Research projects on protein expression and immunostaining. An unknown protein may be localized by double labeling the tissue with a marker whose expression is known. Graduate student research project for a seminar on optical methods in molecular biology. Students were introduced to the imaging core which is equipped with confocal microscopes. The instructors aided with utilization of the computer software, switching between different objectives, and the choice of materials.
Activity-dependent transcriptional dynamics in mouse primary cortical and hum...Darya Vanichkina
Poster I presented at Lorne Genome 2012. Subsequently formed part of the paper
Barry G, Briggs JA, Vanichkina DP, Poth EM, Beveridge NJ, Ratnu VS, Nayler SP, Nones K, Hu J, Bredy TW, Nakagawa S, Rigo F, Taft RJ, Cairns MJ, Blackshaw S, Wolvetang EJ, Mattick JS (2013). The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing. Molecular psychiatry doi: 10.1038/mp.2013.45
Duplicate of http://figshare.com/articles/Activity_dependent_transcriptional_dynamics_in_mouse_primary_cortical_and_human_iPS_derived_neurons/978468
1) The study investigates how localized protein translation in axons regulates presynaptic development at synapses.
2) The researchers developed a method to selectively repress cap-dependent translation in axons using a targeted translational repressor.
3) They found that repressing axonal translation enlarged synaptic vesicle recycling pools, and this effect was partly due to decreased levels of p35, a protein involved in regulating vesicle recycling pools. Local translation of p35 mRNA in axons normally helps regulate vesicle recycling.
SfN 2015 - Anil Sharma - Genetic tools to study sensory motor circuits FINALAnil Sharma
This document describes a study aiming to identify genetic markers for proprioceptive sensory neurons (PSNs) through single cell RNA sequencing of mouse dorsal root ganglia cells. Specifically, the study aims to find markers for Ia PSNs, which relay sensory feedback from muscle spindles and Golgi tendon organs to the spinal cord. The researchers use mouse genetics and viral tracing to label and isolate PSN subtypes, including Ia PSNs, for single cell sequencing. Initial results identify discrete clusters of cells expressing known PSN and cutaneous neuron markers. Differential expression analysis between clusters may reveal new subtype-specific genetic markers.
Ikrar Et Al (Cell Type Specific Regulation Of Cortical Excitability Through T...Taruna Ikrar
This study investigated using the allatostatin receptor (AlstR) system to selectively inhibit neuronal activity in specific cell types in the cortex. The researchers expressed AlstRs in either excitatory or inhibitory neurons in mouse brain slices using Cre-driven expression. They found that applying the allatostatin peptide ligand strongly reduced spiking in AlstR-expressing neurons but not others. When targeting excitatory neurons, it constrained activity propagation, but enhancing it when targeting inhibitory neurons. Expressing AlstRs in excitatory neurons also effectively suppressed induced seizure activity. This work demonstrates the AlstR system can regulate cortical excitability in a cell-type specific manner.
Genome editing methods such as ZFNs, TALENs, and CRISPR/Cas9 use engineered nucleases to create targeted double-stranded breaks in DNA which are then repaired through endogenous cellular processes. These nucleases can be used to modify genomes through techniques like gene knockout, targeted mutation insertion/deletion/correction, and studying gene function. CRISPR/Cas9 uses a guide RNA and Cas9 nuclease to target specific DNA sequences for editing. The four main steps for CRISPR are: 1) selecting target sequences near a PAM site, 2) designing and cloning gRNA, 3) delivering Cas9 and gRNA into cells, and 4) DNA repair after cleavage results in gene modification
This document describes a study that used laser stimulation of neurons expressing channelrhodopsin-2 (ChR2) to study synaptic transmission in cultured hippocampal neurons. Recombinant adeno-associated virus (rAAV) was used to deliver the ChR2 gene to the neurons. Laser stimulation was able to activate action potentials in ChR2-expressing neurons. By voltage-clamping a neuron and scanning a laser, synaptic responses were observed at some locations, indicating spatial localization of stimulation. Pharmacological tests identified responses that were synaptic. While monosynaptic responses could not be entirely distinguished from polysynaptic ones, smaller amplitudes, simpler shapes, and latencies around 8 ms suggested monosynaptic interactions.
