Curcumin and N-Acetylcysteine Nanocarriers Alone or Combined with Deferoxamine Target the Mitochondria and Protect against Neurotoxicity and Oxidative Stress in a Co-Culture Model of Parkinson's Disease.

As the blood-brain barrier (BBB) prevents most compounds from entering the brain, nanocarrier delivery systems are frequently being explored to potentially enhance the passage of drugs due to their nanometer sizes and functional characteristics. This study aims to investigate whether Pluronic® F68 (P68) and dequalinium (DQA) nanocarriers can improve the ability of curcumin, n-acetylcysteine (NAC) and/or deferoxamine (DFO), to access the brain, specifically target mitochondria and protect against rotenone by evaluating their effects in a combined Transwell® hCMEC/D3 BBB and SH-SY5Y based cellular Parkinson's disease (PD) model. P68 + DQA nanoformulations enhanced the mean passage across the BBB model of curcumin, NAC and DFO by 49%, 28% and 49%, respectively (p < 0.01, n = 6). Live cell mitochondrial staining analysis showed consistent co-location of the nanocarriers within the mitochondria. P68 + DQA nanocarriers also increased the ability of curcumin and NAC, alone or combined with DFO, to protect against rotenone induced cytotoxicity and oxidative stress by up to 19% and 14% (p < 0.01, n = 6), as measured by the MTT and mitochondrial hydroxyl radical assays respectively. These results indicate that the P68 + DQA nanocarriers were successful at enhancing the protective effects of curcumin, NAC and/or DFO by increasing the brain penetrance and targeted delivery of the associated bioactives to the mitochondria in this model. This study thus emphasises the potential effectiveness of this nanocarrier strategy in fully utilising the therapeutic benefit of these antioxidants and lays the foundation for further studies in more advanced models of PD.

Enhanced production of biobased, biodegradable, Poly(3-hydroxybutyrate) using an unexplored marine bacterium Pseudohalocynthiibacter aestuariivivens, isolated from highly polluted coastal environment.

The production and disposal of plastics from limited fossil reserves, has prompted research for greener and sustainable alternatives. Polyhydroxyalkanoates (PHAs) are biocompatible, biodegradable, and thermoprocessable polyester produced by microbes. PHAs found several applications but their use is limited due to high production cost and low yields. Herein, for the first time, the isolation and characterization of Pseudohalocynthiibacter aestuariivivens P96, a marine bacterium able to produce surprising amount of PHAs is reported. In the best growth condition P96 was able to reach a maximum production of 4.73 g/L, corresponding to the 87 % of total cell dry-weight. Using scanning and transmission microscopy, lab-scale fermentation, spectroscopic techniques, and genome analysis, the production of thermoprocessable polymer Polyhydroxybutyrate P(3HB), a PHAs class, endowed with mechanical and thermal properties comparable to that of petroleum-based plastics was confirmed. This study represents a milestone toward the use of this unexplored marine bacterium for P(3HB) production.

Micellar Nanocarriers of Hydroxytyrosol Are Protective against Parkinson's Related Oxidative Stress in an In Vitro hCMEC/D3-SH-SY5Y Co-Culture System.

Hydroxytyrosol (HT) is a natural phenolic antioxidant which has neuroprotective effects in models of Parkinson's disease (PD). Due to issues such as rapid metabolism, HT is unlikely to reach the brain at therapeutic concentrations required for a clinical effect. We have previously developed micellar nanocarriers from Pluronic F68® (P68) and dequalinium (DQA) which have suitable characteristics for brain delivery of antioxidants and iron chelators. The aim of this study was to utilise the P68 + DQA nanocarriers for HT alone, or in combination with the iron chelator deferoxamine (DFO), and assess their physical characteristics and ability to pass the blood-brain barrier and protect against rotenone in a cellular hCMEC/D3-SH-SY5Y co-culture system. Both HT and HT + DFO formulations were less than 170 nm in size and demonstrated high encapsulation efficiencies (up to 97%). P68 + DQA nanoformulation enhanced the mean blood-brain barrier (BBB) passage of HT by 50% (p < 0.0001, n = 6). This resulted in increased protection against rotenone induced cytotoxicity and oxidative stress by up to 12% and 9%, respectively, compared to the corresponding free drug treatments (p < 0.01, n = 6). This study demonstrates for the first time the incorporation of HT and HT + DFO into P68 + DQA nanocarriers and successful delivery of these nanocarriers across a BBB model to protect against PD-related oxidative stress. These nanocarriers warrant further investigation to evaluate whether this enhanced neuroprotection is exhibited in in vivo PD models.

