Lower than normal levels of oxygen (hypoxia) is a hallmark of all solid tumours rendering them frequently resistant to both radiotherapy and chemotherapy regimes. Furthermore, tumour hypoxia and activation of the hypoxia inducible factor (HIF) transcriptional pathway is associated with poorer prognosis. Driven by both genetic and epigenetic changes, cancer cells do not only survive but thrive in hypoxic conditions. Detailed knowledge of these changes and their functional consequences is of great clinical utility and is already helping to determine phenotypic plasticity, histological tumour grading and overall prognosis and survival stratification in several cancer types. As epigenetic changes - contrary to genetic changes - are potentially reversible, they may prove to be potent therapeutic targets to add to the cancer physicians' armorarium in the future.Here, we review the therapeutic potential of epigenetic modifications  occurring in hypoxia with particular reference to cancer and tumourigenesis. Control of cellular oxygen concentration is under strict regulation as hyperoxia causes damage secondary to reactive oxygen species and hypoxia leads to activation of a diverse array of downstream transcriptional pathways including angiogenesis, glucose metabolism and apoptosis.It is well understood that tumourigenesis is dependent on the development of microvasculature for micronutrient supply and oxygenation. As this vasculature develops in a chaotic way with structural malformations, regions of hypoxia are present within all solid tumours. Normal oxygen tension in healthy tissue is 7% (53 mmHg), levels of oxygenation in tumours may vary from physiological levels (7%) to severe hypoxia < 1%) which is usually found in areas adjacent to necrotic tissue % which i.Cellular hypoxia is toxic, and when severe leads to cell death in both normal and cancerous cells. However, cancer cells have developed mechanisms through (epi)genetic modifications which allow them to survive and in some cases thrive in hypoxic conditions . PreclinThe direct observation of tumour hypoxia through oxygen electrode recording was first made over ten years ago . This stp53 mediated apoptosis [In vitro studies in several cell culture systems have shown that HIF is activated at approximately 5% O2 (40 mmHg) and activity increases with decreasing oxygenation down to 0.2-1.0% (1.6-0.8 mmHg) O2 nearing anoxia [http://www.biocarta.com). As HIF activation leads to the development of tumour vascularisation, it plays a significant role in tumour progression and metastasis. Over-expression of HIF-1α and HIF-2α has been documented in several primary tumours and associated metastases, the degree of expression correlates with angiogenesis, resistance to treatment and overall patient outcome [VHL or through VHL promoter methylation leading to transcriptional repression. Renal cell carcinoma can develop through all of these mechanisms (reviewed in [The hypoxia inducible factor (HIF) pathway is the key regulatory pathway activated in response to tissue hypoxia. In normoxic conditions HIF is degraded through interaction with the Von-Hippel Lindau (VHL) tumour suppressor protein (pVHL) where it becomes polyubiquinated and undergoes proteosomal degradation. The interaction between VHL and HIF is controlled via post-translational prolyl hydroxylation of HIF through prolyl hydroxylases (PHDs). In hypoxic conditions HIF does not bind to VHL due to the action of PHDs and therefore accumulates. HIF-1α then binds to hypoxia response elements (HRE) along with co-factors HIF-1β ), E1A binding protein p300 (EP300), jun proto-oncogene (c-JUN) and cAMP responsive element binding protein (CREB). This leads to the transcription and up-regulation of over 100 genes involved in angiogenesis, glucose metabolism and transportation, erythropoietin production, cellular proliferation, tumour invasion/metastasis and poptosis ,10. Threpoptosis . In vitrg anoxia . See Fig outcome ,3. The Hiewed in ,14). Foriewed in ,3,15. Asiewed in ).Alongside HIF related transcriptional response to tissue hypoxia, there is increasing evidence to support the development of hypoxia mediated epigenetic modifications which lead to further transcriptional changes and chromosomal instability. This is mediated through changes in DNA methylation at promoter regions and satellite repeats , histonePhenotypic transition can also occur as a result of hypoxia, with evidence of epithelial - mesenchymal transition (EMT)  and the It is clear that the physiological response to hypoxia in normal tissues and organs must involve epigenetic modifications which lead to downstream changes in gene expression which have either protective or adaptive functions. The following studies have found evidence of a global increase in DNA methylation following a period of hypoxia with associated changes in transcriptional activation and repressive histone marks.RNA expression array studies on murine placenta identified increased expression of DNA methyltransferase 3B (Dnmt3b), and methyl-CpG binding domain protein 1 (Mbd1) following a 48 hour period of hypoxia . An incret al determined the epigenetic signature of chronic hypoxia in normal prostate cell lines [2 and then 3 weeks at 1% O2 prior to assessment of global DNA methylation, DNMT and histone deacetylase activity. This found a global increase in DNA methylation with concomitant increase in DNMT3B expression and H3K9 histone acetylation. Gene-specific changes in DNA methylation were also observed at several key loci of imprinted genes.Increased expression of DNA methyltransferases during hypoxia in human tissues was confirmed when Watson ll lines . FollowiHypoxia is known to induce alterations in chromatin, including global deacetylation and also changes in histone methylation and acetylation in the promoter regions of hypoxia related genes. Chromatin precipitation analysis has identified a signature of chromatin modifications which is inducedunder hypoxic stress. It has been suggested that this may play a role in gene regulation in proliferating tumour cells undergoing cyclic periods of hypoxia .The jumonji-domain containing dioxygenases (JMJD) group of histone demethylases (JHDMs) have been recently described and are found to be regulated in hypoxic conditions through HIF-1α . Within Although all of these studies have documented a global increase in DNA methylation and histone modifications, which are associated with changes in downstream transcription, there are no studies to date which document the time course or order of these events. It is very likely that different cell and tissue types would require varying periods of hypoxia before "normal" hypoxic cells become "pre-cancerous" or "cancerous" hypoxic cells.The observation of global hypomethylation was first observed in colorectal cancer cell lines in the 1980's . Over thAlu and other short interspersed nuclear elements (SINEs)) is known to contribute to genomic stability, it has been postulated that chronic hypoxia can lead to global hypomethylation and subsequent increase in genomic instability and aneuploidy which is often observed in tumourigenesis. This is supported by two studies [Alu and other SINE repeat regions within the genome, whereas the study of colorectal cancer and melanoma cell lines used HPLC to quantify 5-methylcytosine (5-mC). These findings are the exact opposite of those observed when culturing non-cancerous cells in hypoxic conditions  and may in part be due to a breakdown in the feedback mechanisms which exist to control the degree of HIF activation occurring in cancer cells and not normal cells during hypoxia.There are few studies which have investigated genome-wide DNA methylation changes occuring during hypoxia. Methylation within DNA repeat elements  and histone methyltranferases (HMTs) . Histoneet al used ChIP-chip and RNA expression profiling of HepG2 (hepatocellular cancer cell line) and U87 (glioma cell line) cells grown in normoxic (ambient) and hypoxic (0.5% O2 for 4 hours) conditions to define HIF-1 chromatin binding targets. In total, 377 HIF-1 binding sites activated by hypoxia were identified across the genome. As predicted, many of the genes associated with these binding sites were from biological pathways known to be activated and controlled by HIF. However, enrichment for the 2-oxoglutarate dioxygenase family of enzymes was also observed including the previously described JHDMs. It was further demonstrated that up-regulation of these JHDMs helps to maintain histone homeostasis during hypoxic tumorigenesis [It is increasingly evident that histone modifying enzymes play a key role in hypoxia-related tumourigenesis. Several research groups have demonstrated that hypoxia regulates the activity of histone demethylases . Xia et igenesis . The JHDigenesis . The JHDigenesis ,40.