RNA-Seq To Identify Novel Markers For Research on Neural Tissue DifferentiationThermo Fisher Scientific
Neural tissue differentiated and cultured from derived stem cells is expected to revolutionize the treatment of brain and spinal injuries and diseases. Critical for these cellular therapies is accurate control and monitoring of differentiation but current methods for such cell typing are limited to qPCR and immunocytochemisty (ICC) which is not sufficient to discriminate between the numerous (likely >100,000) possible neural cell-types. Research using RNA sequencing (RNA-Seq) permits the characterization and discovery of much-needed novel markers. To define the temporal transcriptional signature of neural stem cells, cultured human embryonic stem cells (H9) were compared to induced neural stem cells (NSCs) at d0, d7 and d14. Total RNA was isolated over the time course from the undifferentiated and differentiated cells. Ion Torrent™ libraries were created to profile expression of miRNAs and whole transcriptomes for each cell population. Multiplexed Ion Proton™ sequencing and Torrent Suite™ Software analysis yielded ≥2.5 million small RNA reads and ≥29 million whole transcriptome reads per sample. Cluster analysis of the RNA-Seq profiles indicates that the cell populations have characteristic molecular signatures. Among genes that are decreased in induced cells are OCT4 (POU5F1), JARID2, NANOG, consistent with the differentiation of iPSCs into neurons. Among genes that showed increased expressions are NTRK2, POU3F2, and a number of HOX family genes. Recently, Ion AmpliSeq™ Transcriptome Human Gene Expression Kit has been launched, and the results from this analysis corroborated with whole transcriptome RNA-Seq results.
This study developed an in vitro model for differentiating human pluripotent stem cells into retinal neurons. The differentiation process followed identifiable stages of retinal development:
1) Undifferentiated stem cells expressed pluripotent markers.
2) After 10 days of differentiation, eye field populations expressed neural and eye field transcription factors.
3) By day 30, retinal progenitor neurospheres analogous to the optic vesicle expressed retinal progenitor markers.
4) At day 70, the stem cells yielded retinal ganglion cells and photoreceptor cells, demonstrating the ability to derive major retinal cell types.
Activity-dependent transcriptional dynamics in mouse primary cortical and hum...Darya Vanichkina
Poster I presented at Lorne Genome 2012. Subsequently formed part of the paper
Barry G, Briggs JA, Vanichkina DP, Poth EM, Beveridge NJ, Ratnu VS, Nayler SP, Nones K, Hu J, Bredy TW, Nakagawa S, Rigo F, Taft RJ, Cairns MJ, Blackshaw S, Wolvetang EJ, Mattick JS (2013). The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing. Molecular psychiatry doi: 10.1038/mp.2013.45
Duplicate of http://figshare.com/articles/Activity_dependent_transcriptional_dynamics_in_mouse_primary_cortical_and_human_iPS_derived_neurons/978468
1) The study investigates how localized protein translation in axons regulates presynaptic development at synapses.
2) The researchers developed a method to selectively repress cap-dependent translation in axons using a targeted translational repressor.
3) They found that repressing axonal translation enlarged synaptic vesicle recycling pools, and this effect was partly due to decreased levels of p35, a protein involved in regulating vesicle recycling pools. Local translation of p35 mRNA in axons normally helps regulate vesicle recycling.
SfN 2015 - Anil Sharma - Genetic tools to study sensory motor circuits FINALAnil Sharma
This document describes a study aiming to identify genetic markers for proprioceptive sensory neurons (PSNs) through single cell RNA sequencing of mouse dorsal root ganglia cells. Specifically, the study aims to find markers for Ia PSNs, which relay sensory feedback from muscle spindles and Golgi tendon organs to the spinal cord. The researchers use mouse genetics and viral tracing to label and isolate PSN subtypes, including Ia PSNs, for single cell sequencing. Initial results identify discrete clusters of cells expressing known PSN and cutaneous neuron markers. Differential expression analysis between clusters may reveal new subtype-specific genetic markers.
