The M1311V variant of ATP7A is associated with impaired trafficking and copper homeostasis in models of motor neuron disease.

Disruption in copper homeostasis causes a number of cognitive and motor deficits. Wilson's disease and Menkes disease are neurodevelopmental disorders resulting from mutations in the copper transporters ATP7A and ATP7B, with ATP7A mutations also causing occipital horn syndrome, and distal motor neuropathy. A 65 year old male presenting with brachial amyotrophic diplegia and diagnosed with amyotrophic lateral sclerosis (ALS) was found to harbor a p.Met1311Val (M1311V) substitution variant in ATP7A. ALS is a fatal neurodegenerative disease associated with progressive muscle weakness, synaptic deficits and degeneration of upper and lower motor neurons. To investigate the potential contribution of the ATP7AM1311V variant to neurodegeneration, we obtained and characterized both patient-derived fibroblasts and patient-derived induced pluripotent stem cells differentiated into motor neurons (iPSC-MNs), and compared them to control cell lines. We found reduced localization of ATP7AM1311V to the trans-Golgi network (TGN) at basal copper levels in patient-derived fibroblasts and iPSC-MNs. In addition, redistribution of ATP7AM1311V out of the TGN in response to increased extracellular copper was defective in patient fibroblasts. This manifested in enhanced intracellular copper accumulation and reduced survival of ATP7AM1311V fibroblasts. iPSC-MNs harboring the ATP7AM1311V variant showed decreased dendritic complexity, aberrant spontaneous firing, and decreased survival. Finally, expression of the ATP7AM1311V variant in Drosophila motor neurons resulted in motor deficits. Apilimod, a drug that targets vesicular transport and recently shown to enhance survival of C9orf72-ALS/FTD iPSC-MNs, also increased survival of ATP7AM1311V iPSC-MNs and reduced motor deficits in Drosophila expressing ATP7AM1311V. Taken together, these observations suggest that ATP7AM1311V negatively impacts its role as a copper transporter and impairs several aspects of motor neuron function and morphology.

Optical Clearing of Skeletal Muscle Bundles Engineered in 3-D Printed Templates.

Many techniques for engineering and interrogating three-dimensional (3-D) muscle bundles from animal- or patient-derived myoblasts have recently been developed to overcome the limitations of existing in vitro and in vivo model systems. However, many approaches for engineering 3-D muscle bundles rely on specialized and time-consuming techniques, such as photolithography for fabrication and cryosectioning for histology. Cryosectioning also limits visualization to a single plane instead of the entire 3-D structure. To address these challenges, we first implemented a consumer-grade 3-D-printer to rapidly prototype multiple templates for engineering muscle bundles. We then employed our templates to engineer 3D muscle bundles and identify template geometries that promoted bundle survival over three weeks. Subsequently, we implemented tissue clearing, immunostaining, and confocal imaging to acquire z-stacks of intact muscle bundles labelled for myogenic markers. With this approach, we could select the imaging plane on-demand and visualize the intact 3-D structure of bundles. However, tissue clearing did cause some tissue degradation that should be considered. Together, these advances in muscle tissue engineering and imaging will accelerate the use of these 3-D tissue platforms for disease modeling and therapeutic discovery.

Developing defined substrates for stem cell culture and differentiation.

Over the past few decades, a variety of different reagents for stem cell maintenance and differentiation have been commercialized. These reagents share a common goal in facilitating the manufacture of products suitable for cell therapy while reducing the amount of non-defined components. Lessons from developmental biology have identified signalling molecules that can guide the differentiation process in vitro, but less attention has been paid to the extracellular matrix used. With the introduction of more biologically relevant and defined matrices, that better mimic specific cell niches, researchers now have powerful resources to fine-tune their in vitro differentiation systems, which may allow the manufacture of therapeutically relevant cell types. In this review article, we revisit the basics of the extracellular matrix, and explore the important role of the cell-matrix interaction. We focus on laminin proteins because they help to maintain pluripotency and drive cell fate specification.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons.

An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.

Laminin 521 maintains differentiation potential of mouse and human satellite cell-derived myoblasts during long-term culture expansion.

