Roles of Skeletal Muscle in Development: A Bioinformatics and Systems Biology Overview.

The ability to assess various cellular events consequent to perturbations, such as genetic mutations, disease states and therapies, has been recently revolutionized by technological advances in multiple "omics" fields. The resulting deluge of information has enabled and necessitated the development of tools required to both process and interpret the data. While of tremendous value to basic researchers, the amount and complexity of the data has made it extremely difficult to manually draw inference and identify factors key to the study objectives. The challenges of data reduction and interpretation are being met by the development of increasingly complex tools that integrate disparate knowledge bases and synthesize coherent models based on current biological understanding. This chapter presents an example of how genomics data can be integrated with biological network analyses to gain further insight into the developmental consequences of genetic perturbations. State of the art methods for conducting similar studies are discussed along with modern methods used to analyze and interpret the data.

Immunoassay for Quantitative Detection of Antibody Transcytosis Across the Blood-Brain Barrier In Vitro.

Automated high-throughput immunoassays are emerging as reliable analytic techniques for the quantitative detection of proteins from a variety of sample types. Herein, we describe a method using the Protein Simple Wes capillary-based automated immunoassays platform for the quantification of His- and HA-tagged antibody transcytosis across an in vitro transwell blood-brain barrier (BBB) model. Compared to conventional ELISA, fluorescence, and Mass Spec-based detection approaches, Wes provides comparable datasets with additional information regarding size, aggregation, and potential degradation of samples before and after BBB transcytosis. In this chapter, we have benchmarked our Wes technique against ELISA and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), using known BBB crossing (FC5) and non-crossing (A20.1) single domain antibodies.

Electrophysiological- and Neuropharmacological-Based Benchmarking of Human Induced Pluripotent Stem Cell-Derived and Primary Rodent Neurons.

Human induced pluripotent stem cell (iPSC)-derived neurons are of interest for studying neurological disease mechanisms, developing potential therapies and deepening our understanding of the human nervous system. However, compared to an extensive history of practice with primary rodent neuron cultures, human iPSC-neurons still require more robust characterization of expression of neuronal receptors and ion channels and functional and predictive pharmacological responses. In this study, we differentiated human amniotic fluid-derived iPSCs into a mixed population of neurons (AF-iNs). Functional assessments were performed by evaluating electrophysiological (patch-clamp) properties and the effect of a panel of neuropharmacological agents on spontaneous activity (multi-electrode arrays; MEAs). These electrophysiological data were benchmarked relative to commercially sourced human iPSC-derived neurons (CNS.4U from Ncardia), primary human neurons (ScienCell™) and primary rodent cortical/hippocampal neurons. Patch-clamp whole-cell recordings showed that mature AF-iNs generated repetitive firing of action potentials in response to depolarizations, similar to that of primary rodent cortical/hippocampal neurons, with nearly half of the neurons displaying spontaneous post-synaptic currents. Immunochemical and MEA-based analyses indicated that AF-iNs were composed of functional glutamatergic excitatory and inhibitory GABAergic neurons. Principal component analysis of MEA data indicated that human AF-iN and rat neurons exhibited distinct pharmacological and electrophysiological properties. Collectively, this study establishes a necessary prerequisite for AF-iNs as a human neuron culture model suitable for pharmacological studies.

Striated-for-smooth muscle replacement in the developing mouse esophagus.

The esophagus is a muscular tube which transports swallowed content from the oral cavity and the pharynx to the stomach. Early in mouse development, an entire layer of the esophagus, the muscularis externa, consists of differentiated smooth muscle cells. Starting shortly after mid-gestation till about two weeks after birth, the muscularis externa almost entirely consists of striated muscle. This proximal-to-distal replacement of smooth muscle by the striated muscle depends on a number of factors. To identify the nature of the hypothetical "proximal" (mainly striated muscle originating) and "distal" (mainly smooth muscle originating) signals that govern the striated-for-smooth muscle replacement, we compared the esophagus of Myf5:MyoD null fetuses completely lacking striated muscle to the normal control using cDNA microarray analysis, followed by a comprehensive database search. Here we provide an insight into the nature of "proximal" and "distal" signals that govern the striated-for-smooth muscle replacement in the esophagus.