Ikrar Et Al (Cell Type Specific Regulation Of Cortical Excitability Through T...Taruna Ikrar
This study investigated using the allatostatin receptor (AlstR) system to selectively inhibit neuronal activity in specific cell types in the cortex. The researchers expressed AlstRs in either excitatory or inhibitory neurons in mouse brain slices using Cre-driven expression. They found that applying the allatostatin peptide ligand strongly reduced spiking in AlstR-expressing neurons but not others. When targeting excitatory neurons, it constrained activity propagation, but enhancing it when targeting inhibitory neurons. Expressing AlstRs in excitatory neurons also effectively suppressed induced seizure activity. This work demonstrates the AlstR system can regulate cortical excitability in a cell-type specific manner.
Genome editing methods such as ZFNs, TALENs, and CRISPR/Cas9 use engineered nucleases to create targeted double-stranded breaks in DNA which are then repaired through endogenous cellular processes. These nucleases can be used to modify genomes through techniques like gene knockout, targeted mutation insertion/deletion/correction, and studying gene function. CRISPR/Cas9 uses a guide RNA and Cas9 nuclease to target specific DNA sequences for editing. The four main steps for CRISPR are: 1) selecting target sequences near a PAM site, 2) designing and cloning gRNA, 3) delivering Cas9 and gRNA into cells, and 4) DNA repair after cleavage results in gene modification
This document describes a study that used laser stimulation of neurons expressing channelrhodopsin-2 (ChR2) to study synaptic transmission in cultured hippocampal neurons. Recombinant adeno-associated virus (rAAV) was used to deliver the ChR2 gene to the neurons. Laser stimulation was able to activate action potentials in ChR2-expressing neurons. By voltage-clamping a neuron and scanning a laser, synaptic responses were observed at some locations, indicating spatial localization of stimulation. Pharmacological tests identified responses that were synaptic. While monosynaptic responses could not be entirely distinguished from polysynaptic ones, smaller amplitudes, simpler shapes, and latencies around 8 ms suggested monosynaptic interactions.
RNA-Seq To Identify Novel Markers For Research on Neural Tissue DifferentiationThermo Fisher Scientific
Neural tissue differentiated and cultured from derived stem cells is expected to revolutionize the treatment of brain and spinal injuries and diseases. Critical for these cellular therapies is accurate control and monitoring of differentiation but current methods for such cell typing are limited to qPCR and immunocytochemisty (ICC) which is not sufficient to discriminate between the numerous (likely >100,000) possible neural cell-types. Research using RNA sequencing (RNA-Seq) permits the characterization and discovery of much-needed novel markers. To define the temporal transcriptional signature of neural stem cells, cultured human embryonic stem cells (H9) were compared to induced neural stem cells (NSCs) at d0, d7 and d14. Total RNA was isolated over the time course from the undifferentiated and differentiated cells. Ion Torrent™ libraries were created to profile expression of miRNAs and whole transcriptomes for each cell population. Multiplexed Ion Proton™ sequencing and Torrent Suite™ Software analysis yielded ≥2.5 million small RNA reads and ≥29 million whole transcriptome reads per sample. Cluster analysis of the RNA-Seq profiles indicates that the cell populations have characteristic molecular signatures. Among genes that are decreased in induced cells are OCT4 (POU5F1), JARID2, NANOG, consistent with the differentiation of iPSCs into neurons. Among genes that showed increased expressions are NTRK2, POU3F2, and a number of HOX family genes. Recently, Ion AmpliSeq™ Transcriptome Human Gene Expression Kit has been launched, and the results from this analysis corroborated with whole transcriptome RNA-Seq results.