N-Acetylcysteine Nanocarriers Protect against Oxidative Stress in a Cellular Model of Parkinson's Disease.

Oxidative stress is a key mediator in the development and progression of Parkinson's disease (PD). The antioxidant n-acetylcysteine (NAC) has generated interest as a disease-modifying therapy for PD but is limited due to poor bioavailability, a short half-life, and limited access to the brain. The aim of this study was to formulate and utilise mitochondria-targeted nanocarriers for delivery of NAC alone and in combination with the iron chelator deferoxamine (DFO), and assess their ability to protect against oxidative stress in a cellular rotenone PD model. Pluronic F68 (P68) and dequalinium (DQA) nanocarriers were prepared by a modified thin-film hydration method. An MTT assay assessed cell viability and iron status was measured using a ferrozine assay and ferritin immunoassay. For oxidative stress, a modified cellular antioxidant activity assay and the thiobarbituric acid-reactive substances assay and mitochondrial hydroxyl assay were utilised. Overall, this study demonstrates, for the first time, successful formulation of NAC and NAC + DFO into P68 + DQA nanocarriers for neuronal delivery. The results indicate that NAC and NAC + DFO nanocarriers have the potential characteristics to access the brain and that 1000 µM P68 + DQA NAC exhibited the strongest ability to protect against reduced cell viability (p = 0.0001), increased iron (p = 0.0033) and oxidative stress (p ≤ 0.0003). These NAC nanocarriers therefore demonstrate significant potential to be transitioned for further preclinical testing for PD.

Techniques for the Study of Apoptosis in Bone.

Osteocyte apoptosis has been associated with a number of clinical conditions in bone and with the targeted turnover of specific skeletal areas. There has been great interest in the identification of the mechanisms by which apoptosis is regulated in bone and in the biological role that this process plays in bone metabolism and associated bone disease or loss of structural integrity. Here we describe several methods for the detection of apoptosis in bone sections and in bone cell cultures.

Directed osteogenic differentiation of human mesenchymal stem/precursor cells on silicate substituted calcium phosphate.

Insufficient, underactive, or inappropriate osteoblast function results in serious clinical conditions such as osteoporosis, osteogenesis imperfecta and fracture nonunion and therefore the control of osteogenesis is a medical priority. In vitro mesenchymal stem cells (MSCs) can be directed to form osteoblasts through the addition of soluble factors such as ß-glycerophosphate, ascorbic acid, and dexamethasone; however this is unlikely to be practical in the clinical setting. An alternative approach would be to use a scaffold or matrix engineered to provide cues for differentiation without the need for soluble factors. Here we describe studies using Silicate-substituted calcium phosphate (Si-CaP) and unmodified hydroxyapatite (HA) to test whether these materials are capable of promoting osteogenic differentiation of MSCs in the absence of soluble factors. Si-CaP supported attachment and proliferation of MSCs and induced osteogenesis to a greater extent than HA, as evidenced through upregulation of the osteoblast-related genes: Runx2 (1.2 fold), Col1a1 (2 fold), Pth1r (1.5 fold), and Bglap (1.7 fold) Dmp1 (1.1 fold), respectively. Osteogenic-associated proteins, alkaline phosphatase (1.4 fold), RUNX2, COL1A1, and BGLAP, were also upregulated and there was an increased production of mineralized bone matrix (1.75 fold), as detected by the Von Kossa Assay. These data indicate that inorganic substrates are capable of directing the differentiation programme of stem cells in the absence of known chemical drivers and therefore may provide the basis for bone repair in the clinical setting.

Osteocyte apoptosis.

Apoptotic death of osteocytes was recognized over 15 years ago, but its significance for bone homeostasis has remained elusive. A new paradigm has emerged that invokes osteocyte apoptosis as a critical event in the recruitment of osteoclasts to a specific site in response to skeletal unloading, fatigue damage, estrogen deficiency and perhaps in other states where bone must be removed. This is accomplished by yet to be defined signals emanating from dying osteocytes, which stimulate neighboring viable osteocytes to produce osteoclastogenic cytokines. The osteocyte apoptosis caused by chronic glucocorticoid administration does not increase osteoclasts; however, it does negatively impact maintenance of bone hydration, vascularity, and strength.

3-D printouts of the tracheobronchial tree generated from CT images as an aid to management in a case of tracheobronchial chondromalacia caused by relapsing polychondritis.