Characterizing Adversity of Lysosomal Accumulation in Nonclinical Toxicity Studies: Results from the 5th ESTP International Expert Workshop.

Lysosomes have a central role in cellular catabolism, trafficking, and processing of foreign particles. Accumulation of endogenous and exogenous materials in lysosomes represents a common finding in nonclinical toxicity studies. Histologically, these accumulations often lack distinctive features indicative of lysosomal or cellular dysfunction, making it difficult to consistently interpret and assign adverse dose levels. To help address this issue, the European Society of Toxicologic Pathology organized a workshop where representative types of lysosomal accumulation induced by pharmaceuticals and environmental chemicals were presented and discussed. The expert working group agreed that the diversity of lysosomal accumulations requires a case-by-case weight-of-evidence approach and outlined several factors to consider in the adversity assessment, including location and type of cell affected, lysosomal contents, severity of the accumulation, and related pathological effects as evidence of cellular or organ dysfunction. Lysosomal accumulations associated with cytotoxicity, inflammation, or fibrosis were generally considered to be adverse, while those found in isolation (without morphologic or functional consequences) were not. Workshop examples highlighted the importance of thoroughly characterizing the biological context of lysosomal effects, including mechanistic data and functional in vitro readouts if available. The information provided here should facilitate greater consistency and transparency in the interpretation of lysosomal effects.

Analysis of the pharmacological properties of JWH-122 isomers and THJ-2201, RCS-4 and AB-CHMINACA in HEK293T cells and hippocampal neurons.

Synthetic cannabinoids are marketed as legal alternatives to Δ9-THC, and are a growing worldwide concern as these drugs are associated with severe adverse effects. Unfortunately, insufficient information regarding the physiological and pharmacological effects of emerging synthetic cannabinoids (ESCs) makes their regulation by government authorities difficult. One strategy used to evade regulation is to distribute isomers of regulated synthetic cannabinoids. This study characterized the pharmacological properties of a panel of ESCs in comparison to Δ9-THC, as well as six JWH-122 isomers relative to its parent compound (JWH-122-4). Two cell-based assays were used to determine the potency and efficacy of ESCs and a panel of reference cannabinoids. HEK293T cells were transfected with human cannabinoid receptor 1 (CB1) and pGloSensor-22F, and the inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels was monitored in live cells. All ESCs examined were classified as agonists, with the following rank order of potency: Win 55,212-2 > CP 55,940 > JWH-122-4 > Δ9-THC ≈ RCS-4 ≈ THJ-2201 > JWH-122-5 > JWH-122-7 > JWH-122-2 ≈ AB-CHMINACA > JWH-122-8 > JWH-122-6 > JWH-122-3. Evaluation of ESC-stimulated Ca2+ transients in cultured rat primary hippocampal neurons confirmed the efficacy of four of the most potent ESCs (JWH-122-4, JWH-122-5, JWH-122-7 and AB-CHMINACA). This work helps regulatory agencies make informed decisions concerning these poorly characterized recreational drugs.

More complications in uncemented compared to cemented hemiarthroplasty for displaced femoral neck fractures: a randomized controlled trial of 201 patients, with one year follow-up.