This study developed an in vitro model for differentiating human pluripotent stem cells into retinal neurons. The differentiation process followed identifiable stages of retinal development:
1) Undifferentiated stem cells expressed pluripotent markers.
2) After 10 days of differentiation, eye field populations expressed neural and eye field transcription factors.
3) By day 30, retinal progenitor neurospheres analogous to the optic vesicle expressed retinal progenitor markers.
4) At day 70, the stem cells yielded retinal ganglion cells and photoreceptor cells, demonstrating the ability to derive major retinal cell types.
- The study analyzed Alzheimer's disease pathology and expression of the nuclear receptor Nurr1 in 5XFAD mice, a model of severe amyloidosis and neuronal death.
- In the subiculum region of the hippocampus, amyloid plaques and microgliosis increased with age in 5XFAD mice. Significant neuronal loss occurred by 4 months of age.
- Nurr1 expression, which plays a neuroprotective role, decreased in the hippocampus of 5XFAD mice compared to controls by 4 months. Nurr1 was mainly expressed in neurons.
This presentation summarizes some of the most popular neural differentiation protocols. It also contains some of the most recent developments in these protocols including small molecule based methods.
This document discusses the role of the guanine nucleotide exchange factor C3G in neuronal differentiation. It finds that C3G protein levels increase when human neuroblastoma cells are induced to differentiate through serum starvation or treatment with forskolin or nerve growth factor. Overexpression of C3G stimulates neurite growth and increases responsiveness to differentiation signals, in a process dependent on C3G's catalytic domain and the functions of Rap1 and Cdc42. Knockdown of C3G inhibits forskolin- and nerve growth factor-induced differentiation and enhances cell death from serum starvation. C3G phosphorylation and localization to the Golgi are increased by forskolin and nerve growth factor treatment, and C3G
Abstract In the mammalian neocortex, excitatory neurons provide excitation in both columnar and laminar dimensions, which is modulated further by inhibitory neurons. However, our understanding of intracortical excitatory and inhibitory synaptic inputs in relation to principal excitatory neurons remains incomplete, and it is unclear how local excitatory and inhibitory synaptic connections to excitatory neurons are spatially organized on a layer-by-layer basis. In the present study, we combined whole cell recordings with laser scanning photostimulation via glutamate uncaging to map excitatory and inhibitory synaptic inputs to single excitatory neurons throughout cortical layers 2/3–6 in the mouse primary visual cortex (V1). We find that synaptic input sources of excitatory neurons span the radial columns of laminar microcircuits, and excitatory neurons in different V1 laminae exhibit distinct patterns of layer-specific organizationofexcitatoryinputs.Remarkably,thespatialextentofinhibitoryinputsofexcitatory neurons for a given layer closely mirrors that of their excitatory input sources, indicating that excitatory and inhibitory synaptic connectivity is spatially balanced across excitatory neuronal networks. Strong interlaminar inhibitory inputs are found, particularly for excitatory neurons in layers 2/3 and 5. This differs from earlier studies reporting that inhibitory cortical connections to excitatory neurons are generally localized within the same cortical layer. On the basis of the functional mapping assays, we conducted a quantitative assessment of both excitatory and inhibitory synaptic laminar connections to excitatory cells at single cell resolution, establishing precise layer-by-layer synaptic wiring diagrams of excitatory neurons in the visual cortex.
1) The document summarizes research on the effect of the Wiskott-Aldrich syndrome protein (Wasp) in the developing central nervous system of Drosophila.
2) Wasp is expressed uniformly in Drosophila embryos and enriched in certain tissues like the stomach and posterior midgut. Its role includes axonogenesis, actin polymerization, and regulation of synaptic growth.
3) Experiments were conducted to examine Wasp expression and localization in Drosophila embryos and cells. Co-transfection of cells showed co-localization between Wasp and the Frazzled receptor intracellular domain, indicating a potential protein-protein interaction.