BACKGROUND: Large-scale expansion of myogenic progenitors is necessary to support the development of high-throughput cellular assays in vitro and to advance genetic engineering approaches necessary to develop cellular therapies for rare muscle diseases. However, optimization has not been performed in order to maintain the differentiation capacity of myogenic cells undergoing long-term cell culture. Multiple extracellular matrices have been utilized for myogenic cell studies, but it remains unclear how different matrices influence long-term myogenic activity in culture. To address this challenge, we have evaluated multiple extracellular matrices in myogenic studies over long-term expansion. METHODS: We evaluated the consequence of propagating mouse and human myogenic stem cell progenitors on various extracellular matrices to determine if they could enhance long-term myogenic potential. For the first time reported, we comprehensively examine the effect of physiologically relevant laminins, laminin 211 and laminin 521, compared to traditionally utilized ECMs (e.g., laminin 111, gelatin, and Matrigel) to assess their capacity to preserve myogenic differentiation potential. RESULTS: Laminin 521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin 211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed excellent in short-term mouse studies but showed high amounts of variability following long-term expansion. CONCLUSIONS: These results demonstrate laminin 521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Since Matrigel cannot be used for clinical applications, we propose that laminin 521 could possibly be employed in the future to provide myoblasts for cellular therapy directed clinical studies.

A human in vitro model of Duchenne muscular dystrophy muscle formation and contractility.

Tongue weakness, like all weakness in Duchenne muscular dystrophy (DMD), occurs as a result of contraction-induced muscle damage and deficient muscular repair. Although membrane fragility is known to potentiate injury in DMD, whether muscle stem cells are implicated in deficient muscular repair remains unclear. We hypothesized that DMD myoblasts are less sensitive to cues in the extracellular matrix designed to potentiate structure-function relationships of healthy muscle. To test this hypothesis, we drew inspiration from the tongue and engineered contractile human muscle tissues on thin films. On this platform, DMD myoblasts formed fewer and smaller myotubes and exhibited impaired polarization of the cell nucleus and contractile cytoskeleton when compared with healthy cells. These structural aberrations were reflected in their functional behavior, as engineered tongues from DMD myoblasts failed to achieve the same contractile strength as healthy tongue structures. These data suggest that dystrophic muscle may fail to organize with respect to extracellular cues necessary to potentiate adaptive growth and remodeling.

In vitro myelin formation using embryonic stem cells.

Myelination in the central nervous system is the process by which oligodendrocytes form myelin sheaths around the axons of neurons. Myelination enables neurons to transmit information more quickly and more efficiently and allows for more complex brain functions; yet, remarkably, the underlying mechanism by which myelination occurs is still not fully understood. A reliable in vitro assay is essential to dissect oligodendrocyte and myelin biology. Hence, we developed a protocol to generate myelinating oligodendrocytes from mouse embryonic stem cells and established a myelin formation assay with embryonic stem cell-derived neurons in microfluidic devices. Myelin formation was quantified using a custom semi-automated method that is suitable for larger scale analysis. Finally, early myelination was followed in real time over several days and the results have led us to propose a new model for myelin formation.

High-content phenotypic screening and triaging strategy to identify small molecules driving oligodendrocyte progenitor cell differentiation.

Multiple Sclerosis is a demyelinating disease of the CNS and the primary cause of neurological disability in young adults. Loss of myelinating oligodendrocytes leads to neuronal dysfunction and death and is an important contributing factor to this disease. Endogenous oligodendrocyte precursor cells (OPCs), which on differentiation are responsible for replacing myelin, are present in the adult CNS. As such, therapeutic agents that can stimulate OPCs to differentiate and remyelinate demyelinated axons under pathologic conditions may improve neuronal function and clinical outcome. We describe the details of an automated, cell-based, morphometric-based, high-content screen that is used to identify small molecules eliciting the differentiation of OPCs after 3 days. Primary screening was performed using rat CG-4 cells maintained in culture conditions that normally support a progenitor cell-like state. From a library of 73,000 diverse small molecules within the Sanofi collection, 342 compounds were identified that increased OPC morphological complexity as an indicator of oligodendrocyte maturation. Subsequent to the primary high-content screen, a suite of cellular assays was established that identified 22 nontoxic compounds that selectively stimulated primary rat OPCs but not C2C12 muscle cell differentiation. This rigorous triaging yielded several chemical series for further expansion and bio- or cheminformatics studies, and their compelling biological activity merits further investigation.

Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Carbon dioxide laser treatment of cutaneous neurofibromas.

