Growing gold nanostructures for shape-selective cellular uptake.

With development in the synthesis of shape- and size-dependent gold (Au) nanostructures (NSs) and their applications in nanomedicine, one of the biggest challenges is to understand the interaction of these shapes with cancer cells. Herein, we study the interaction of Au NSs of five different shapes with glioblastoma-astrocytoma cells. Three different shapes (nanorods, tetrahexahedra, and bipyramids), possessing tunable optical properties, have been synthesized by a single-step seed-mediated growth approach employing binary surfactant mixtures of CTAB and a secondary surfactant. By the use of two-step seed-mediated approach, we obtained new NSs, named nanomakura (Makura is a Japanese word used for pillow) which is reported for the first time here. Spherical Au nanoparticles were prepared by the Turkevich method. To study NS-cell interactions, we functionalized the NSs using thiolated PEG followed by 11-Mercaptoundecanoic acid. The influence of shape and concentration of NSs on the cytotoxicity were assessed with a LIVE/DEAD assay in glioblastoma-astrocytoma cells. Furthermore, the time-dependent uptake of nanomakura was studied with TEM. Our results indicate that unlike the other shapes studied here, the nanomakura were taken up both via receptor-mediated endocytosis and macropinocytosis. Thus, from our library of different NSs with similar surface functionality, the shape is found to be an important parameter for cellular uptake

Glucose transporter-1 deficiency syndrome: the expanding clinical and genetic spectrum of a treatable disorder

Glucose transporter-1 deficiency syndrome is caused by mutations in the SLC2A1 gene in the majority of patients and results in impaired glucose transport into the brain. From 2004-2008, 132 requests for mutational analysis of the SLC2A1 gene were studied by automated Sanger sequencing and multiplex ligation-dependent probe amplification. Mutations in the SLC2A1 gene were detected in 54 patients (41%) and subsequently in three clinically affected family members. In these 57 patients we identified 49 different mutations, including six multiple exon deletions, six known mutations and 37 novel mutations (13 missense, five nonsense, 13 frame shift, four splice site and two translation initiation mutations). Clinical data were retrospectively collected from referring physicians by means of a questionnaire. Three different phenotypes were recognized: (i) the classical phenotype (84%), subdivided into early-onset (<2 years) (65%) and late-onset (18%); (ii) a non-classical phenotype, with mental retardation and movement disorder, without epilepsy (15%); and (iii) one adult case of glucose transporter-1 deficiency syndrome with minimal symptoms. Recognizing glucose transporter-1 deficiency syndrome is important, since a ketogenic diet was effective in most of the patients with epilepsy (86%) and also reduced movement disorders in 48% of the patients with a classical phenotype and 71% of the patients with a non-classical phenotype. The average delay in diagnosing classical glucose transporter-1 deficiency syndrome was 6.6 years (range 1 month-16 years). Cerebrospinal fluid glucose was below 2.5 mmol/l (range 0.9-2.4 mmol/l) in all patients and cerebrospinal fluid : blood glucose ratio was below 0.50 in all but one patient (range 0.19-0.52). Cerebrospinal fluid lactate was low to normal in all patients. Our relatively large series of 57 patients with glucose transporter-1 deficiency syndrome allowed us to identify correlations between genotype, phenotype and biochemical data. Type of mutation was related to the severity of mental retardation and the presence of complex movement disorders. Cerebrospinal fluid : blood glucose ratio was related to type of mutation and phenotype. In conclusion, a substantial number of the patients with glucose transporter-1 deficiency syndrome do not have epilepsy. Our study demonstrates that a lumbar puncture provides the diagnostic clue to glucose transporter-1 deficiency syndrome and can thereby dramatically reduce diagnostic delay to allow early start of the ketogenic die

Connectomics of morphogenetically engineered neurons as a predictor of functional integration in the ischemic brain

Recent advances in cell reprogramming technologies enable the in vitro generation of theoretically unlimited numbers of cells, including cells of neural lineage and specific neuronal subtypes from human, including patient-specific, somatic cells. Similarly, as demonstrated in recent animal studies, by applying morphogenetic neuroengineering principles in situ, it is possible to reprogram resident brain cells to the desired phenotype. These developments open new exciting possibilities for cell replacement therapy in stroke, albeit not without caveats. Main challenges include the successful integration of engineered cells in the ischemic brain to promote functional restoration as well as the fact that the underlying mechanisms of action are not fully understood. In this review, we aim to provide new insights to the above in the context of connectomics of morphogenetically engineered neural networks. Specifically, we discuss the relevance of combining advanced interdisciplinary approaches to: validate the functionality of engineered neurons by studying their self-organizing behavior into neural networks as well as responses to stroke-related pathology in vitro; derive structural and functional connectomes from these networks in healthy and perturbed conditions; and identify and extract key elements regulating neural network dynamics, which might predict the behavior of grafted engineered neurons post-transplantation in the stroke-injured brain