An Evolutionarily Conserved Long Noncoding RNA TUNA Controls Pluripotency and...Zach Rana
Here are 3 sentences summarizing the key points of the document:
1) An unbiased genome-scale RNAi screen in mouse embryonic stem cells identified 20 long noncoding RNAs (lincRNAs) required for maintenance of pluripotency, including linc86023 (also known as TUNA or megamind).
2) TUNA was found to form a complex with RNA-binding proteins at the promoters of pluripotency genes Nanog, Sox2, and Fgf4, and its depletion inhibited neural differentiation and impaired proliferation of mouse ESCs.
3) TUNA showed evolutionary conservation and central nervous system-restricted expression in vertebrates, and its knockdown
This study investigated the effects of developmental exposure to the organophosphate chlorpyrifos on social play behavior and endocannabinoid system activation in rats. The researchers found that rats exposed to chlorpyrifos engaged in more social play behaviors compared to controls. However, chlorpyrifos exposure did not affect phosphorylation of cannabinoid receptors in brain regions involved in social play, suggesting that increased social play is not related to changes in endocannabinoid system activation. While endocannabinoid signaling may play a role in altered social behavior, chlorpyrifos may induce changes in other neurotransmitter systems like dopamine that are important for social play.
LRRK2 is a protein implicated in Parkinson's disease. The study found that LRRK2 is more highly expressed in striosome compartments of the mouse striatum. The study used mice with LRRK2 knocked out and mice with the R1441C mutation to examine how loss of LRRK2 or this mutation affects the structure of striosome compartments. While no significant differences in striosome volume were found, the study observed potential enlargement of striosome compartments in knockout and mutant mice, warranting further investigation.
The document discusses apoptosis or programmed cell death. It provides background on the history of apoptosis, definitions, key morphological changes, major players involved like caspases and Bcl-2 proteins, and the two main pathways of apoptosis - the intrinsic mitochondrial pathway and extrinsic death receptor pathway. Detection methods for apoptotic cells are also covered, including electron microscopy, DNA fragmentation analysis, TUNEL assay, and flow cytometry. Therapeutic implications for targeting apoptosis in diseases like cancer, neurodegeneration and myocardial infarction are also mentioned.
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
Differentiation of neural_cells_in_human_embryonic_stemHoney Cheng
The document discusses neural differentiation from human and mouse embryonic stem cells. It outlines inducing neural differentiation from hESCs through various stages, including rosette formation and differentiation into neural precursor cells, neurons, astrocytes, and oligodendrocytes. Methods of identifying differentiated cell types using immunocytochemistry and markers are also presented. The document also briefly discusses the linkage between neural cells and retinal cells during embryonic development.
The document discusses neural differentiation from human and mouse embryonic stem cells. It outlines inducing neural differentiation from hESCs through various stages, including rosette formation and differentiation into neural precursor cells, neurons, astrocytes, and oligodendrocytes. Methods for identifying and characterizing the differentiated neural cell types are also presented, such as immunocytochemical staining and electrophysiological recording. The linkage between neural cells and retinal cells is briefly discussed.
1) Noncoding RNAs (ncRNAs), such as microRNAs and long noncoding RNAs, play important physiological roles that have been revealed by analyzing mutant mice lacking these ncRNAs.
2) One such ncRNA is NEAT1, which is required for the formation of nuclear paraspeckles. Mice lacking NEAT1 are infertile due to a failure of corpus luteum development and progesterone production.
3) Another ncRNA is 4.5SH RNA, which is essential for mouse embryonic development. Mice completely lacking the 4.5SH RNA cluster are embryonic lethal.
Human genetic variation and its contribution to complex traitsgroovescience
This document summarizes a meeting discussing the human genome and human genetic variation.
The key points are:
1) In June 2000, the first draft of the human genome was announced, with fully sequenced publications in 2001. This marked a major milestone in understanding the human genome.
2) Since then, over 11 million SNPs and thousands of structural variants have been discovered through projects like HapMap and large sequencing studies. However, the majority of rare variants are still unknown.