INTRODUCTION: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder, with multisystem involvement, including cutaneous manifestations of hyperpigmentation and neurofibromas. Multiple cutaneous lesions are often disfiguring and lead to emotional distress and social isolation. Treatment of NF1 is predominantly surgical but alternative treatments should be considered for patients with large numbers of lesions as cold steel excision of multiple lesions can be cumbersome and may not be practical. The authors report a series of patients with multiple neurofibromas successfully treated using a CO(2) laser. METHODS: Data on CO(2) laser treatments, follow-up, and recurrence following treatment was collected retrospectively. A post-treatment telephone survey was carried out to assess patient satisfaction using a standardized set of questions and a scoring tool. RESULTS: Five of seven patients who underwent CO(2) laser treatment of their multiple neurofibromas responded to the post-treatment survey. All five patients (age range 36-56 years, mean age 45.2 years, three men:two women) had multiple variable-sized neurofibromas. The mean number of lesions per patient was 114 (range 20-200 lesions). The mean number of treatment sessions was 2.2 (range 1-4 sessions) and mean follow-up was 14.4 months (range 6-24 months). Three patients (60%) reported no recurrence up to 2 years post-laser treatment. Two patients (40%) had recurrences of a few lesions (≤10% of treated lesions per patient). The mean patient satisfaction score was 9.2 out of 10 (range 8-10). All patients mentioned that they would recommend CO(2) laser treatment to others with multiple neurofibromas. Hypopigmentation or depigmentation at treatment sites were the only reported adverse effects. CONCLUSION: Based on current results, the authors feel that CO(2) laser treatment achieves a high level of patient satisfaction with a low recurrence of treated lesions.

Compound screening platform using human induced pluripotent stem cells to identify small molecules that promote chondrogenesis.

Articular cartilage, which is mainly composed of collagen II, enables smooth skeletal movement. Degeneration of collagen II can be caused by various events, such as injury, but degeneration especially increases over the course of normal aging. Unfortunately, the body does not fully repair itself from this type of degeneration, resulting in impaired movement. Microfracture, an articular cartilage repair surgical technique, has been commonly used in the clinic to induce the repair of tissue at damage sites. Mesenchymal stem cells (MSC) have also been used as cell therapy to repair degenerated cartilage. However, the therapeutic outcomes of all these techniques vary in different patients depending on their age, health, lesion size and the extent of damage to the cartilage. The repairing tissues either form fibrocartilage or go into a hypertrophic stage, both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage. One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC. Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone, or combined with other techniques to greatly assist the therapeutic outcomes. The recent development of human induced pluripotent stem cell (iPSCs), which are able to self-renew and differentiate into multiple cell types, provides a potentially valuable cell resource for drug screening in a "more relevant" cell type. Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis.

Lentigo maligna--outcomes of treatment with Q-switched Nd:YAG and alexandrite lasers.

BACKGROUND: Surgical excision is considered the criterion standard treatment for lentigo maligna (LM) but may sometimes be unsuitable for elderly people or in the treatment of large lesions. OBJECTIVE: To evaluate the role of Q-switched neodymium-doped yttrium aluminium garnet (QS:Nd:YAG) and alexandrite lasers in the treatment of LM. METHODS: QS:NdYAG and alexandrite lasers were used to treat histologically proven LM in 22 patients who were considered unsuitable for wide surgical excision or declined it. Patients were assessed at 6-month intervals for a maximum period of 5 years. RESULTS: Complete clinical response was achieved in 12 patients after one to four treatments and a follow-up of 2 to 5 years after last treatment. Excellent cosmesis was achieved in all patients. LM melanoma developed in two lesions, which were subsequently excised. Recurrence occurred in four patients. CONCLUSION: Although QS:Nd:YAG and alexandrite laser treatment of LM produced long-term clearance in 12 of the 22 patients, the ease and speed of this treatment and excellent cosmetic outcome achieved make this a suitable alternative to surgical excision, especially in elderly people, in treatment of large lesions, or in patients who refuse surgical treatment.

Overcoming health inequity: potential benefits of a patient-centered open-source public health infostructure.

The past 20 years have witnessed an astonishing increase in computational power and an incredible reduction in the cost of contemporary computer systems, but the public health infostructure in most countries has not changed significantly. This article discusses the potential benefits of applying patient-centered infostructure at the primary medical "points-of-care" services, based on networked integrated open-source technology and programming standards to develop tools to detect and reduce health inequalities. Such systems, which could be implemented from the local to the national level, would enable the expansion of evidence-based medicine, clearer identification of health inequalities, and more accurate cost-benefit analyses. In addition, the public health sector could link such databases to traditional Electronic Patient Record (EPR) systems at a greatly reduced cost by promoting the use of standards-based formats for data transfer and storage. Ultimately, the new health infostructure would help decrease health inequity. In fact, developing countries like Brazil, India, and South Africa are well-positioned to take advantage of the open-source movement and "leapfrog" countries burdened by legacy systems.