BACKGROUND: It is unclear whether cemented or uncemented hemiarthroplasty is the best treatment option in elderly patients with displaced femoral neck fractures. Previous randomized trials comparing cemented and uncemented hemiarthroplasty have conflicting results. We conducted a randomized controlled trial to compare cemented and uncemented hemiarthroplasty. METHODS: This multicenter parallel-randomized controlled trial included patients of 70 years and older with a displaced femoral neck fracture (Garden type III or IV). Inclusion was between August 2008 and June 2012. Patients were randomized between a cemented hemiarthroplasty, type Müller Straight Stem or an uncemented hemiarthroplasty, type DB-10. Primary outcomes were complications, operation time, functional outcome (measured by Timed-Up-and-Go (TUG) and Groningen Activity Restriction Scale (GARS)) and mid-thigh pain. Health Related Quality of Life (HRQoL, expressed with the SF-12) was measured as an secondary outcome. Follow up was 1 year. RESULTS: In total 201 patients were included in the study (91 uncemented, 110 cemented hemiarthroplasties) The uncemented group showed more major local complications (intra- and postoperative fractures and dislocations) odds ratio (95% confidence interval) 3.36 (1.40 to 8.11). There was no difference in mean operation time (57.3 vs 55.4 min). There were no differences in functional outcomes (TUG 12.8 (9.4) vs. 13.9 (9.0), GARS 43.2 (19.7) vs. 39.2 (16.5)) and mid-thigh pain (18.6 vs 21.6%). Physical component SF-12 HRQoLwas lower in the uncemented group (30.3 vs. 35.3 p < 0.05 after six weeks, 33.8 vs 38.5 p < 0.05 after 12 weeks). CONCLUSION: A cemented hemiarthroplasty in elderly patients with a displaced femoral neck fracture results in less complications compared to an uncemented hemiarthroplasty. TRIAL REGISTRATION: Netherlands Trial Registry; NTR 1508 , accepted date 27 okt 2008.

Role of skeletal muscle in ear development.

The current paper is a continuation of our work described in Rot and Kablar, 2010. Here, we show lists of 10 up- and 87 down-regulated genes obtained by a cDNA microarray analysis that compared developing Myf5-/-:Myod-/- (and Mrf4-/-) petrous part of the temporal bone, containing middle and inner ear, to the control, at embryonic day 18.5. Myf5-/-:Myod-/- fetuses entirely lack skeletal myoblasts and muscles. They are unable to move their head, which interferes with the perception of angular acceleration. Previously, we showed that the inner ear areas most affected in Myf5-/-:Myod-/- fetuses were the vestibular cristae ampullaris, sensitive to angular acceleration. Our finding that the type I hair cells were absent in the mutants' cristae was further used here to identify a profile of genes specific to the lacking cell type. Microarrays followed by a detailed consultation of web-accessible mouse databases allowed us to identify 6 candidate genes with a possible role in the development of the inner ear sensory organs: Actc1, Pgam2, Ldb3, Eno3, Hspb7 and Smpx. Additionally, we searched for human homologues of the candidate genes since a number of syndromes in humans have associated inner ear abnormalities. Mutations in one of our candidate genes, Smpx, have been reported as the cause of X-linked deafness in humans. Our current study suggests an epigenetic role that mechanical, and potentially other, stimuli originating from muscle, play in organogenesis, and offers an approach to finding novel genes responsible for altered inner ear phenotypes.

Pharmacological characterization of emerging synthetic cannabinoids in HEK293T cells and hippocampal neurons.

There has been a worldwide proliferation of synthetic cannabinoids that have become marketed as legal alternatives to cannabis (marijuana). Unfortunately, there is a dearth of information about the pharmacological effects of many of these emerging synthetic cannabinoids (ESCs), which presents a challenge for regulatory authorities that need to take such scientific evidence into consideration in order to regulate ECSs as controlled substances. We aimed to characterize the pharmacological properties of ten ESCs using two cell based assays that enabled the determination of potency and efficacy relative to a panel of well-characterized cannabinoids. Agonist-mediated inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels was monitored in live HEK293T cells transfected with human cannabinoid receptor 1 gene (CNR1) and pGloSensor-22F. Pharmacological analysis of this data indicated that all of the ESCs tested were full agonists, with the following rank order of potency: Win 55212-2≈5F-PB-22≈AB-PINACA≈EAM-2201≈MAM-2201>JWH-250≈ PB-22>AKB48 N-(5FP)>AKB-48≈STS-135>XLR-11. Assessment of agonist-stimulated depression of Ca(2+) transients was also used to confirm the efficacy of five ESCs (XLR-11, JWH-250, AB-PINACA, 5F-PB-22, and MAM-2201) in cultured primary hippocampal neurons. This work aims to help inform decisions made by regulatory agencies concerned with the profusion of these poorly characterized recreational drugs.

Effect of synthetic cannabinoids on spontaneous neuronal activity: Evaluation using Ca(2+) spiking and multi-electrode arrays.