3) Genome-wide association studies since 2006 have identified over 300 genetic loci associated with over 80 human traits and diseases. However, determining the functional consequences and molecular mechanisms remains a major challenge.
This document summarizes research on the subcellular distribution of the RNA-splicing factor NeuN/Fox-3 in Alzheimer's disease. The study found that in control brains, Fox-3 was largely nuclear, but in Alzheimer's brains it exhibited increased cytoplasmic localization. This may be due to alternative splicing of Fox-3 isoforms with different nuclear localization signals. The results suggest that stress factors in Alzheimer's disease may affect the subcellular distribution of Fox-3 and disrupt its RNA-splicing activity, contributing to disease pathogenesis.
This document describes a study investigating the mechanisms underlying X chromosome counting in female embryonic stem cells (ESCs). The researchers conducted an RNA interference screen targeting proteins involved in chromosome structure to identify those necessary for the unique organization of X-linked genes observed in XX ESCs, which is a signature of counting. Knockdown of the loading factor Nipbl for the cohesin complex decreased doublet signals and increased singlet signals without changing cell morphology. Knockdown of Smc2, a subunit of the condensin complex, had no effect. In total, 15 targets were identified that may affect counting.
The document discusses various techniques used for nucleic acid hybridization, including Southern blotting, Northern blotting, dot blot hybridization, and in situ hybridization. Southern blotting involves separating DNA fragments by size, transferring them to a membrane, and using a labeled probe to detect complementary DNA sequences. It can be used to detect mutations. Northern blotting is similar but detects RNA. Dot blot hybridization spots DNA/RNA samples directly onto a membrane. In situ hybridization detects nucleic acids within intact cells using labeled probes. Microarrays allow simultaneous screening of thousands of genes using hybridization on an array.
How Does Inflammation in the Adipose Tissue Contribute to DiabetesKiana Khosravian
This document summarizes research into how inflammation in adipose tissue contributes to diabetes. Mouse models were used where certain inflammatory signaling genes were conditionally knocked out in adipose tissue. Histological analysis found that obese knockout mice had similar numbers of fat cells as obese wildtype mice, but a higher level of apoptotic cells in fat tissue. The results suggest the knocked out gene affects cell viability and survival in fat tissue. Conditional gene knockout was achieved using the Cre-Lox system to selectively delete genes in adipose tissue.
- The study analyzed Alzheimer's disease pathology and expression of the nuclear receptor Nurr1 in 5XFAD mice, a model of severe amyloidosis and neuronal death.
- In the subiculum region of the hippocampus, amyloid plaques and microgliosis increased with age in 5XFAD mice. Significant neuronal loss occurred by 4 months of age.
- Nurr1 expression, which plays a neuroprotective role, decreased in the hippocampus of 5XFAD mice compared to controls by 4 months. Nurr1 was mainly expressed in neurons.
This presentation summarizes some of the most popular neural differentiation protocols. It also contains some of the most recent developments in these protocols including small molecule based methods.