This report concerns a 67 year old male patient with known advanced relapsing polychondritis complicated by tracheobronchial chondromalacia who is increasingly symptomatic and therapeutic options such as tracheostomy and stenting procedures are being considered. The DICOM files from the patient's dynamic chest CT in its inspiratory and expiratory phases were used to generate stereolithography (STL) files and hence print out 3-D models of the patient's trachea and central airways. The 4 full-sized models allowed better understanding of the extent and location of any stenosis or malacic change and should aid any planned future stenting procedures. The future possibility of using the models as scaffolding to generate a new cartilaginous upper airway using regenerative medical techniques is also discussed.

Techniques for the study of apoptosis in bone.

There has been great interest in the identifying the mechanisms by which apoptosis is regulated in bone over recent years and in the biological role that this process plays in bone metabolism and bone disease. Here, we describe several methods for the detection of apoptosis in bone sections and in bone cell cultures.

Collaborative project to co-ordinate care for patients with dementia.

Health leaders from across Suffolk joined together in a collaborative action-learning project to identify ways of offering more productive and personalised care for patients with dementia and their carers. The project revealed a range of factors necessary for success, notably professional collaboration and effective facilitation. The outcome was a range of evidenced-based recommendations to improve care and efficiency, as well as ensuring that the quality, innovation, productivity and prevention (QIPP) agenda was met. The lessons can be applied not just in dementia care, but to other long-term and complex care situations.

Laser microdissection and pressure catapulting of single human motor neurons for RNA editing analysis.

Glutamate is the major excitatory neurotransmitter in the mammalian nervous system. The properties of their ionotropic glutamate receptors largely determine how different neurons respond to glutamate. RNA editing in pre-mRNAs encoding subunits of glutamate receptors, particularly the GluR 2 subunit of AMPA receptors, controls calcium permeability, response time, and total ion flow in individual receptors as well as the density of AMPA receptors at synapses through effects on ER assembly, sorting, and plasma membrane insertion. When RNA editing fails in a neuron, calcium influx through AMPA receptors may cause neuron death by glutamate excitotoxicity, as in the case of vulnerable hippocampal CA1 pyramidal neurons that die after transient forebrain ischemia. Elevated cerebrospinal glutamate is common in ALS and loss of GluR 2 Q/R site RNA editing has been reported to occur selectively in lower motor neurons in a majority of Japanese sporadic ALS patients. We describe our methods for laser microdissection followed by RT-PCR analysis to study RNA editing in single motor neurons.

TGFbeta3 and loading increases osteocyte survival in human cancellous bone cultured ex vivo.

The goal of this study was to assess the effect of the addition of TGFbeta(3), alone or in combination with loading, on the survival of osteocytes in 3D human explant cancellous bone during long-term culture in an ex vivo loading bioreactor. Human cancellous bone explants were cultured for up to 14 days with or without TGFbeta(3) (15 ng ml(-1)) and with or without loading (300 cycles, at 1 Hz, producing 4000 microstrain). Bone core response was visualized using undecalcified histology with morphological methods after embedding with Technovit 9100 New resin. Histological examination revealed normal gross level bone structure with or without the application of load or the addition of TGFbeta(3). The viability of the osteocytes within the bone was assessed by lactate dehydrogenase (LDH) activity. We demonstrate that this ex vivo loading bioreactor is able to maintain a high percentage (over 50%) of viable osteocytes throughout the bone explants after 14 days in ex vivo culture. Further to this, the combination of daily loading and TGFbeta(3) administration produced superior osteocyte survival at the core centres when compared to loading or TGFbeta alone.

The osteocyte lineage.

The osteocyte resides in the lacuna/canalicular system in bone and has been hypothesized to orchestrate local bone remodeling. Certainly the identification of the osteocyte as the source of Sclerostin, a molecule that regulates osteoblast function, has supported this possibility. As our understanding of this cell increases it has become clear that it has more far reaching influence than simply local bone turnover activity. The osteocyte is also the source of DMP-1 and FGF-23, the later being a hormone that regulates kidney function in terms of phosphate uptake. We now see the osteocyte as having important roles both locally in the skeleton and also in other distant tissues. The study of osteocyte biology has reached a particularly exiting level of maturity and illustrates the value of this cell type as a drug discovery target.

Apoptotic bodies convey activity capable of initiating osteoclastogenesis and localized bone destruction.