Activation of cannabinoid receptor 1 (CB1) inhibits synaptic transmission in hippocampal neurons. The goal of this study was to evaluate the ability of benchmark and emerging synthetic cannabinoids to suppress neuronal activity in vitro using two complementary techniques, Ca(2+) spiking and multi-electrode arrays (MEAs). Neuron culture and fluorescence imaging conditions were extensively optimized to provide maximum sensitivity for detection of suppression of neural activity by cannabinoids. The neuronal Ca(2+) spiking frequency was significantly suppressed within 10min by the prototypic aminoalkylindole cannabinoid, WIN 55,212-2 (10µM). Suppression by WIN 55,212-2 was not improved by pharmacological intervention with signaling pathways known to interfere with CB1 signaling. The naphthoylindole CB1 agonist, JWH-018 suppressed Ca(2+) spiking at a lower concentration (2.5µM), and the CB1 antagonist rimonabant (5µM), reversed this suppression. In the MEA assay, the ability of synthetic CB1 agonists to suppress spontaneous electrical activity of hippocampal neurons was evaluated over 80min sessions. All benchmark (WIN 55,212-2, HU-210, CP 55,940 and JWH-018) and emerging synthetic cannabinoids (XLR-11, JWH-250, 5F-PB-22, AB-PINACA and MAM-2201) suppressed neural activity at a concentration of 10µM; furthermore, several of these compounds also significantly suppressed activity at 1µM concentrations. Rimonabant partially reversed spiking suppression of 5F-PB-22 and, to a lesser extent, of MAM-2201, supporting CB1-mediated involvement, although the inactive WIN 55,212-3 also partially suppressed activity. Taken together, synthetic cannabinoid CB1-mediated suppression of neuronal activity was detected using Ca(2+) spiking and MEAs.

Role of skeletal muscle in motor neuron development.

The current paper is a continuation of our work most recently described in Kablar, 2011. Here, we show lists of up- and down-regulated genes obtained by a cDNA microarray analysis that compared developing mouse MyoD-/- limb musculature (MyoD-dependent, innervated by Lateral Motor Column motor neurons) and Myf5-/- back (epaxial) musculature (Myf5-dependent, innervated by Medial Motor Column motor neurons) to the control and to each other, at embryonic day 13.5 which coincides with the robust programmed cell death of motor neurons and the inability of myogenesis to undergo its normal progression in the absence of Myf5 and MyoD that at this embryonic day cannot substitute for each other. We wanted to see if/how the myogenic program couples with the neurotrophic one, and also to separate Lateral from Medial column trophic requirements, potentially relevant to Motor Neuron Diseases with the predilection for the Lateral column. Several follow-up steps revealed that Kif5c, Stxbp1 and Polb, differentially expressed in the MyoD-/- limb muscle, and Ppargc1a, Glrb and Hoxd10, differentially expressed in the Myf5-/- back muscle, are actually regulators of motor neuron numbers. We propose a series of follow-up experiments and various ways to consider our current data.

Synthesis and photochemical properties of PEGylated coumarin-caged ceramides for cell studies.

Caged ceramide analogues (C6-, C16-, C18-, C22- and C24-Cer) have been prepared by introducing a hydrophilic coumarin-based cage bearing a short polyethylene glycol (PEG) chain. (6-Bromo-7-mTEGylated-coumarin-4-yl)methyl (Btc) caged ceramide showed efficient photo-uncaging to release the parent ceramide upon direct exposure to 350 nm UV light; in contrast (7-mTEGylated-coumarin-4-yl)methyl (Tc) caged ceramide was photolysed more slowly. In preliminary experiments, Btc-caged ceramides were taken up by cells and their photolysis led to decreases in cell viability, but not to activation of caspase enzymes, suggesting that either reactive oxygen species or an alternate caspase-independent pathway may be responsible for the decreases in cell viability caused by photolysis of caged ceramides.

Comparison of S-nitrosoglutathione- and staurosporine-induced apoptosis in human neural cells.