(SETD2), a histone H3 lysine 36 methyltransferase, Jumonji/ARID domain-containing protein 1C (JARID1C), a histone H3 lysine 4 demethylase and ubiquitously transcribed X chromosome tetratricopeptide repeat protein (UTX) a histone H3 lysine 27 demethylase [SETD2 or JARID1C, these mutations are potentially of high relevance in hypoxia since 88% of samples with SETD2 and JARID1C inactivating mutations also harboured mutations in VHL and/or over-expression of egl nine homolog 3 (ELGN3) which is also known as HIF-prolyl hydroxylase 3 (PHD3) [JARID1B, a histone H3 lysine 4 demethylase is found to be up-regulated in prostate cancer [In addition, inactivating mutations in enzymes controlling histone modification have been detected in clear cell renal carcinoma (ccRCC), clear cell ovarian cancer  and pancethylase ,44. Even3 (PHD3) . JARID1Be cancer .RUNX3 through histone H3-lysine 9 dimethylation and decreased H3 acetylation during disease progression has been observed [RUNX3 is also known to be silenced in several cancers through promoter methylation. This study performed on gastric cancer cell lines revealed that following treatment with the histone deacetylase inhibitor trichostatin A and the cytosine-methylation inhibitor 5-aza-2-deoxycytidine, down-regulation of RUNX3 was reversed. Bisulphite-specific PCR demonstrated that promoter region methylation was unchanged in hypoxia, whereas chromatin immunoprecipitation (CHIP) found an increase in the repressive histone mark H3K9me2 and a decrease in the transcriptionally active H3K9 acetylation mark. It was also found that the G9a histone methyltransferase and HDAC1 were up-regulated following hypoxia, alongside diminished nuclear localisation and expression of RUNX3, demonstrating an epigenetic mechanism for the observed decrease in RUNX3 expression.In gastric cancer, a hypoxia-induced epigenetic silencing of the tumour suppressor observed . RUNX3 iHypoxic induction of histone demethylases therefore play a pivotal role in tumourigenesis, one such mechanism being HIF- α mediated induction of JHDMs leading to cancer progression. The role of histone methyltransferases which may oppose the effects of histone demethylases during hypoxia is less well understood. If the effects of these two opposing families of histone modifying enzymes during hypoxia are determined, a vital connection between environmental stressors and epigentically regulated tumourigenesis would be made and furthermore, novel epigenetic cancer therapeutic targets identified.Micro-RNAs (miRNAs) are short (20-24) non-coding nucleotide RNA molecules . They aret al have identified a group of hypoxia regulated miRNAs (HRMs). Several of these miRNAs regulate apoptotic signalling during hypoxic periods. The majority of the identified HRMs were found to be over-expressed in human cancers and were consistently induced in breast and colorectal cancer cell lines during culture in hypoxic conditions [Kulshreshtha nditions ,52. Othenditions  but it iin vitro studies on the lung adenocarcinoma cell line A549 demonstrated that miR-210 expression results in caspase 3/7 activity, followed by mitochondrial dysfunction. In the same study, subunit D of succinate dehydrogenase complex (SDHD) was validated as a miR-210 target [miR-210 is known to be regulated by hypoxia and has found to be a diagnostic marker in pancreatic adenocarcinoma  and prog0 target .et al first demonstrated the impact of the VHL tumour suppressor gene on miRNAs in renal clear cell carcinoma [VHL mutation or VHL promoter methylation), miR-210 expression is significantly increased and potentially targets the iron-sulphur cluster protein (ISCU). ISCU is implicated in the mitochondrial electron transport chain and potentially, in anaerobic respiration of tumours. A further group of miRNAs has been identified which regulate VEGF  [Neal arcinoma . This st20, 321) .The over expression of miR-21 (a HRM) has been implicated in the development of glioblastoma (which is known to have extensive regions of hypoxia and necrosis), and knock-down of this miRNA in glioblastoma cell lines lead to apoptosis, suggesting a role in tumour survival .Although none of the hypoxia regulated miRNAs described above are used routinely for clinical diagnosis and prognosis to date, these biomarkers once validated, are easily accessible from both tissue biopsies, resections and are found in circulating plasma enabling clinical access for both diagnostic and prognostic purposes.VHL in clear cell renal carcinoma in vitro and in vivo through inhibition of DNA methylation in the VHL promoter region [DNA methyltransferase (DMNT) inhibitors have been investigated for some years as a novel class of anti-cancer drug. There is substantial evidence to support the use of the DNMT inhibitors 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine) for certain haematological malignancies, resulting in their approval by the FDA. Following a number of phase I and II trials the evidence for their use in solid tumours is less encouraging. In light of this, these agents have not been trialled with specific reference to tumour hypoxia. Treatment with decitabine has been shown to lead to re-expression of r region . This ler region .HDAC inhibitors are compounds which function through inhibition of deacetylases, they are a diverse group of compounds which are divided in to classes I-IV. HDAC inhibitors do not act solely as inhibitors of HDACs as a large number of non-histone transcription factors and transcriptional co-regulators are known to be modified by acetylation.A clear mechanism of action of these drugs is yet to be elucidated. To date, they have been found to have pro-apoptopic and anti-angiogenic properties. Several HDAC inhibitors are currently under evaluation in clinical trials both a single agents and in combination with chemotherapry and targeted therapy -64. The HIF-1α and HIF-2α require modification by type I/II deacetylases in order to become transcriptionally active, it is through this mechanism that HDAC inhibitors are thought to reduce HIF transcription [et al further demonstrated that the inhibition of HIF-1α and HIF-2α transactivation mediated by HDAC inhibitors was independent of VHL and p53 function. This is a significant finding as it implies that HDAC inhibitors may have a therapeutic effect in VHL deficient tumours . The class III HDAC inhibitor SirtI has also been found to deacetylate HIF-1α and HIF-2α thus repressing HIF activity [Recently it has been shown that the class I and II HDAC inhibitors disrupt HIF both directly through reduction of HIF-1α levels and indirectly through repressing transcription, the exact mechanism of this remains unknown . A numbecription . Fath etactivity .HIF1α induction and inhibited in vitro and in vivo angiogenesis [Sodium butyrate, a novel HDAC inhibitor has been shown to inhibit hypoxia induced ogenesis . The stuet al., demonstrated in prostate and renal cancer cell lines that combination treatment with the mTOR inhibitor rapamycin and the HDAC inhibitor LBH589, significantly reduced HIF-1α protein expression and consequently suppressed tumour induced angiogenesis on comparison with either agent used singularly [Verheul ngularly .The effects of HDAC inhibitors on both HIF degradation and transcription are therapeutically very appealing and further clinical evaluation should be considered trialing other targeted treatments such as mTOR and VEGF inhibitors alongside HDAC inhibition. It should be noted however that long term systemic administration of HDAC inhibitors may lead to downstream effects on erythropoeisis and ischaemia (including coronary ischaemia) through inhibition of the physiological role that HIF plays in erythoropoietin production, and adaptive metabolic changes that occur during hypoxia. Therefore scheduling and timing of these therapies requires robust phase I trial assessment.Through the identification of specific miRNAs involved in tumorigenesis (in both normoxic and hypoxic conditions) it is deemed feasible to inhibit specific miRNAs in the future through anti-miRNA oligonucleotides , locked et al [There is recent evidence supporting the potential use of anti-oxidants and naturally occurring compounds to regulate miRNA expression in cancer cells . Li et aet al  demonstret al ,76, histet al  and NAC The role of miR-210 in relation to tumour hypoxia has been recently reviewed . AlongsiIt is clear that epigenetic changes occurring during hypoxia have a significant downstream effect physiologically in normal tissue, in the transition from normal to cancerous tissue and in the progression of solid tumours. These changes can be used as biomarkers in diagnosis and prognosis, for example the micro-RNA miR-210 in pancreatic and breast cancer.However, in order to fully understand these changes and harness their clinical benefit for diagnostics and therapeutics, several key areas need to be addressed which are discussed below.de novo. What we do not know is whether the global DNA hypomethylation observed in hypoxic cancer tissue is somehow protective enabling survival in hypoxic conditions.In normal tissue global DNA hypermethylation is observed following a period of hypoxia whereas in cancer cell lines global DNA hypomethylation is observed leading to increased genomic instability. This is likely due to decreased DNA methyltransferase activity - both maintenance and It is not known whether a stepwise chain of epigenetic events occurs during hypoxia, ie changes in DNA methylation occurring before histone modifications and alteration in expression of downstream mi-RNAs. If this sequence of events could be determined it might highlight the best diagnostic and therapeutic strategies to employ in different cancer types. In reality it is likely that there is interaction and positive and negative feedback between all epigenetic modifications.HIF transcription and degradation making them promising therapeutic agents for use in vascular tumours and tumours with severe regions of hypoxia. If used in the right clinical setting for selected tumour types this therapeutic intervention could prove to be clinically beneficial. Future therapeutic strategies could include the integration of HDAC inhibition with other targeted therapies such mTOR and VEGF/c-kit/platelet derived growth factor (PDGF) tyrosine kinase inhibitors. Sequencing and duration of treatment with such therapies in combination is yet to be determined.Therapeutically, DNMT inhibitors have not been trialled with respect to tumour hypoxia. However HDAC inhibitors appear to have a direct effect on both With the recent massive expansion of molecular biology tools available for studying the epigenome (both array and sequencing based) , we are The authors declare that they have no competing interests.CT drafted and revised the manuscript, LKES contributed to content relating to miRNAs and HKD contributed to content relating to histone modifications. SB provided guidance for the overall structure and content of the manuscript. All authours read and approved the final manuscript.