Overcoming health inequity: potential benefits of a patient-centered open-source public health infostructure / Superando a falta de eqüidade em saúde: benefícios potenciais de uma estrutura de informação em saúde pública, centrada no paciente e de domínio público

The past 20 years have witnessed an astonishing increase in computational power and an incredible reduction in the cost of contemporary computer systems, but the public health infostructure in most countries has not changed significantly. This article discusses the potential benefits of applying patient-centered infostructure at the primary medical "points-of-care" services, based on networked integrated open-source technology and programming standards to develop tools to detect and reduce health inequalities. Such systems, which could be implemented from the local to the national level, would enable the expansion of evidence-based medicine, clearer identification of health inequalities, and more accurate cost-benefit analyses. In addition, the public health sector could link such databases to traditional Electronic Patient Record (EPR) systems at a greatly reduced cost by promoting the use of standards-based formats for data transfer and storage. Ultimately, the new health infostructure would help decrease health inequity. In fact, developing countries like Brazil, India, and South Africa are well-positioned to take advantage of the open-source movement and "leapfrog" countries burdened by legacy systems.

Este artigo discute os benefícios de se desenvolver um sistema de informação em rede, centrado nos pacientes dos serviços médicos primários, usando tecnologias open-source e definições de padrões de programação e o desenvolvimento de ferramentas capazes de detectar desigualdades. Esses sistemas, que podem ser implementados em nível local e gradualmente se expandirem para uso nacional, capacitariam a expansão da prática da medicina baseada em evidência, a identificação mais clara das desigualdades e análises mais precisas de custos-benefícios. Os setores de saúde pública também poderiam interligar esse sistema aos prontuários eletrônicos tradicionais a custos muito reduzidos por meio da promoção do uso dos sistemas padrões de estocagem e transferência de dados nos produtos de informática comerciais usados nos serviços de saúde. Em sua forma final, esse novo sistema de informação em saúde seria capaz de assistir à gestão da redução das desigualdades nas medidas de saúde pública. De fato, países em desenvolvimento como Brasil, Índia e África do Sul estão muito bem posicionados para darem um salto na frente de outros países que já estão comprometidos com sistemas de informações antiquados.

A functional screen in human cells identifies UBF2 as an RNA polymerase II transcription factor that enhances the beta-catenin signaling pathway.

beta-Catenin signaling plays an important role in the development of many organisms and has a key part in driving the malignant transformation of epithelial cells comprising a variety of cancers. beta-Catenin can activate gene expression through its association with transcription factors of the lymphoid enhancer factor 1 (LEF-1)/T-cell factor (TCF) family. We designed a screen in human cells to identify novel genes that activate a beta-catenin-LEF/TCF-responsive promoter and isolated the high-mobility group box transcription factor, UBF2. UBF1 and UBF2 are splice variants of a common precursor RNA. Although UBF1 has been shown to activate RNA polymerase I-regulated genes, the function of UBF2 has remained obscure. Here, we show for the first time that both UBF1 and UBF2 activate RNA polymerase II-regulated promoters. UBF2 associates with LEF-1, as shown by coimmunoprecipitation experiments, and potentiates transcriptional activation stimulated by LEF-1/beta-catenin from a synthetic promoter with multimerized LEF/TCF binding sites and a natural cyclin D1 promoter with consensus LEF/TCF binding sites. Downregulation of endogenous UBF expression using an RNA interference approach reduces transcriptional activation of a beta-catenin-LEF/TCF-responsive promoter by means of overexpressed beta-catenin, further implicating UBF as a transcriptional enhancer of the beta-catenin pathway.

Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products.

To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone "shotgun" environmental DNA library by using an Escherichia coli-Streptomyces lividans shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.

Efficient cloning of full-length cDNAs based on cDNA size fractionation.

The ability to generate and obtain full-length (FL) cDNAs is of critical importance to the field of genomics. Most cDNAs in a traditional cDNA library lack the initiating 5' ATG, making it difficult to obtain a FL clone. We report here on an improved protocol for the preparation of FL enriched cDNA libraries. We demonstrate that if good quality RNA is used in the cDNA synthesis, high-quality, FL cDNA can be generated for messages upward of 7 kb. In addition, we demonstrate the utility of size fractionation as a means to produce libraries containing a high percentage of initiating 5' ATG containing clones with insert sizes greater than 4 kb. The method is simple, cost efficient, and can be performed in most laboratories equipped to perform molecular biology. Lastly, the novel methodologies used in the analysis of the cDNA and library should prove useful to others working to create high-quality cDNA libraries.