INTRODUCTION: Osteocyte apoptosis co-localizes with sites of osteoclastic bone resorption in vivo, but to date, no causal molecular or signaling link has been identified between these two processes. MATERIALS AND METHODS: Osteocyte apoptotic bodies (OABs) derived from the MLO-Y4 osteocyte-like cell line and primary murine osteocytes and apoptotic bodies (ABs) derived from primary murine osteoblasts were introduced onto the right parietal bone of murine calvariae, and osteoclastic bone resorption was examined 5 days after treatment. In addition, the ability of primary murine and cell line-derived OABs to support osteoclastogenesis was examined in vitro in co-culture with murine bone marrow hematopoietic progenitors in the absence of RANKL or macrophage-colony stimulating factor. RESULTS: For the first time, we show that OABs are capable of initiating de novo osteoclastic bone resorption on quiescent bone surfaces in vivo. Furthermore, the addition of OABs to mononuclear osteoclast precursors (OPs) in vitro resulted in the maintenance of OP cell numbers and an increase in the proportion and activity of TRACP(+) cells. In contrast, application of ABs from osteoblasts showed no osteoclastogenic activity either in vivo or in vitro. The osteoclastogenic capacity of OABs was shown to be independent of the known osteoclastogenic factor RANKL but dependent on the induction of TNF-alpha production by OP. CONCLUSIONS: These data point to a mechanism by which dying osteocytes might target bone destruction through the distribution of OAB-associated signals and give further physiological meaning to the apoptotic process in bone.

Ex Vivo bone formation in bovine trabecular bone cultured in a dynamic 3D bioreactor is enhanced by compressive mechanical strain.

Our aim was to test cell and trabecular responses to mechanical loading in vitro in a tissue bone explant culture model. We used a new three-dimensional culture model, the ZetOS system, which provides the ability to exert cyclic compression on cancellous bone cylinders (bovine sternum) cultured in forced flow circumfusion chambers, and allows to assess mechanical parameters of the cultivated samples. We evaluated bone cellular parameters through osteocyte viability test, gene and protein expression, and histomorphometric bone formation rate, in nonloaded versus loaded samples. The microarchitecture of bone cores was appraised by in vivo micro-CT imaging. After 3 weeks, the samples receiving daily cyclic compression exhibited increased osteoblast differentiation and activity associated with thicker, more plate-like-shaped trabeculae and higher Young's modulus and ultimate force as compared to unloaded samples. Osteoclast activity was not affected by mechanical strain, although it was responsive to drug treatments (retinoic acid and bisphosphonate) during the first 2 weeks of culture. Thus, in the ZetOS apparatus, we reproduce in vitro the osteogenic effects of mechanical strain known in vivo, making this system a unique and an essential laboratory aid for ex vivo testing of lamellar bone remodeling.

The biology of osteocytes.

Osteocytes, the most abundant cell type in bone, remain the least characterized. Several theories have been proposed regarding their function, including osteolysis, sensing the strains produced in response to mechanical loading of bones, and producing signals that affect the function of osteoblasts and osteoclasts and hence, bone turnover. This review also discusses the role of osteocyte apoptosis in targeted bone remodeling and proposes that the occurrence of osteocyte apoptosis is consistent with the description of apoptosis as an essential homeostatic mechanism for the healthy maintenance of tissues.

The antioxidant effect of estrogen and Selective Estrogen Receptor Modulators in the inhibition of osteocyte apoptosis in vitro.

Withdrawal of estrogen represents the primary factor determining post menopausal bone loss and has been associated with negative indicators of bone quality including the apoptotic death of osteocytes in vivo. While hormone replacement therapy in the form of Estrogen or Selective Estrogen Receptor Modulators (SERMs) demonstrates clear estrogen receptor (ER)-mediated benefits to bone mass, less is known regarding the mechanism of action of these compounds in the maintenance of bone cell populations. We have investigated the potential antioxidant effects of estrogen, estrogen derivatives and the SERMs Raloxifene and LY117018 in the prevention of oxidative stress induced apoptosis in the osteocyte like cell line MLO-Y4. Treatment of MLO-Y4 with 0.3 mM H(2)O(2) induced apoptosis that was significantly inhibited (p< or =0.002) when the cells were pre-treated for 1 h with either 17beta-estradiol, Raloxifene or LY117018 (10 nM). The stereoisomer 17alpha-estradiol also prevented H(2)O(2) induced apoptosis in MLO-Y4. Importantly, pre-treatment of ER-negative HEK293 cells with either 1 microM, 100 nM or 10 nM 17beta-estradiol, Raloxifene or LY117018 significantly inhibited H(2)O(2) induced apoptosis in these cells (p< or =4.2x10(-5)) indicating an estrogen receptor-independent effect of these compounds. Comparisons of 17beta-estradiol and similar molecules containing the putative free radical scavenger C3-OH moiety on the steroid A-ring (17alpha-estradiol, 17alpha-ethinylestradiol; 10 nM) with structurally related molecules lacking the C3-OH grouping (Mestranol and Quinestrol; 10 nM) demonstrated that only compounds containing the C3-OH moiety showed anti-apoptotic behavior in these studies (p< or =0.0033). Similarly the identification of the presence of reactive oxygen species (ROS) in cells as evidenced by the free radical indicator 2'7'-dichlorodihydrofluorescein diacetate demonstrated that 17beta-estradiol, SERMs and related molecules with C3-OH moiety were capable of blocking ROS generated in cells by H(2)O(2) (p< or =0.002) while Mestranol and Quinestrol showed no such blockade. It is possible that the loss of osteocytes during estrogen insufficiency may occur through a failure to suppress the activity of naturally occurring or disease associated oxidant molecules. These data suggest that the osteocyte protective effects of estrogen and SERMs may operate through a common receptor-independent mechanism which may be related to the antioxidant activity of these molecules.