B cells and humoral immune responses play an important role in the pathogenesis and diagnosis of multiple sclerosis (MS). A characteristic finding in patients with MS is a polyspecific intrathecal B cell response against neurotropic viruses, specifically against measles virus, rubella virus, and varicella zoster virus, also known as an MRZ reaction (MRZR). Here, we correlated from the routine clinical diagnostics individual IgG antibody indices (AIs) of MRZR with magnetic resonance imaging (MRI) findings in patients with first MS diagnosis.U test) and the sum of lesions on cMRI and sMRI (p = 0.0413). Varicella zoster virus AI also showed a correlation with T2 lesion load on cMRI but did not reach statistical significance (p = 0.2893).MRZR was determined in 68 patients with a clinically isolated syndrome (CIS) or early relapsing-remitting MS (RRMS). Absolute AI values for measles virus, rubella virus, and varicella zoster virus were correlated with T2 lesion load and gadolinium enhancing lesions on cerebral MRI (cMRI) and cMRI combined with spinal MRI (sMRI). Measles virus AI correlated significantly with T2 lesion load on cMRI (p = 0.0312, Mann-Whitney The results confirm MRZR as part of the polyspecific immune reaction in MS with possible prognostic impact on MRI and clinical parameters.Furthermore, the data indicate that intrathecal measles virus IgG production correlates with disease activity on cMRI and sMRI in patients with early MS. Great effort has done into defining prognostic markers in multiple sclerosis (MS) over recent years. To date, a combination of clinical, cerebrospinal fluid (CSF) and MRI variables are used to predict disease course. Research has shown high T2 lesion load on cMRI at diagnosis is associated with a more active disease course CSF is a promising source of biochemical markers in MS, as its compounds are relevant not only in the diagnosis of MS but may also reflect disease activity. Detection of oligoclonal bands in CSF and at least two positive AI against measles virus, rubella virus and varicella zoster virus of ≥1.5 (MRZR), are important markers in the diagnosis of MS. In 80–100% of patients with MS, a polyspecific intrathecal B cell response in the form of a positive MRZR is detectable in CSF and is accepted as being highly specific for this disease In the present study, we correlated from the routine clinical diagnostics absolute AIs for measles virus, rubella virus, and varicella zoster virus (VZV) with magnetic resonance imaging (MRI) parameters of disease activity in patients with a first diagnosis of clinically isolated syndrome (CIS) or early RRMS.61patients with relapsing-remitting MS according to the revised McDonald criteria from 2010 Antibodies against measles virus and VZV were measured in serum and CSF samples using commercial enzyme-linked immunosorbent assays  according to the manufacturer's instructions. Antibodies against rubella virus were also measured by ELISA . A standard curve consisting of serial dilutions of a standard human plasma  was included on each ELISA plate. The same batch of SHP was used for all experiments. Total albumin and IgG concentration in serum (IgG (serum)) and CSF were measured nephelometrically .spec CSF/IgGspec serum)/ = Qspec/QIgG if QIgG<QLim, or AI = Qspec/QLim if QIgG>QLim. QLim was calculated as described previously . AI values ≥1.5 were considered as indicative of an intrathecal virus-specific antibody production.AI values were calculated either with the formula: AI =  axial slices with and without application of gadolinium as well as T2-weighted SE axial slices. Hyperintense lesion on T2-weighted MRI lesions >3 mmStatistical analysis: Statistical significance of differences in absolute AI values between patients with high and low lesion loads in T2-weighted images and patients with and without Gd-enhancing lesions were analysed by Mann-Whitney U test using GraphPad Prism 5 software. P values <0.05 were considered significant.The demographic data of all patients included in this study are shown in But rubella virus AI also showed a correlation with Gd-enhancing lesions on combined cerebral and spinal MRI but did not reach statistical significance (p = 0.0611). Furthermore, none of the other AIs correlated with the presence of Gd-enhancing lesions see .We also performed a Spearman Rank Correlation for single AIs and the number of T2 or Gd-enhancing lesions, which did not yield any statistically significant results see .We investigated the relationship between the levels of the individual IgG AIs that form the MRZ reaction and quantitative MRI outcomes in patients with MS. Higher anti-measles AI levels were associated with a higher T2 lesion load in cMRI and sum of T2 lesions in cerebral and spinal MRI. There was no link between individual MRZ-IgG AIs and Gd-enhancing lesions as a marker of acute inflammation on cMRI.MRZ reaction in CSF is positive in chronic CNS autoimmune diseases (e.g. neurolupus or CNS-vasculitis), but has been proposed as a specific marker for autoimmunity in MS On the other hand MRI T2 lesion load has an important clinical impact in diagnosis of MS and therapeutic decisions. The number of lesions in CIS correlates with the risk of conversion to clinically definite MS and is used to monitor disease activity In conclusion, our data suggest that measles AIs correlate with MRI markers in early MS and increased measles AI levels are a marker of increased disease activity. The study underlines the relevance of humoral immunity in MS pathogenesis and supports MRZR as an important tool not only in MS diagnosis but also to estimate disease activity. Additional longitudinal studies are needed to investigate the relationship between MRZ AIs and MRI parameters on one hand and clinical aspects like neurocognitive function and disability progression on the other.Figure S1Measle virus IgG AI and T2 lesion load in MRI. Correlation between number of T2 lesions in cerebral MRI (A), including 67 patients, and sum of lesions in cerebral and spinal MRI (sMRI) (B) from 43 patients with IgG AI for measles virus.(TIF)Click here for additional data file.Figure S2Correlation between symptom duration and total intrathecal IgG synthesis. Correlation of time between first symptoms (FS) and lumbal puncture (LP) and total intrathecal IgG synthesis from 65 patients.(TIF)Click here for additional data file.Figure S3Total intrathecal IgG and T2 and Gd-enhancing lesion load in MRI. Comparison of mean total intrathecal IgG for patients with <6 lesions and ≥6 lesions in cMRI (A) and both cMRI and sMRT (B). Comparison of mean total intrathecal IgG for patients with Gd-enhancing and no Gd-enhancing lesions in cMRI (C) and both cMRI and sMRI (D).(TIF)Click here for additional data file.


Primary open angle glaucoma affects more than 67 million people. Elevated intraocular pressure (IOP) is a risk factor for glaucoma and may reduce nutrient availability by decreasing ocular perfusion pressure (OPP). An interaction between arterial blood pressure and IOP determines OPP; but the exact contribution that these factors have for retinal function is not fully understood. Here we sought to determine how acute modifications of arterial pressure will affect the susceptibility of neuronal function and blood flow to IOP challenge. Anaesthetized (ketamine:xylazine) Long-Evan rats with low , moderate , or high levels  of mean arterial pressure  were subjected to IOP challenge . Electroretinograms were measured at each IOP step to assess bipolar cell (b-wave) and inner retinal function (scotopic threshold response or STR). Ocular blood flow was measured using laser-Doppler flowmetry in groups with similar MAP level and the same IOP challenge protocol. Both b-wave and STR amplitudes decreased with IOP elevation. Retinal function was less susceptible to IOP challenge when MAP was high, whereas the converse was true for low MAP. Consistent with the effects on retinal function, higher IOP was needed to attenuated ocular blood flow in animals with higher MAP. The susceptibility of retinal function to IOP challenge can be ameliorated by acute high BP, and exacerbated by low BP. This is partially mediated by modifications in ocular blood flow. Glaucoma is the second leading cause of blindness in those of working age and is characterized by a progressive death of the cells that make up the optic nerve Several studies suggest that blood pressure plays an important role in glaucoma. More specifically, nocturnal hypotension may exacerbate the progression of visual field loss in patients with glaucoma Given the above, a better understanding of how IOP and BP interact to influence retinal function is needed. An acute experimental approach that affords accurate simultaneous control of both IOP and BP is useful as it avoids long term cardiovascular confounds. Few studies have measured retinal function during simultaneous manipulation of IOP and BP ad libitum. All experimental procedures were conducted under general anaesthesia . As ketamine is known to affect blood pressure in rodents All experimental procedures were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the NHMRC Australian Code of Practice for the care and use of animals for scientific purposes. Animal ethics approval was obtained from the Animal Ethics Committee, Science Faculty, University of Melbourne. Animals used in this study were male Long-Evans rats . Rats were housed in a 12-hour light (50 lux) /12-hour dark environment with normal rat chow and water available To assess ocular susceptibility across a gradient of OPPs, animals underwent stepwise IOP elevation from 10 to 120 mmHg (increment of 5 mmHg for 3 min) while MAP was held stable at low, moderate or high level . Each BPBlood pressure was monitored via an indwelling cannula in the left femoral artery. Blood pressure was modulated by pharmacological intervention. Blood pressure reduction and elevation was achieved by variable infusion into the femoral vein of sodium nitroprusside  and angiotensin II , respectively.To cannulate the femoral arteries and veins, the left groin area was shaved and disinfected with 70% ethanol. The left femoral artery and vein were exposed with a 2 cm skin incision and separated from the surrounding connective tissue by blunt dissection. A small incision was made through the arterial wall. A heparinised polyethylene cannula (inner and outer diameter 0.28 and 0.61 mm respectively) was inserted 3 cm proximally into the femoral artery. The exterior end of the cannula was connected to a pressure transducer  to give direct and continuous BP monitoring.Using the same method, the femoral vein was also cannulated, which allowed systemic BP to be maintained at low, moderate and high levels by infusion of SNP , normal saline (5 µl/min) or AngII , respectively. The rate of infusion was constantly adjusted within these ranges using an electronic syringe pump  to produce a stable BP level during the simultaneous stepwise IOP elevation . The aveWhile one eye underwent IOP challenge, the fellow eye served as control with IOP set manometrically to 10 mmHg via vitreal chamber cannulation. Retinal function was found to remain unchanged in these sham control eyes . This suIn this study, OPP is calculated as the difference between mean arterial pressure and IOP (OPP = MAP−IOP), which differs slightly from the relationship used in humans (OPP = 2/3 MAP−IOP). The ‘2/3’ MAP adjustment factor is only applicable to primates in an upright position and therefore not incorporated here, as rat OPP is measured in a supine position. This estimate of OPP for rats is consistent with previous estimates from pigs To quantify retinal susceptibility to IOP challenge, scotopic electroretinograms (ERG) were recorded at each IOP step. In rodents, the scotopic b-wave reflects rod bipolar cell function −2 was presented via a Ganzfeld integrating sphere , which elicited the b-wave and the STR (STR averaged over 20 repeats), respectively. Signals were digitized at 4 kHz with ×1000 amplification and a band-pass of 0.3 to 1000 Hz (−3 dB). Both b-wave and STR were quantified in terms of the peak-to-trough amplitude.Prior to functional measurement, animals were dark-adapted overnight (12 hours). To maintain dark-adaptation, all procedures including simultaneous acute IOP elevation and BP manipulations were performed in darkness with the aid of night vision goggles  and an infra-red light source . Anaesthetized rats were placed on a heated platform. The active electrode (custom-made chlorided silver electrode) was placed on the cornea, and the ring-shaped reference electrode positioned around the limbus. The ground electrode was inserted subcutaneously in the tail. At each IOP step, a pair of stimuli of −1.12 and −5.25 log cd.s.mLaser-Doppler flowmetry  was employed to measure ocular blood flow by inserting an invasive needle probe  into the vitreal chamber. The working principle of LDF and its applications in the eye are detailed elsewhere For any given eye, the LDF provides a reliable intra-subject comparison and is excellent for continuous blood flow monitoring, which makes it ideal for assessing a relative change in response to IOP challenge 50 represents the IOP for a 50% reduction in retinal function, which provides a measure of susceptibility to IOP elevation. To consider the association between blood pressure and function or blood flow susceptibility to IOP challenge, the correlation between MAP and IOP50 was examined with a Deming regression, which is a robust method of regression that accounts for the variability in both axes (MAP and IOP50). Equation 1 was also fitted to relative retinal function plotted against OPP, where OPP50 is the index of susceptibility.All group data were expressed as mean ± SEM. At baseline, ERG and LDF measurements were found to be normally distributed  with equal variance. ERG amplitudes were also expressed relative to baseline (IOP of 10 mmHg). The relationship between IOP and relative retinal function (b-wave and STR amplitude) was described using a cumulative normal function The same control animal  shown in per se (AngII and SNP) had any detectable effect on the ERG. Therefore b-wave and STR amplitude were normalized to their own baseline  should be the same for all animals regardless of blood pressure. However, this was not the case. The presence of a significant positive correlation between OPP50 and MAP  substantially influences retinal function. In animals with low BP, milder IOP elevations were needed to compromise retinal function compared with controls. In contrast, animals with an acute increase in BP endured a greater IOP challenge before retinal dysfunction occurred . These r50) is linearly related to BP .In addition, the current study extends previous work ed to BP . However and MAP  would haThat IOP is more important than BP in determining retinal function was further illustrated in Measurement of ocular blood flow by laser Doppler indicates that the IOP-flow relationship was rightward shifted with increasing BP , indicatAs it is technically difficult to monitor retinal function and blood flow simultaneously whilst concurrently manipulating IOP and BP, we adopt an alternate approach. We measured these outcomes in separate groups of animals under closely matched experimental conditions  and vitreal chamber (B) show a strong and similar linear relationship with IOP elevation induced via anterior chamber cannulation.(TIF)Click here for additional data file.Figure S2Effect of SNP, saline and AngII on retinal function. Relative retinal function remained stable in the sham control eye (IOP = 10 mmHg) during continuous infusion of SNP (A & D), saline (B & E) or AngII (C & F) to sustain low, moderate or high blood pressure. Time “0” represents the beginning of stepwise IOP elevation in the fellow eye. A, B & C: relative b-wave amplitude; D, E & F: relative STR amplitude; Shaded area: 95% confidence intervals for b-wave and STR amplitudes at time “0”.(TIF)Click here for additional data file.Figure S32 breathing on ocular blood flow. Backscatter (A), MAP (B) and ocular blood flow (C) were measured before, during and after100% oxygen breathing.Effect of 100% O Error bars: SEM; n = 6. Shaded area: duration (2 minutes) of 100% oxygen administration. Dashed line: baseline blood flow (100%).(TIF)Click here for additional data file.


Mycobacterium tuberculosis (Mtb) is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. 13C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb's central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt) Mtb. When both glucokinase genes, ppgK and glkA, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose in vivo and relies on glucose phosphorylation to survive during chronic mouse infections. Mycobacterium tuberculosis (Mtb) will benefit from a better understanding of the mechanisms by which this pathogen establishes and maintains chronic infections. Mtb has to adapt its metabolic needs to the nutritional environment in the host. We investigated the role of glucose phosphorylation and discovered that Mtb expresses two functional glucokinases. Using 13C-tracing experiments we demonstrated that both enzymes are competent to incorporate glucose into central carbon metabolism. In agreement with the view that Mtb metabolizes fatty acids to grow in vivo, both enzymes were dispensable for Mtb replication in mouse lungs and spleens. Surprisingly, however, the glucokinase double mutant was attenuated during the chronic phase of mouse infections. These studies suggest that Mtb metabolizes glucose in vivo and that its survival in chronically infected mice depends on glucose phosphorylation.The development of new drugs targeting  Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a disease that killed 1.5 million people in 2010 worldwide in vivo growth and persistence of Mtb remains uncertain. Mtb possesses carbohydrate transporters and the enzymes required to metabolize sugars in vivo. Mtb is not thought to encounter a sugar-rich environment inside the host, given that expression of sugar catabolism genes was not induced during infection of macrophages and mice ppgK (Rv2702) coding for a polyphosphate glucokinase ppgK deletion mutant in Mtb H37Rv and found that it was severely attenuated for growth with glucose. The mutant, however, retained the ability to grow slowly with glucose as sole carbon source and to incorporate glucose into central carbon metabolism. This led to the identification of a second glucokinase, GLKA, encoded by Rv0650. We demonstrate that both PPGK and GLKA are functional glucokinases that allow Mtb to metabolize glucose. Glucose-6-phosphate production by GLKA was, however, low and not sufficient for normal growth of Mtb on glucose. Thus, Mtb primarily relies on PPGK activity for growth on glucose as the sole carbon source in vitro. Mtb missing both PPGK and GLKA failed to persist normally in mouse lungs suggesting that glucose is available to Mtb in vivo and that Mtb depends on glucose phosphorylation to survive during chronic mouse infection.We sought to investigate the role of glucose metabolism for Mtb pathogenesis and approached this by studying glucokinase, the enzyme catalyzing the phosphorylation of glucose to produce glucose-6-phosphate. This phosphorylation reaction constitutes the first committed step in glucose metabolism activating glucose for oxidation through glycolysis. The Mtb genome contains ppgK deletion mutant (ppgK) was severely attenuated compared to wt with glucose as the sole carbon source  and triose-P), pentose phosphate pathway (pentose-P) and the tricaboxylic acid (TCA) cycle . As previously described 13C-glucose-derived carbons into all the examined central carbon metabolism intermediates mediates . Incorpomediates . Express13C glucose into intermediates of central carbon metabolism was reduced but not abolished in ΔglkA compared to wt (13C glucose incorporation when Mtb was missing PPGK (13C-glucose-derived carbon into intermediates of glycolysis (hexose-P and triose-P) and the pentose phosphate pathway (pentose-P) was significantly reduced in Δppgk and we could not detect 13C incorporation into the TCA cycle metabolites α-ketoglutarate, malate or aspartate . This phenotype was fully complemented when ppgK expression was restored using an integrative plasmid indicating that the impaired survival after exposure to H2O2 was solely due to loss of glucokinase activity. This hypersusceptibility was not due inability to induce expression of genes involved resistance to oxidative stress . Thus, removal of oxidative species production by the host NADPH oxidase bypassed the need of catalase for Mtb pathogenesis. To determine if attenuation of ΔppgKΔglkA was dependent on the host respiratory burst we infected gp91Phox−/− mice. Similar to the phenotype in wt mice, viability of ΔppgKΔglkA decreased by 1 log between days 21 and 56 in gp91Phox−/− mouse lungs  demonstrating that loss of glucokinases does not sensitize Mtb to all forms of oxidative stress. The reason for the specific hypersusceptibility to hydrogen peroxide is unclear.We assessed if the persistence defect was associated with increased susceptibility of ΔBacteroides fragilis has been shown to phosphorylate several substrates including glucose, mannose and N-acetyl-D-glucosamine (NAG) in vivo attenuation of ΔppgKΔglkA to loss of NAG kinase activity, both enzymes would need to be able to phosphorylate NAG, which seems unlikely, given the high affinity of PPGK for glucose ppgK and glkA affected permeability of the mycobacterial cell envelope.We cannot exclude that Mtb's glucokinases phosphorylate an alternative substrate. Broad-specificity hexokinases have mostly been identified in Archea and eukaryotes and are less common in eubacteria Glucose-6-P has many important cellular functions such as energy production through glycolysis, production of reducing power through the pentose phosphate pathway, and it serves