagnosis are non-specific, which may delay diagnosis and have a negative impact on patient outcome. To overcome these issues, research is continuing to identify AL diagnostic or predictive biomarkers. In this review, we highlight promising candidate biomarkers including ischaemic metabolites, inflammatory markers and bacteria. Although research has focused on the use of blood or peritoneal fluid samples, we describe the use of implantable medical devices that have been designed to measure biomarkers in peri-anastomotic tissue. Biomarkers that can be used in conjunction with clinical status, routine haematological and biochemical analysis and imaging have the potential to help to deliver a precision medicine package that could significantly enhance a patient's post-operative care and improve outcomes. Although no AL biomarker has yet been validated in large-scale clinical trials, there is confidence that personalised medicine, through biomarker analysis, could be realised for colorectal cancer intestinal resection and anastomosis patients in the years to come.

Post-Operative Monitoring of Intestinal Tissue Oxygenation Using an Implantable Microfabricated Oxygen Sensor.

Anastomotic leakage (AL) is a common and dangerous post-operative complication following intestinal resection, causing substantial morbidity and mortality. Ischaemia in the tissue surrounding the anastomosis is a major risk-factor for AL development. Continuous tissue oxygenation monitoring during the post-operative recovery period would provide early and accurate early identification of AL risk. We describe the construction and testing of a miniature implantable electrochemical oxygen sensor that addresses this need. It consisted of an array of platinum microelectrodes, microfabricated on a silicon substrate, with a poly(2-hydroxyethyl methacrylate) hydrogel membrane to protect the sensor surface. The sensor was encapsulated in a biocompatible package with a wired connection to external instrumentation. It gave a sensitive and highly linear response to variations in oxygen partial pressure in vitro, although over time its sensitivity was partially decreased by protein biofouling. Using a pre-clinical in vivo pig model, acute intestinal ischaemia was robustly and accurately detected by the sensor. Graded changes in tissue oxygenation were also measurable, with relative differences detected more accurately than absolute differences. Finally, we demonstrated its suitability for continuous monitoring of tissue oxygenation at a colorectal anastomosis over a period of at least 45 h. This study provides evidence to support the development and use of implantable electrochemical oxygen sensors for post-operative monitoring of anastomosis oxygenation.

Biocompatibility of common implantable sensor materials in a tumor xenograft model.

Real-time monitoring of tumor microenvironment parameters using an implanted biosensor could provide valuable information on the dynamic nature of a tumor's biology and its response to treatment. However, following implantation biosensors may lose functionality due to biofouling caused by the foreign body response (FBR). This study developed a novel tumor xenograft model to evaluate the potential of six biomaterials (silicon dioxide, silicon nitride, Parylene-C, Nafion, biocompatible EPOTEK epoxy resin, and platinum) to trigger a FBR when implanted into a solid tumor. Biomaterials were chosen based on their use in the construction of a novel biosensor, designed to measure spatial and temporal changes in intra-tumoral O2 , and pH. None of the biomaterials had any detrimental effect on tumor growth or body weight of the murine host. Immunohistochemistry showed no significant changes in tumor necrosis, hypoxic cell number, proliferation, apoptosis, immune cell infiltration, or collagen deposition. The absence of biofouling supports the use of these materials in biosensors; future investigations in preclinical cancer models are required, with a view to eventual applications in humans. To our knowledge this is the first documented investigation of the effects of modern biomaterials, used in the production of implantable sensors, on tumor tissue after implantation. © 2018 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc. J Biomed Mater Res Part B, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1620-1633, 2019.

A Novel Translational Ovine Pulmonary Adenocarcinoma Model for Human Lung Cancer.

In vitro cell line and in vivo murine models have historically dominated pre-clinical cancer research. These models can be expensive and time consuming and lead to only a small percentage of anti-cancer drugs gaining a license for human use. Large animal models that reflect human disease have high translational value; these can be used to overcome current pre-clinical research limitations through the integration of drug development techniques with surgical procedures and anesthetic protocols, along with emerging fields such as implantable medical devices. Ovine pulmonary adenocarcinoma (OPA) is a naturally-occurring lung cancer that is caused by the jaagsiekte sheep retrovirus. The disease has similar histological classification and oncogenic pathway activation to that of human lung adenocarcinomas making it a valuable model for studying human lung cancer. Developing OPA models to include techniques used in the treatment of human lung cancer would enhance its translational potential, making it an excellent research tool in assessing cancer therapeutics. In this study we developed a novel OPA model to validate the ability of miniaturized implantable O2 and pH sensors to monitor the tumor microenvironment. Naturally-occurring pre-clinical OPA cases were obtained through an on-farm ultrasound screening programme. Sensors were implanted into OPA tumors of anesthetized sheep using a CT-guided trans-thoracic percutaneous implantation procedure. This study reports the findings from 9 sheep that received sensor implantations. Time taken from initial CT scans to the placement of a single sensor into an OPA tumor was 45 ± 5 min, with all implantations resulting in the successful delivery of sensors into tumors. Immediate post-implantation mild pneumothoraces occurred in 4 sheep, which was successfully managed in all cases. This is, to the best of our knowledge, the first description of the use of naturally-occurring OPA cases as a pre-clinical surgical model. Through the integration of techniques used in the treatment of human lung cancer patients, including ultrasound, general anesthesia, CT and surgery into the OPA model, we have demonstrated its translational potential. Although our research was tailored specifically for the implantation of sensors into lung tumors, we believe the model could also be developed for other pre-clinical applications.