S-nitrosoglutathione (GSNO) is an endogenously produced S-nitrosylating compound that controls the function of various proteins. While a number of rodent cell lines have been used to study GSNO-induced apoptosis, the mechanisms of action remain to be evaluated in human cells and in parallel with other common apoptosis-inducing agents. In this study, we compared the pro-apoptotic effects of GSNO and staurosporine (STS) on human neural progenitors (NT2, hNP1) and neuroblasts (SH-SY5Y). We show that these cells exhibit comparable levels of susceptibility to GSNO- and STS-induced apoptotic cell death, as demonstrated by condensed nuclei and CASP3 activation. Mechanistic differences in apoptotic responses were observed as differential patterns of DNA fragmentation and levels of BAX, BCL-XL, CASP8, and p-ERK in response to GSNO and STS treatment. Mitochondrial membrane potential analysis revealed that NT2 and hNP1 cells, but not SH-SY5Y cells, undergo mitochondrial hyperpolarization in response to short-term exposure to STS prior to undergoing subsequent depolarization. This is the first study to report differences in apoptotic responses to GSNO and STS in 3 complementary human neural cell lines. Furthermore, these cells represent useful tools in cell pharmacological paradigms in which susceptibility to apoptosis-inducing agents needs to be assessed at different stages of neural cell fate commitment and differentiation.

Role of skeletal muscle in mandible development.

As a continuation of the previous study on palate development (Rot and Kablar, 2013), here we explore the relationship between the secondary cartilage mandibular condyles (parts of the temporomandibular joint) and the contributions (mechanical and secretory) from the adjacent skeletal musculature. Previous analysis of Myf5-/-:MyoD-/- mouse fetuses lacking skeletal muscle demonstrated the importance of muscle contraction and static loading in mouse skeletogenesis. Among abnormal skeletal features, micrognathia (mandibular hypoplasia) was detected: small, bent and posteriorly displaced mandible. As an example of Waddingtonian epigenetics, we suggest that muscle, in addition to acting via mechanochemical signal transduction pathways, networks and promoters, also exerts secretory stimuli on skeleton. Our goal is to identify candidate molecules at that muscle-mandible interface. By employing Systematic Subtractive Microarray Analysis approach, we compared gene expression between mandibles of amyogenic and wild type mouse fetuses and we identified up- and down-regulated genes. This step was followed by a bioinformatics approach and consultation of web-accessible mouse databases. We searched for individual tissue-specific gene expression and distribution, and for the functional effects of mutations in a particular gene. The database search tools allowed us to generate a set of candidate genes with involvement in mandibular development: Cacna1s, Ckm, Des, Mir300, Myog and Tnnc1. We also performed mouse-to-human translational experiments and found analogies. In the light of our findings we discuss various players in mandibular morphogenesis and make an argument for the need to consider mandibular development as a consequence of reciprocal epigenetic interactions of both skeletal and non-skeletal compartments.

Plasma surface modification of electrospun fibers for adhesion-based cancer cell sorting.

Personalized cancer therapies drive the need for devices that rapidly and accurately segregate cancer cells from solid tumors. One potential sorting strategy is to segregate populations of cells based on their relative strength of adhesion. To investigate the effect of surface hydrophilicity and cell phenotype on adhesion, primary human breast skin fibroblasts and keratinocytes and MCF-7 breast cancer cells were seeded onto air and CF(4) plasma-treated nanofibers followed by exposure to three shear stresses (200, 275 and 350 dynes per cm(2)) 1 hour after inoculation. No difference in strength of adhesion was measured in either fibroblasts or keratinocytes on either plasma treated-surface: all exhibited >60% of the initial cell count after a 5 minute exposure to 350 dynes per cm(2) of shear stress. In contrast, a significant difference between relative strength of adhesion on air versus CF(4) plasma-treated surfaces was observed for MCF-7 cells: 26% and 6.6% of cells remained on the air and CF(4) plasma-treated surfaces, respectively. The ability to sort this cancer cell line from two non-cancerous primary human cells was evaluated by inoculating a mixture of all three cell types simultaneously onto CF(4) treated nanofibers followed by 1 hour of culture and exposure to 350 dynes per cm(2) shear stress. The majority of MCF-7 cells were removed (0.7% remained) while a majority of fibroblasts and keratinocytes remained adhered (74 and 57%). Post-sorted MCF-7 viability and morphology remained unchanged, preserving the possibility of post-separation and analysis. These data suggest that the plasma treatment of electrospun scaffolds provides a tool useful in sorting cancer cells from a mixed cell population based on adhesion strength.