as an important precursor for anabolic processes, especially cell wall and nucleotide biosynthesis. Gluconeogenesis could potentially produce the necessary glucose-6-P to meet cellular demands. However, glucose-6-P generation directly from glucose via glucokinase consumes one molecule of ATP while its production through gluconeogenesis is more energetically costly. Exposure to diverse stresses associated with the host immune response might compromise ATP production in Mtb through the electron transport chain, such that ATP production through substrate level phosphorylation using glucose-6-P as a precursor might become important.M. smegmatis and Mtb contains a homolog The glucokinase mutants replicated normally with trehalose as sole carbon source indicating that trehalose metabolism does not depend on trehalase. Trehalase hydrolyzes trehalose into two molecules of glucose, while trehalose phosphorylase catalyzes the phosphorolytic cleavage of trehalose into glucose1-P and glucose M. smegmatis which was predicted to encode 28 carbohydrate transporters, only five such transporters were identified in the Mtb genome How Mtb takes up glucose remains to be determined. In contrast to in vivo attenuation of Mtb lacking both glucokinases, PPGK and GLKA, suggests that Mtb has access to glucose and relies on glucose phosphorylation to survive during chronic infection. While the physiological function of the generated glucose-6-P remains unclear, the impact of loss of glucokinase activity emphasizes that Mtb's metabolic flexibility is crucial to its pathogenesis and that the pathogen does not exclusively rely on metabolism of host lipids.Our work identified a previously unrecognized role for glucose phosphorylation in Mtb pathogenesis. Recent work showed that Mtb is metabolically flexible and capable of utilizing multiple carbon substrates simultaneously All procedures including animal studies were conducted following the National Institutes of Health guidelines for housing and care of laboratory animals and performed in accordance with institutional regulations after protocol review and approval by the Institutional Animal Care and Use Committee of Weill Cornell Medical College .M. tuberculosis (Erdman) strains were routinely grown at 37°C in liquid Middelbrook 7H9 medium (Difco) containing 0.2% glycerol, 0.5% bovine serum albumin, 0.2% dextrose, 0.085% NaCl, and 0.05% Tween 80 or on Middlebrook 7H10 agar plates containing 10% OADC supplement (Becton Dickinson) and 0.5% glycerol. For growth in carbon defined media, we used 7H9 with 0.05% Tyloxapol and a carbon substrate at the indicated concentration (% wt/vol). When required, hygromycin B was used at a concentration of 50 µg/ml and kanamycin at a concentration of 25 µg/ml.ppgK genome by expressing the Cre recombinase from an unstable plasmid. The glkA gene was subsequently deleted in the ΔppgK strain that was cured of both the hygromycing cassette and the Cre expression plasmid. All Mtb knockout strains were confirmed using Southern Blot Analysis. Complementation plasmids were constructed using the Gateway Cloning Technology (Invitrogen) as described previously ppgK gene was cloned containing its putative promoter (350 nucleotides upstream of the start codon) into a plasmid that integrates into the chromosomal attB site. To complement ΔppgK with glkA, the glkA gene was cloned into an episomal plasmid behind the strong mycobacterial promoter PsmycMtb knockout strains were constructed via allelic exchange using the conditionally replicating mycobacteriophage method as described elsewhere 10 dynamic range. Detected ions were deemed metabolites on the basis of unique accurate mass-retention time (AMRT) identifiers for masses exhibiting the expected distribution of accompanying isotopomers. Metabolite identities were established by querying against a pre-populated AMRT library of metabolite standards and demonstrating chromatographic co-elution of candidate metabolites with pure chemical standards spiked into representative biological samples.Metabolites were separated on a Cogent Diamond Hydride Type C column (Microsolve Technologies). The mass spectrometer used was Agilent Accurate Mass 6220 TOF coupled to an Agilent 1200 LC system. Dynamic mass axis calibration was achieved by continuous infusion of a reference mass solution using an isocratic pump with a 100∶1 splitter. This configuration achieved mass errors of approximately 5 parts-per-million (ppm), mass resolution ranging from 10,000–25,000 (over m/z 121–955 amu), and 5 log13C species (i. e. [M+1] and [M+2]). The relative abundance of each isotopically labeled species was determined by dividing the peak height ion intensity of each isotopic form  by the summed peak height ion intensity of all labeled species.The extent of isotopic labeling for each metabolite was determined by dividing the summed peak height ion intensities of all labeled species by the ion intensity of both labeled and unlabeled species, expressed in percent. Label-specific ion counts were corrected for naturally occurring phox−/− mice (Jackson Laboratory) were infected with Mtb by aerosol as described Female 7–8 week old C57BL/6 and gp916 bacteria in PBS+0.5% Tyloxapol or in Sauton's base medium  supplemented with 0.4% glucose or no carbon. Bacterial survival in these media was determined by periodically plating serial dilutions on 7H10 plates to estimate CFU/ml. Susceptibility to nitrosative, oxidative stress and low pH was determined as described previously Starvation survival experiments were carried by inoculating ∼5×10Figure S1Southern blot analysis of glucokinase mutants. (A) Schematic representation of the ppgK genomic region in wt, ΔppgK and ΔppgKΔglkA, in which the hygromycin cassette was removed. (B) Southern blot of chromosomal DNA digested with PvuI+PvuII from wt (lane1), ΔppgK (lane 2) and ΔppgKΔglkA (lane 3). (C) Schematic representation of glkA genomic region in wt and ΔglkA. (D) Southern blot of chromosomal DNA digested with SacI from wt (lane 1), ΔglkA (lane 2) and ΔppgKΔglkA (lane 3).(PDF)Click here for additional data file.Figure S2Comparison of Mtb's glucokinases, GLKA and PPGK. (A) Amino acid sequence alignment of PPGK and GLKA. The overall sequence identity is 22%. (B) Schematic representation of PPGK and GLKA. Residues involved in glucose binding are indicated in red with corresponding residue number shown below. Grey boxes represent ATP binding domains and the catalytic aspartate residues are marked with an asterisk.(PDF)Click here for additional data file.Figure S3Quantification of ppgK and glkA (Rv0650) transcript levels in wt Mtb and mutants. Absolute mRNA amounts were determined by quantitative real time PCR. Serial dilutions of defined copy numbers of the Mtb chromosome were included for each real-time PCR to generate standard curves, which were used to calculate the absolute copy number of each gene. Data are means from 3 independent replicates ± SD.(TIFF)Click here for additional data file.Figure S4KatG and SodA amounts in cell extracts. KatG and SodA were detected by immunoblotting in lysates from wt, ΔppgK, ΔglkA and ΔppgKΔglkA.(TIFF)Click here for additional data file.Table S1Enzymatic quantification of glucose-6-P and fructose-6-P metabolite pools in cell extracts from wt and glucokinase mutants. Metabolite amounts were measured as described in (PDF)Click here for additional data file.Table S2Expression of antioxidant genes in wt and ΔppgKΔglkA. mRNA amounts were quantified by quantitative real time PCR before and after exposure to 5 mM H2O2 for 30 min and normalized to sigA. Data represent the mean of three experimental replicates ± SD.(PDF)Click here for additional data file.


Direct electrode/neuron interfacing is a key challenge to achieve high resolution of neuronal stimulation required for visual prostheses. Neuronal interfacing on biomaterials commonly requires the presence of glial cells and/or protein coating. Nanocrystalline diamond is a highly mechanically stable biomaterial with a remarkably large potential window for the electrical stimulation of tissues. Using adult retinal cell cultures from rats, we found that glial cells and retinal neurons grew equally well on glass and nanocrystalline diamond. The use of a protein coating increased cell survival, particularly for glial cells. However, bipolar neurons appeared to grow even in direct contact with bare diamond. We investigated whether the presence of glial cells contributed to this direct neuron/diamond interface, by using purified adult retinal ganglion cells to seed diamond and glass surfaces with and without protein coatings. Surprisingly, these fully differentiated spiking neurons survived better on nanocrystalline diamond without any protein coating. This greater survival was indicated by larger cell numbers and the presence of longer neurites. When a protein pattern was drawn on diamond, neurons did not grow preferentially on the coated area, by contrast to their behavior on a patterned glass. This study highlights the interesting biocompatibility properties of nanocrystalline diamond, allowing direct neuronal interfacing, whereas a protein coating was required for glial cell growth. The development of neuroprostheses and brain-machine interfaces has increased exponentially following the success of cochlear implants and deep brain stimulation for Parkinsonian patients in vivo applications, due to the risk of mobility within the body. Finally, the presence of glial cells in embryonic and stem cell cultures complicates the assessment of direct diamond/neuron interfacing.Alternatively, resolution could be achieved by direct neuron/material interfacing. Indeed, the implant is often in contact with a surface glial layer, as in the retinal implants described above. Penetrating electrodes have been used in attempts to resolve this problem, but the glial cells grow around them In this study, we addressed the biocompatibility of nanocrystalline diamond layers, using retinal cells or purified RGC neurons from both adult and postnatal animals.2SO4/KNO3).Nanocrystalline diamond films were produced as previously described A UV-Vis Spectroscopic Ellipsometer , at a fixed angle of 70°, was used to characterize the roughness and thickness of the diamond films before and after incubation  with pure deionized water or water containing 0.1 mg/ml poly-D-lysine (PDL) (Sigma-Aldrich). The films were then rinsed in water and dried under argon. We used a Tauc-Laurentz dispersion law 7/2 peak located at 83.6 eV. A curve fitting procedure was applied to smoothed spectra, to extract the components in the N1s spectrum, using Voigt functions with Lorentzian half-widths of 0.2 eV and 0.6 eV.In X-ray photoelectron spectroscopy (XPS), binding energies were expressed relative to the Au4f2 sedation and cervical elongation, and all efforts were made to minimize suffering.All experiments were carried out in accordance with European Community Council Directives (86/609/EEC) and with