Skeletal muscle IGF-I isoform expression in healthy women after isometric exercise.

CONTEXT: Little is known about the expression of skeletal muscle-specific isoforms of the insulin-like growth factor-I (IGF-I) gene in response to exercise in humans. Data for women are sparse and the influences of age and physical training status are unknown. Here we aimed to describe the expression of mRNA for the IGF-I isoforms in the quadriceps muscles of women at rest and after isometric exercise. OBJECTIVE: To categorize mRNA expression of the IGF-I splice variants IGF-IEa and IGF-IEc (MGF) in healthy women, determine whether isometric exercise stimulates expression, and to determine the duration of the response. DESIGN: Observational study. SETTING: Clinical research facility. PARTICIPANTS: Six healthy women (median age 38 years, range 25-43 years). INTERVENTION: Single bout of maximal isometric knee extension undertaken as 20 sets of 6 contractions. Muscle samples from the lateral mass of the quadriceps were obtained by needle biopsy at baseline, 2.5h and 50h after exercise. MAIN OUTCOME MEASURES: Quantification of mRNAs for IGF-IEa and MGF. RESULTS: mRNA for the IGF-IEa transcript was significantly elevated by 50h post-exercise compared to baseline (p=0.005) and mRNA for MGF was significantly elevated by 2.5h (p=0.026). There were no statistically significant differences between measurements at 2.5h and 50h for either transcript. CONCLUSIONS: We have shown that the upregulation of the muscle specific IGF-IEa and MGF isoforms occurs within 2.5h of a single bout of isometric exercise in women. The upregulation persists for at least 2 days after exercise. We have also demonstrated a potentially safe and effective way of studying the responsiveness of these isoforms to resistance exercise in future studies of older and/or frail individuals.

Physiologic oxygen enhances human embryonic stem cell clonal recovery and reduces chromosomal abnormalities.

Human embryonic stem cells (hESC) have great potential in regenerative medicine, provided that culture systems are established that maintain genomic integrity. Here we describe a comparison of the effects of culture in either physiologic oxygen (2%) or room oxygen (21%) on the hESC lines, H1, H9, and RH1. Physiologic oxygen enabled an average sixfold increase in clone recovery across the hESC lines tested (p < 0.001). FACS analysis showed that cells cultured in physiologic oxygen were significantly smaller and less granular. No significant changes had occurred in levels of SSEA4, SSEA1, TRA-1-60, or TRA-1-81. While karyotypic normalcy was maintained in both H1 and H9, the frequency of spontaneous chromosomal aberrations was significantly increased in room oxygen. This increase was not observed in physiologic oxygen. These results clearly demonstrate that physiologic oxygen culture conditions are indispensable for robust hES clone recovery and may enhance the isolation of novel hES lines and transgenic clones.

Microdamage and apoptosis.

Microdamage of healthy bone leads to targeted removal and repair of the damage. This process must involve the production of specific targeting signals. The identity of these signals is unknown but constitutes a legitimate research goal since it is this targeting process which appears to become impaired in ageing and disease. Here we discuss the potential role of the matrix bound osteocyte in the sensing and targeting of microdamage. In particular we will review current understanding concerning the apoptotic death of osteocytes at sites of microdamage and discuss the potential physiological significance of these findings in the light of knowledge of the significance of apoptosis in other cell systems.