Metal ion levels and functional results after either resurfacing hip arthroplasty or conventional metal-on-metal hip arthroplasty.

BACKGROUND: Modern metal-on-metal hip resurfacing was introduced as a bone-preserving method of joint reconstruction for young and active patients; however, the large diameter of the bearing surfaces is of concern for potentially increased metal ion release. PATIENTS AND METHODS: 71 patients (< 65 years old) were randomly assigned to receive either a resurfacing (R) hip arthroplasty (n = 38) or a conventional metal-on-metal (C) hip arthroplasty (n = 33). Functional outcomes were assessed preoperatively and at 6, 12, and 24 months. Cobalt and chromium blood levels were analyzed preoperatively and at 3, 6, 12, and 24 months. RESULTS: All functional outcome scores improved for both groups. At 12 and 24 months, the median UCLA activity score was 8 in the R patients and 7 in the C patients (p < 0.05). At 24 months, OHS was median 16 in C patients and 13 in R patients (p < 0.05). However, in spite of randomization, UCLA scores also appeared to be higher in R patients at baseline. Satisfaction was similar in both groups at 24 months. Cobalt concentrations were statistically significantly higher for R patients only at 3 and 6 months. Chromium levels remained significantly higher for R patients until 24 months. No pseudotumors were encountered in either group. One R patient was revised for early aseptic loosening and in 2 C patients a cup insert was exchanged for recurrent dislocation. INTERPRETATION: R patients scored higher on UCLA, OHS, and satisfaction at some time points; however, as for the UCLA, preoperative levels were already in favor of R. The differences, although statistically significant, were of minor clinical importance. Chromium blood levels were statistically significantly higher for R patients at all follow-up measurements, whereas for cobalt this was only observed up to 6 months. The true value of resurfacing hip arthroplasty over conventional metal-on-metal hip arthroplasty will be determined by longer follow-up and a possible shift of balance between their respective (dis)advantages.

A new tool to assess ceramide bioactivity: 6-bromo-7-hydroxycoumarinyl-caged ceramide.

The bioactivity of natural, long-chain ceramides has until now been studied after its delivery to cells in organic solvent mixtures containing dodecane. We have synthesized ceramides conjugated to a (6-bromo-7-hydroxycoumarin-4-yl)methyl group. The photocaged ceramide is efficiently released with 350 nm light in aqueous solution at neutral pH, thus providing a promising new tool to study ceramide's properties.

[A boy with a painless swelling of the wrist]. / Een jongen met een pijnloze zwelling aan de pols.

A 3-year-old boy presented with a painless swelling of his right wrist, which was diagnosed as dysplasia epiphysealis hemimelica.

Proteomic analysis of synaptosomal protein expression reveals that cerebral ischemia alters lysosomal Psap processing.

Cerebral ischemia (CI) induces dramatic changes in synaptic structure and function that precedes delayed post-ischemic neuronal death. Here, a proteomic analysis was used to identify the effects of focal CI on synaptosomal protein levels. Contralateral and ipsilateral synaptosomes, prepared from adult mice subjected to 60 min middle cerebral artery occlusion, were isolated following 3, 6 and 20 h of reperfusion. Synaptosomal protein samples (n=3) were labeled using the cleavable ICAT system prior to analysis with nanoLC-MS/MS. Each sample was analyzed by LC-MS to identify differential expressions using InDEPT software and differentially expressed peptides were identified by targeted LC-MS/MS. A total of 62 differentially expressed proteins were identified and Gene Ontology classification (cellular component) indicated that the majority of the proteins were located in the mitochondria and other components consistent with synaptic localization. The observed alterations in synaptic protein levels poorly correlated with gene expression, indicating the involvement of post-transcriptional regulatory mechanisms in determining post-ischemic synaptic protein content. Additionally, immunohistochemistry analysis of prosaposin (Psap) and saposin C (SapC) indicates that CI disrupts Psap processing and glycosphingolipid metabolism. These results demonstrate that the synapse is adversely affected by CI and may play a role in mediating post-ischemic neuronal viability.