the ARVO  statement for the use of animals in ophthalmic and visual research. Ethics committee approval is not required for cell culture experiments, as stated in European Union Directives (2010/63/UE). Animals were killed by CO2, 45 min) then laminin (1 μg/cm2 overnight) (Sigma-Aldrich). For protein patterns, microstamps were produced by photolithography and molding Mixed retinal cell cultures and purified retinal ganglion cells (RGCs) were prepared from adult and postnatal Long Evans rats , as previously described 5 cells/cm2 in Neurobasal-A medium containing L-glutamine (2 mM) and B27 (2%) (Life Technologies). Pure adult and postnatal RGCs were used to seed substrates at a density of 2×104 cells/cm2 in the same NBA+ medium or a ND-G medium, respectively. Cell viability was indicated by calcein green fluorescence following incubation with the “live-dead” test kit (Life Technologies) in phosphate buffer saline  for one hour in a humidified chamber . RGC survival was calculated by normalizing the density of calcein AM-positive cells at 6 days in vitro (DIV) by that measured at 1 DIV in the same experiment. Neurite lengths and colocalization on the protein patterns were quantified with MetaMorph software (Roper Scientific). For the control conditions in the quantification of pattern colocalization, a virtual grid was superimposed to the images of cells on the unpatterned materials in order to calculate a theoretical colocalization percentage for these control conditions. Cells were identified after fixation  in PBS, 15 min, room temperature), by immunolabeling with primary anti-G0α  and anti-GFAP  antibodies in mixed culture, and with anti-NF200 primary antibody  for purified RGCs on protein patterns. Cell quantification was performed automatically with the Metamorph software. Glial cell counts were estimated by surface coverage whereas bipolar cells were counted individually with a 5 μm-diameter threshold and an additional circular criterion to avoid cell debris.Mixed adult cells were used to seed the substrate at a density of 2×10Neuron survival in culture requires the substrate considered to be coated with proteins or extracellular matrix proteins before cell seeding 2) 3+) We characterized the chemical nature of the protein coating on diamonds (nitrogen atoms of poly-D-lysine) further, by performing X-ray photoelectron spectroscopy (XPS). The survey spectra revealed that carbon, oxygen and nitrogen were the predominant elements on the coated H-terminated diamond. As no nitrogen was observed on uncoated H-terminated diamond or on coated and uncoated O-terminated diamond, we studied core nitrogen levels to characterize the protein coating. We then investigated cell survival on diamond, initially with mixed retinal cell cultures on two diamonds with the two different terminations: hydrogen-terminated diamond and chemically oxidized diamonds. Retinal neurons have also been reported to require a protein coating to adhere and survive To further assess the nature of these surviving cells on diamond, different cell markers were applied on the cultures. We determined which cells survived by immunolabeling for glial cells . Thus, isolated adult RGCs can survive on diamond, but the growth of their neurites is reduced by coating the surface with a protein.We assessed neuronal survival on diamond further and investigated the behavior of RGCs on diamond, using purified adult RGCs n = 3) were then quantified automatically, and were clearly different from the substrate areas occupied by the protein pattern (15%) (p<0.001) from that obtained on glass  . This inrn (15%) , the difrn (15%)  instead rn (15%)  or from rn (15%) . The fraon glass . Thus, RDiamond is, potentially, an excellent candidate biomaterial for use in neuroprosthesis electrodes, because it is highly mechanically stable and has semiconductor properties when doped with boron, providing a large electrochemical potential window for electrical stimulations. The use of nanocrystalline diamond for such medical applications will require evidence of its biocompatibility for neuronal tissues. Several studies addressed this question with cultures of embryonic cortical neuronal cells Previous studies have shown that glial cells especially retinal Müller glial cells grow best on protein-coated substrates, in particular with laminin Embryonic neuronal cultures survive well on dispersed nanodiamond particles, indicating that such particles may constitute a potential alternative to protein coating This study not only confirms the biocompatibility of diamond for neurons and glial cells, it also highlights the clear need for protein-coating for some types of expected diamond/cell interface. It therefore supports the consideration of diamond as an attractive electrode material for medical devices interfacing with neural tissues.


There was no difference in the low immunocyte presence for untreated tumors, nude mice, and matrigel-only injections in both strains. These findings suggest IRE therapy may have greater therapeutic efficacy in immunocompetent patients than what has been suggested by immunodeficient models, and that IRE may invoke a systemic response beyond the targeted ablation region.Irreversible electroporation (IRE) is a non-thermal focal ablation technique that uses a series of brief but intense electric pulses delivered into a targeted region of tissue, killing the cells by irrecoverably disrupting cellular membrane integrity. This study investigates if there is an improved local anti-tumor response in immunocompetent (IC) BALB/c versus immunodeficient (ID) nude mice, including the potential for a systemic protective effect against rechallenge. Subcutaneous murine renal carcinoma tumors were treated with an IRE pulsing protocol that used 60% of the predicted voltage required to invoke complete regressions in the ID mice. Tumors were followed for 34 days following treatment for 11 treated mice from each strain, and 7 controls from each strain. Mouse survival based on tumor burden and the progression-free disease period was substantially longer in the treated IC mice relative to the treated ID mice and sham controls for both strains. Treated IC mice were rechallenged with the same cell line 18 days after treatment, where growth of the second tumors was shown to be significantly reduced or prevented entirely. There was robust CD3 Focal ablative therapies are an important alternative to traditional surgical excision in clinical oncology. Many of these therapies utilize thermally-mediated tissue effects (heating or cooling) to kill the targeted volumes of cells, such as radiofrequency ablation, cryoablation, and high intensity focused ultrasound IRE procedures involve placing small (∼1 mm diameter) needle electrodes into the targeted region to deliver a series of brief (∼100 µs long) electric pulses. These pulses alter the cellular transmembrane potential, resulting in irrecoverable membrane defects that kill the tumor’s cells in a controllable manner with sub-millimeter demarcation between treated and normal regions in situ following treatment, there is a release of antigens and the various signals of cellular distress  in the treated and peripheral volumes that may promote local and systemic immune responses Most ablation requires inserting a probe directly into the tumor, and the therapies are regarded as therapeutic only within the targeted region. However, since the tissue remains in vivo gene-based immunotherapy, which has obtained local and distant protective effects experimentally and clinically using cytokine encoding plasmids Further studies have combined focal ablation with adjuvant immunomodulating materials experimentally and clinically to augment the systemic effects observed in ablation therapies in vivo+/CD8+ ratio, cytokine IFN-γ and IL-4-positive splenocytes, and serum sIL-2R and IL-10 The ability for IRE to kill cells through a non-thermal mechanism may release substantial intact antigens and stress signals from the affected cells, resulting in an immune response that enhances therapeutic outcome locally and distant from the treated region. Although IRE-induced cell death has been shown to occur independently from an immune response, immunocyte infiltration and lymphatic drainage have been observed following tissue ablations To determine the potential effect of a functioning immune system on IRE tumor treatment outcomes, we performed identical IRE treatments on immunodeficient (ID) athymic (nude) mice and immunocompetent (IC) BALB/c mice bearing subcutaneous murine renal carcinoma tumors. In order to delineate any difference in treatment outcome between the two mouse groups, we treated the tumors with an IRE pulsing protocol that used a voltage-to-distance ratio 40% lower than one that obtained a 92% regression rate in ID mice This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Virginia Tech institutional animal care and use committee . All surgery was performed under isofluorane anesthesia followed by buprenorphine analgesic, and all efforts were made to minimize suffering.ad libitum, and were kept in the animal facility for approximately 2 weeks, reaching an age of 7–12 weeks and weighing 15–25 g prior to tumor implantation.Two strains of female mice, immunodeficient (ID) Nu/Nu athymic nude and immunocompetent (IC) BALB/c,  were housed in individually ventilated cages in groups of five under specific pathogen free conditions. Mice were allowed access to sterilized water and feed Renca murine kidney cancer cells (CRL-2947) were obtained from the ATCC  and verified by Short Tandem Repeat DNA profiling. The Renca cell line was derived from a spontaneous renal cortical adenocarcinoma in BALB/cCr mice as previously described 7 cells/ml in a 50/50 mixture of PBS and Matrigel High Concentration . Mice were anesthetized by inhalation of 3% isoflourane  for induction and 2% for maintenance. The hair on the BALB/c mice was clipped at the injection site, and the flank of both mice strains was sterilized with isopropyl alcohol prior to injecting 200 µl aliquots of the cell suspension  subcutaneously on the rear right flank of 18 nude and 18 BALB/c mice. An additional 8 mice from each strain received 200 µl of the matrigel and PBS mixture only, without any tumor cells, in order to histologically evaluate baseline immunocyte response to the inoculation matrix.Once reaching approximately 80% confluence, cells were twice washed with phosphate buffered saline (PBS) and treated with 0.25% trypsin EDTA (Fisher) for detachment from culture flasks. Cells were resuspended at a concentration of 1×10Tumor growth was measured using calipers. Once reaching a minimum of 5×5 mm in top cross-sectional area and approximately 4 mm deep, tumors were treated in 11 mice from each strain. Mice were anesthetized following the same isofluorane inhalation protocol from tumor implantation. The skin over the tumor was prepped with 70% isopropyl alcohol, and 2 insulated metal needles