Connectomics of Morphogenetically Engineered Neurons as a Predictor of Functional Integration in the Ischemic Brain

Recent advances in cell reprogramming technologies enable the in vitro generation of theoretically unlimited numbers of cells, including cells of neural lineage and specific neuronal subtypes from human, including patient-specific, somatic cells. Similarly, as demonstrated in recent animal studies, by applying morphogenetic neuroengineering principles in situ, it is possible to reprogram resident brain cells to the desired phenotype. These developments open new exciting possibilities for cell replacement therapy in stroke, albeit not without caveats. Main challenges include the successful integration of engineered cells in the ischemic brain to promote functional restoration as well as the fact that the underlying mechanisms of action are not fully understood. In this review, we aim to provide new insights to the above in the context of connectomics of morphogenetically engineered neural networks. Specifically, we discuss the relevance of combining advanced interdisciplinary approaches to: validate the functionality of engineered neurons by studying their self-organizing behavior into neural networks as well as responses to stroke-related pathology in vitro; derive structural and functional connectomes from these networks in healthy and perturbed conditions; and identify and extract key elements regulating neural network dynamics, which might predict the behavior of grafted engineered neurons post-transplantation in the stroke-injured brain

Silencing of Activity During Hypoxia Improves Functional Outcomes in Motor Neuron Networks in vitro

The effects of hypoxia, or reduced oxygen supply, to brain tissue can be disastrous, leading to extensive loss of function. Deoxygenated tissue becomes unable to maintain healthy metabolism, which leads to increased production of reactive oxygen species (ROS) and loss of calcium homoeostasis, with damaging downstream effects. Neurons are a highly energy demanding cell type, and as such they are highly sensitive to reductions in oxygenation and some types of neurons such as motor neurons are even more susceptible to hypoxic damage. In addition to the immediate deleterious effects hypoxia can have on neurons, there can be delayed effects which lead to increased risk of developing neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), even if no immediate consequences are apparent. Furthermore, impairment of the function of various hypoxia-responsive factors has been shown to increase the risk of developing several neurodegenerative disorders. Longitudinal assessment of electrophysiological network activity is underutilised in assessing the effects of hypoxia on neurons and how their activity and communication change over time following a hypoxic challenge. This study utilised multielectrode arrays and motor neuron networks to study the response to hypoxia and the subsequent development of the neuronal activity over time, as well as the effect of silencing network activity during the hypoxic challenge. We found that motor neuron networks exposed to hypoxic challenge exhibited a delayed fluctuation in multiple network activity parameters compared to normoxic networks. Silencing of activity during the hypoxic challenge leads to maintained bursting activity, suggesting that functional outcomes are better maintained in these networks and that there are activity-dependent mechanisms involved in the network damage following hypoxia

Silencing of activity during hypoxia improves functional outcomes in motor neuron networks in vitro

The effects of hypoxia, or reduced oxygen supply, to brain tissue can be disastrous, leading to extensive loss of function. Deoxygenated tissue becomes unable to maintain healthy metabolism, which leads to increased production of reactive oxygen species (ROS) and loss of calcium homoeostasis, with damaging downstream effects. Neurons are a highly energy demanding cell type, and as such they are highly sensitive to reductions in oxygenation and some types of neurons such as motor neurons are even more susceptible to hypoxic damage. In addition to the immediate deleterious effects hypoxia can have on neurons, there can be delayed effects which lead to increased risk of developing neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), even if no immediate consequences are apparent. Furthermore, impairment of the function of various hypoxia-responsive factors has been shown to increase the risk of developing several neurodegenerative disorders. Longitudinal assessment of electrophysiological network activity is underutilised in assessing the effects of hypoxia on neurons and how their activity and communication change over time following a hypoxic challenge. This study utilised multielectrode arrays and motor neuron networks to study the response to hypoxia and the subsequent development of the neuronal activity over time, as well as the effect of silencing network activity during the hypoxic challenge. We found that motor neuron networks exposed to hypoxic challenge exhibited a delayed fluctuation in multiple network activity parameters compared to normoxic networks. Silencing of activity during the hypoxic challenge leads to maintained bursting activity, suggesting that functional outcomes are better maintained in these networks and that there are activity-dependent mechanisms involved in the network damage following hypoxia