Ovine Pulmonary Adenocarcinoma: A Unique Model to Improve Lung Cancer Research.

Lung cancer represents a major worldwide health concern; although advances in patient management have improved outcomes for some patients, overall 5-year survival rates are only around 15%. In vitro studies and mouse models are commonly used to study lung cancer and their use has increased the molecular understanding of the disease. Unfortunately, mouse models are poor predictors of clinical outcome and seldom mimic advanced stages of the human disease. Animal models that more accurately reflect human disease are required for progress to be made in improving treatment outcomes and prognosis. Similarities in pulmonary anatomy and physiology potentially make sheep better models for studying human lung function and disease. Ovine pulmonary adenocarcinoma (OPA) is a naturally occurring lung cancer that is caused by the jaagsiekte sheep retrovirus. The disease is endemic in many countries throughout the world and has several features in common with human lung adenocarcinomas, including histological classification and activation of common cellular signaling pathways. Here we discuss the in vivo and in vitro OPA models that are currently available and describe the advantages of using pre-clinical naturally occurring OPA cases as a translational animal model for human lung adenocarcinoma. The challenges and options for obtaining these OPA cases for research purposes, along with their use in developing novel techniques for the evaluation of chemotherapeutic agents or for monitoring the tumor microenvironment in response to treatment, are also discussed.

Synaptic Vesicle Recycling Is Unaffected in the Ts65Dn Mouse Model of Down Syndrome.

Down syndrome (DS) is the most common genetic cause of intellectual disability, and arises from trisomy of human chromosome 21. Accumulating evidence from studies of both DS patient tissue and mouse models has suggested that synaptic dysfunction is a key factor in the disorder. The presence of several genes within the DS trisomy that are either directly or indirectly linked to synaptic vesicle (SV) endocytosis suggested that presynaptic dysfunction could underlie some of these synaptic defects. Therefore we determined whether SV recycling was altered in neurons from the Ts65Dn mouse, the best characterised model of DS to date. We found that SV exocytosis, the size of the SV recycling pool, clathrin-mediated endocytosis, activity-dependent bulk endocytosis and SV generation from bulk endosomes were all unaffected by the presence of the Ts65Dn trisomy. These results were obtained using battery of complementary assays employing genetically-encoded fluorescent reporters of SV cargo trafficking, and fluorescent and morphological assays of fluid-phase uptake in primary neuronal culture. The absence of presynaptic dysfunction in central nerve terminals of the Ts65Dn mouse suggests that future research should focus on the established alterations in excitatory / inhibitory balance as a potential route for future pharmacotherapy.

Nanog retrotransposed genes with functionally conserved open reading frames.

The Nanog gene plays a key role in the pluripotency of early embryonic cells in vitro and in vivo. In this article retrotransposed copies of Nanog, termed NanogPc and NanogPd, are identified on mouse Chromosomes 4 and 7, respectively. In contrast to the two previously characterized mouse Nanog retrogenes that contain multiple frameshifts and point mutations, NanogPc and NanogPd are 98% identical to NANOG within the open reading frame and encode proteins with activity in an embryonic stem cell self-renewal assay. Mutations common to all four retrotransposed genes but distinct from Nanog suggest divergence from a common progenitor that appears likely to be Nanog because transcripts derived from Nanog but not from the retrogenes are detected in germ-line cells. The possibility that expression of Nanog could be erroneously attributed to novel cellular sources is suggested by the high homology among Nanog, NanogPc, and NanogPd. Analysis of distinct Mus species suggests that NanogPc and NanogPd arose between divergence of M. caroli and M. spretus and indicates that Nanog retrotransposition events continue to occur at a high frequency, a property likely to extend to other germ-line transcripts.