with 1 mm exposed length were inserted into the tumor to measure the voltage drop between them to ensure effective delivery of the electric pulses to the tumors. The needles were 0.3 mm in diameter and separated by 1 mm center-to-center. A layer of conductive gel (4.15 S/m)  was applied to opposing ends of the tumor, perpendicular to needle orientation. In addition, a thin layer of gel was applied to the inward-facing plate portions on custom-built electrodes made from a modified BTX Caliper Electrode . The electrode was modified to use custom-built plates contoured to the mouse’s body, providing a larger surface area for pulse delivery, and which were insulated on the non-contact regions using a thin layer of epoxy.The custom-built pulse delivery plate electrodes were placed on opposing sides of the tumor, perpendicular to needle orientation , and tigTo ensure similar pulse delivery between the experimental groups, electrical current was measured using a Hall effect probe (Tektronix TCP 303) and amplifier (Tektronix TCPA300) interfaced with an oscilloscope . In addition, the needles inserted into the middle of the tumor were connected to an oscilloscope, where the electric potential difference between the electrodes was measured. By knowing the voltage drop and separation distance between the electrodes, we were able to calculate the delivered electric field to the tumor.Seven mice with tumors from each strain received sham treatments once the tumors reached a treatable size, where the protocol setup was repeated without delivery of the electric pulses. Matrigel/PBS-only inoculations received pulsed treatments over the acellular Matrigel/PBS plugs. Following all procedures, topical antibiotic ointment was applied to the needle insertion wounds, mice were removed from anesthesia and given subcutaneous injections of 0.05mg/kg buprenorphine analgesic diluted in 2 ml sterile saline. All mice received analgesic daily for an additional 2 days.·2V = 4/3·π·a·b, where a is the long dimension of the tumor ellipsoid, and b is the dimension perpendicular to the axis along a.Following observance of a visible tumor, tumors and weights were recorded three and two times weekly, respectively. Tumor volumes were calculated according to the equation for an ellipsoid, Mice were re-challenged with a second inoculation in their contralateral left flank 18 days following treatment. These mice were followed for an additional 15–16 days after reinoculation unless either the primary or rechallenge tumor reached a diameter of 18 mm in any dimension, after which they were euthanized. Matrigel/PBS-only inoculations of each mouse strain were euthanized at 18 days  and 34 days  after pulse delivery.Once the mice reached a point to require euthanasia, samples of any present tumor tissue were excised and sectioned for processing. Representative tissues were preserved in 10% neutral buffered formalin and embedded in paraffin. Formalin preserved paraffin embedded samples were sectioned and processed for histology using Haematoxylin and Eosin and anti-CD3 antibody  on a Ventana automated immunostainer  using a fast red detection system. All photomicrographs were obtained with a Leitz Wetzlar Orthoplan microscope using a Nikon DS-Fi1 charge coupled digital camera . Digital images were imported into Nikon Elements AR image analysis software  and saved on a personal computer.Tumor response was evaluated from individual tumor dimension measurements taken at least 3 times weekly. Due to the nature of treating the mice based on tumor burden rather than time post-inoculation, this resulted in some variation among which days were evaluated. Therefore, the most recent observation for each mouse was imputed until a new observation was recorded. Median tumor size was evaluated for comparison according to the four basic protocol groups of A: ID/Control, B: ID/Treated, C: IC/Control, and D: IC/Treatment. Two-way ANOVA and Mann-Whitney post-hoc tests were performed on the square root of the median tumor size to test the statistical significance of tumor size differences between the groups at days 10, 15, 20, and 25 post-treatment. In addition, the potential for IRE treatment to induce a systemic protective effect against rechallenge was evaluated using a Wilcoxon signed rank test. This test assessed the significance in time required for tumors to grow to a treatable size for the rechallenge inoculation compared to the initial inoculation.3 on the day of treatment. Treatment associated complications were limited to superficial skin necrosis induced by transcutaneous pulse delivery, with resulting skin wounds resolving within 1 week of treatment. Electric field exposure within the tumor of the treatment groups had median and quartile (1st/3rd) ranges of 839 V/cm (749/1185 V/cm) for the IC treated mice and 838 V/cm (479/1055 V/cm) for the ID mice, showing similar tumor exposure to IRE despite the clipped fur on the skin of IC mice. Most variation likely resulted from variable bowing of the needle electrodes that measured the drop in voltage upon insertion into the tumor. The measured current between the plate electrodes had median and quartile (1st/3rd) currents of 4.1 A (3.6/6.1 A) for the IC mice and 4.6 A (4.0/6.2 A) for the ID mice.Initial inoculations produced tumors of treatable size after an average of 9.4 days for the nude mice and 9.7 days for the BALB/c mice, reaching average tumor volumes of 1728±868 mmA Kaplan-Meier plot  shows thImputed median  and indiStatistical analysis of differences in median and mean tumor volumes between the four treatment groups were carried out using two-way rank ANOVA (data not shown) and Mann-Whitney post-hoc tests  at 10, 1Treatment group mice were rechallenged with a second tumor inoculation of the same cell line 18 days after IRE treatment. Only 5/11 ID mice were able to reach this reinoculation time point; with just 2/5 surviving 4 days beyond rechallenge without a tumor reaching 18 mm, and none reaching the full 16 days. Therefore, reinoculation data on the ID nude mice have been excluded from analysis.Tumor growth curves for the original and rechallenge inoculations in the IC mice  clearly 3, the average volume prior to fulfilling treatment criterion. The test was performed on the smallest difference between the two observed time intervals for the tumors to reach 700 mm3, where the difference was found to be statistically significant (p = 0.0214).Statistical analysis of differential growth rates between the initial inoculations and rechallenge was performed using Wilcoxon one-sample test for the time required for the tumors to reach a volume of 700 mm+ immunohistochemistry   and CD3+hemistry . There wThis study provides evidence supporting an improved irreversible electroporation localized tumor therapeutic response in an immunocompetent (IC) versus immunodeficient (ID) mouse tumor model. An additional protective effect against tumor rechallenge also is observed in IRE treated IC but not ID mice. While we provide empirical evidence to this improved and protective effect in IC mice based on tumor growth curves and histology, future work should examine the exact mechanisms inducing these enhanced and protective effects.In the first portion of this investigation, we examine if tumor treatment response to IRE is greater in the presence of a complete, functioning immune system, versus immunodeficient athymic nude mice which lack functional T-cells. Studies have shown immunocyte presence in ablated regions and lymphatic drainage following healthy tissue ablations The treated IC group responded significantly better than the treated ID group, despite no inherent difference in tumor susceptibility between the two immune-status groups. Similar susceptibility is indicated by tumors reaching a treatable size for both groups within the same number of days, and similar tumor growth response to sham treatments . This suBecause the ID Nu/Nu mouse model is an athymic-only immune deficiency, one mechanism for improved anti-tumor efficacy of IRE in the BALB/C IC mice may relate to adaptive immunity. Focal ablation causes local damage to the tissue that promotes an innate immune response, including inflammation and immunocyte infiltration, to resorb cellular debris. However, the functioning innate immunity in ID mice suggests that this alone is not as effective as both aspects of the immune system (innate and adaptive) in cooperation. This may relate to electroporation-induced tumor antigen release into the interstitium, initiating an immune response capable of locally improving tumor response and exhibiting a systemic protective effect. This immunostimulation would also occur for reversibly electroporated cells.+ T-cell peripheral tolerance As noted in The third danger signal can come in the form of exogenous markers for invading organisms, or endogenous signals based on the mechanism of cell death. Several studies have demonstrated that necrotic cells will lead to increased dendritic cell (DC) and macrophage activation Apoptotic cells do not release their contents like necrotic cells, reducing the presence of danger signals, and some studies show that apoptotic cells actually mitigate immune recognition It should be noted that the effect regarding apoptotic cells is not entirely clear, and some studies have shown apoptotic cells to produce a superior immune effect relative to necrotic cells The IC murine model showed slowed or completely inhibited growth of a rechallenge inoculation with the same cancer cell line. It should be noted that too few control IC and both groups of ID mice survived the initial tumor burden remaining less than 18 mm in diameter long enough to examine if this effect would occur for them as well. However, the observed response in the rechallenged IRE treated IC mice supports the possibility for future studies to investigate the promotion of a systemic response capable of targeting distant regions of cancerous cells undetectable by modern techniques, both experimentally and clinically, when using IRE to treat the visible regions of tumor. This capability remains speculative, and would likely benefit from the inclusion of adjuvant immunostimulants to fully exploit such a potential. Such techniques may be particularly beneficial when using immunostimulants that encourage adaptive immunity.The tumor immune response observed in this study may derive itself from IREs non-thermal method of causing cell death. By remaining below temperatures that cause protein coagulation and molecular denaturation, dead electroporated cells will maintain identifying molecular antigens that may be used by antigen-presenting cells to activate systemic immunity, when presented in conjunction with additional required immune co-stimulating molecules. Further, in addition to evidence for IRE-induced tumor cell apoptosis, the evidence for IRE to cause immediate necrosis of the ablated region may present the body’s immune system with all three signals required to incite a tumor-specific adaptive immune response. IRE ablation promotes near-immediate edema and an inflammatory response, rapidly attracting lymphocytes to the treated region, which may acquire antigens as well as kill remaining cancer cells that survived the IRE electric pulses. These aspects of improved IRE local therapeutic response may also further benefit from the addition of adjuvant immunostimulants, which should be examined in future work.+ cell infiltration in some, but not all, treated immunocompetent BALB/C mouse tumors; without significant infiltration in untreated controls or either treatment group of immunodeficient Nu/Nu mice. This study provides evidence for enhancement of local and distant therapeutic outcome in immunocompetent cancer patients following IRE treatment.This investigation examined the potential improved anti-tumor response in immunocompetent versus immunodeficient mice from IRE focal ablation tumor therapy. Subcutaneous tumors in immunocompetent and immunodeficient mice were treated with a low IRE pulsing protocol designed to avoid complete regressions in immunodeficient mice. Tumor response in the treated immunocompetent mice was significantly stronger than the treated immunodeficient mice, or either of the sham controls from each mouse strain. Rechallenge with the same cancer cell line 18 days after IRE resulted in limited, if any, second tumor growth in the treated immunocompetent mice. Histology showed robust CD3


MPV17 – a gene implicated in severe, infantile hepatocerebral mtDNA depletion disorders characterised by a loss of mtDNA copies – can also cause clonally-expanded mtDNA deletion and focal cytochrome c oxidase (COX) deficiency in skeletal muscle associated with an adult presentation of neuropathy and leukoencephalopathy. The mpv17 protein is therefore intimately involved in both the mtDNA replication and repair processes and associated with both quantitative and qualitative mtDNA abnormalities.Disorders of mitochondrial DNA (mtDNA) maintenance are clinically and genetically heterogeneous, embracing recessive mtDNA depletion syndromes affecting children and adult-onset multiple mtDNA deletion disorders. Here we show that mutation of  These can be due to either mitochondrial DNA (mtDNA) mutations, or one of an increasing number of Mendelian disorders in which the primary genetic defect has a secondary consequence on mtDNA stability, resulting in defective nuclear–mitochondrial intergenomic communication POLG, PEO1, RRM2B and recently TK2 – have been described as causes of both infantile mtDNA depletion syndromes and adult-onset, multiple mtDNA deletion disorders MPV17 gene mutation leading to multiple mtDNA deletions and focal COX deficiency in skeletal muscle.To date, mutations in just four of these genes – 22.1The patient is a 21 year-old Pakistani male, born to non-consanguineous parents. He was diagnosed with Hepatitis A (IgM positive) at the age of 3 years and made a full and uneventful recovery. He first developed neurological symptoms at age 14 years, with gradual onset of walking difficulties that progressed over several years to bilateral foot drops requiring orthoses. Over the same period he also developed symmetrical distal numbness and subjective coldness in both feet ascending to the knees, and more mildly in his hands. There was no significant cognitive impairment.Examination revealed muscle wasting of hands and distal lower limbs. He had clawed toes, but no pes cavus or nerve thickening. He walked with a slow, unsteady gait. Tone was decreased and muscle power was severely weak in a number of distal muscle groups, with no detectable power (MRC grade 0/5) in left abductor pollicis brevis, both first dorsal interossei and both anterior tibialis muscles. While proximal power was normal in the upper limbs, there was symmetrical weakness (MRC grade 4-/5) of hip flexion. All tendon reflexes were absent. Sensory examination was normal in the upper limbs, but revealed decreased perception of pin-prick and light touch distal to the knees, impaired vibration sense to the ankles and absent joint position sense at the toes. There was mild hepatomegaly.Liver ultrasound revealed abnormal echogenicity, serum liver enzymes were minimally abnormal , CT showed heterogeneous liver enhancement, and liver biopsy confirmed patchy steatosis (∼20%) with early bridging fibrosis, but overall clinically there was no significant chronic liver disease. CSF analysis showed an increased protein of 1963 mg/L  and raised lactate of 4.3 mmol/L . Cranial MRI revealed diffuse hyperintense T2 weighted signal in the cerebral white matter  and cereThe patient’s sister developed a progressive, fatal hepatitis at age 9 years. Their father had suffered an episode of hepatitis as a child but made a full recovery. The patient’s mother and older brother are both well and there are no other family members with either liver disease or neuropathy.2.2Left quadriceps needle muscle biopsy was performed with informed consent. Histological and histochemical analyses of mitochondrial enzyme activities, including the sequential reaction for COX and succinate dehydrogenase (SDH) activities, were performed using 10 μm serial cross-sections according to standard procedures NC_012920.1). The level of deleted mtDNA in individual COX-deficient and COX-positive reacting muscle fibres was determined by quantitative real-time PCR of the MTND1 and MTND4 regions of the mitochondrial genome as previously described MTND1) and a nuclear housekeeping gene (18S rRNA) Total DNA was extracted from the muscle biopsy by standard procedures and screened for common mtDNA point mutations. A long-range PCR assay employing a pair of primers (L3965 (nucleotides 3965–3984) and H129 (nucleotides 129–110)) amplified a ∼12.7 kb product in wild-type mtDNA across the major arc of mtDNA to screen for mtDNA rearrangements (GenBank Accession No. 2.3POLG (NM_002693), DGUOK (NM_080918) and MPV17 (NM_002437.4) genes were amplified using intronic M13-tailed primers by standard PCR. Purified PCR products  were sequenced with BigDye Terminator cycle sequencing chemistries on an ABI3130xl Genetic Analyzer (Applied Biosystems) and analysed using Mutation Surveyor software (Softgenetics).The entire coding regions, including intron–exon boundaries, of the 3H&E staining of the muscle biopsy was generally unremarkable, revealing only scattered small fibres; there was no evidence of necrosis, phagocytosis, inclusions, fibre splitting or inflammatory infiltrate although occasional fibres displayed internal nucleation a. ModifiMTND4 gene  implicated in mtDNA maintenance disorders with hepatocerebral involvement revealed a homozygous missense variant  in MPV17 which has previously been reported as a recessive mutation associated with mtDNA depletion in liver , a disorder affecting Native American Navajo children, who present with liver disease, failure to thrive, recurrent metabolic acidosis at times of intercurrent infection, corneal scarring, severe sensory and motor nerve impairment and cerebral leukoencephalopathy MPV17 mutations and hepatocerebral depletion syndrome Previously, a p.Arg50Gln ‘founder’ mutation in MPV17 mutations.The case we present, although phenotypically similar to the classic form of NNH, is unusual in several respects. Importantly, histochemical analysis demonstrated clear evidence of muscle involvement with ragged red fibres and a mosaic pattern of COX deficiency. The latter implicated mtDNA in the pathogenetic mechanism of disease in this patient, but rather than depletion of mtDNA, we identified multiple mtDNA deletions in skeletal muscle, a finding not previously noted in patients with Although the parents were non-consanguineous, they were from the same Asian ethnicity and the presence of a homozygous p.Pro98Leu mutation SYM1, the yeast orthologue of the MPV17 gene, causes mitochondrial bioenergetic defects and mtDNA instability MPV17−/− knockout mouse MPV17 mutation leading to multiple mtDNA deletions and associated COX abnormalities in muscle, the clinical and molecular findings in patients with hepatocerebral mtDNA depletion have recently been summarised to investigate possible phenotype–genotype correlations associated with autosomal recessive MPV17 abnormalities The mpv17 protein is an inner mitochondrial membrane comprising four transmembrane domains yet its function and role in the pathogenesis of autosomal recessive disorders of mtDNA maintenance are far from clear in spite of studies in disease models. Deletion of In conclusion, we have identified the presence of clonally-expanded mtDNA deletions in muscle, a tissue not previously recognised to be involved in the pathological process of mpv17-related disease. Although the precise function of this remains undetermined, we confirm this protein as being intimately involved in both the mtDNA replication and repair processes and associated with both quantitative and qualitative mtDNA abnormalities.


AMPK has been recognized as an important upstream signaling intermediate intimately involved in the regulation of the mTOR pathway [722/Ser792 which it is essential for inhibition of the raptor-containing mTOR complex I (mTORCI). Moreover, AMPK is activated by the adenosine analogue, 5-aminoimidazole-4-carboxamide (AICA)-riboside (AICAR) and metformin. These drugs have been used to inhibit the growth and survival of breast cancer and glioblastomas cells [AMPK is a physiological cellular energy sensor that is activated by phosphorylation at Thr473 of Akt/PKB, which is essential for full Akt/PKB activation. We have evidence in TSC-deficient proximal tubular cells accumulates significant amounts of ROS compared to wild type cells (data not shown). Accumulation of ROS is associated with activation/phosphorylation of Akt at Ser473, inactivation/phosphorylation of tuberin at Thr1462 and activation mTOR/rictor complex II [1135 by p70S6K, which negatively regulates mTORC2 protein complex as part of a negative feedback mechanism controlling Akt activity [mTOR is a large protein kinase with two different complexes. One complex contains mTOR, GβL and raptor, which is a target of rapamycin . The othTargeting mTOR is emerging as an important approach in cancer therapeutics . Early c1387 and suppressed the mTOR activity. Moreover, rapamycin treatment led to a robust phosphorylation and activation of AMPK and ACC to a degree similar to that observed with AICAR stimulation in TSC2+/− treated with HG further supporting the importance of rapamycin as an activator of AMPK. In addition, treatment of TSC-deficient mice with rapamycin showed inhibition of mTORC1 through increasing AMPK phosphorylation at Thr172 and it is downstream, ACC at Ser79 and accompanied by increased OGG1 activity [722/Ser792 and inhibit mTOR that lead to activate the DNA repair enzyme OGG1 in cancer cells.Upregulation of OGG1 was detected in several cancer cells treated with AMPK activator, AICAR . Activat