This document discusses the role of the guanine nucleotide exchange factor C3G in neuronal differentiation. It finds that C3G protein levels increase when human neuroblastoma cells are induced to differentiate through serum starvation or treatment with forskolin or nerve growth factor. Overexpression of C3G stimulates neurite growth and increases responsiveness to differentiation signals, in a process dependent on C3G's catalytic domain and the functions of Rap1 and Cdc42. Knockdown of C3G inhibits forskolin- and nerve growth factor-induced differentiation and enhances cell death from serum starvation. C3G phosphorylation and localization to the Golgi are increased by forskolin and nerve growth factor treatment, and C3G
Abstract In the mammalian neocortex, excitatory neurons provide excitation in both columnar and laminar dimensions, which is modulated further by inhibitory neurons. However, our understanding of intracortical excitatory and inhibitory synaptic inputs in relation to principal excitatory neurons remains incomplete, and it is unclear how local excitatory and inhibitory synaptic connections to excitatory neurons are spatially organized on a layer-by-layer basis. In the present study, we combined whole cell recordings with laser scanning photostimulation via glutamate uncaging to map excitatory and inhibitory synaptic inputs to single excitatory neurons throughout cortical layers 2/3–6 in the mouse primary visual cortex (V1). We find that synaptic input sources of excitatory neurons span the radial columns of laminar microcircuits, and excitatory neurons in different V1 laminae exhibit distinct patterns of layer-specific organizationofexcitatoryinputs.Remarkably,thespatialextentofinhibitoryinputsofexcitatory neurons for a given layer closely mirrors that of their excitatory input sources, indicating that excitatory and inhibitory synaptic connectivity is spatially balanced across excitatory neuronal networks. Strong interlaminar inhibitory inputs are found, particularly for excitatory neurons in layers 2/3 and 5. This differs from earlier studies reporting that inhibitory cortical connections to excitatory neurons are generally localized within the same cortical layer. On the basis of the functional mapping assays, we conducted a quantitative assessment of both excitatory and inhibitory synaptic laminar connections to excitatory cells at single cell resolution, establishing precise layer-by-layer synaptic wiring diagrams of excitatory neurons in the visual cortex.
1) The document summarizes research on the effect of the Wiskott-Aldrich syndrome protein (Wasp) in the developing central nervous system of Drosophila.
2) Wasp is expressed uniformly in Drosophila embryos and enriched in certain tissues like the stomach and posterior midgut. Its role includes axonogenesis, actin polymerization, and regulation of synaptic growth.
3) Experiments were conducted to examine Wasp expression and localization in Drosophila embryos and cells. Co-transfection of cells showed co-localization between Wasp and the Frazzled receptor intracellular domain, indicating a potential protein-protein interaction.
An Evolutionarily Conserved Long Noncoding RNA TUNA Controls Pluripotency and...Zach Rana
Here are 3 sentences summarizing the key points of the document:
1) An unbiased genome-scale RNAi screen in mouse embryonic stem cells identified 20 long noncoding RNAs (lincRNAs) required for maintenance of pluripotency, including linc86023 (also known as TUNA or megamind).
2) TUNA was found to form a complex with RNA-binding proteins at the promoters of pluripotency genes Nanog, Sox2, and Fgf4, and its depletion inhibited neural differentiation and impaired proliferation of mouse ESCs.
3) TUNA showed evolutionary conservation and central nervous system-restricted expression in vertebrates, and its knockdown
This study investigated the effects of developmental exposure to the organophosphate chlorpyrifos on social play behavior and endocannabinoid system activation in rats. The researchers found that rats exposed to chlorpyrifos engaged in more social play behaviors compared to controls. However, chlorpyrifos exposure did not affect phosphorylation of cannabinoid receptors in brain regions involved in social play, suggesting that increased social play is not related to changes in endocannabinoid system activation. While endocannabinoid signaling may play a role in altered social behavior, chlorpyrifos may induce changes in other neurotransmitter systems like dopamine that are important for social play.
LRRK2 is a protein implicated in Parkinson's disease. The study found that LRRK2 is more highly expressed in striosome compartments of the mouse striatum. The study used mice with LRRK2 knocked out and mice with the R1441C mutation to examine how loss of LRRK2 or this mutation affects the structure of striosome compartments. While no significant differences in striosome volume were found, the study observed potential enlargement of striosome compartments in knockout and mutant mice, warranting further investigation.
The document discusses apoptosis or programmed cell death. It provides background on the history of apoptosis, definitions, key morphological changes, major players involved like caspases and Bcl-2 proteins, and the two main pathways of apoptosis - the intrinsic mitochondrial pathway and extrinsic death receptor pathway. Detection methods for apoptotic cells are also covered, including electron microscopy, DNA fragmentation analysis, TUNEL assay, and flow cytometry. Therapeutic implications for targeting apoptosis in diseases like cancer, neurodegeneration and myocardial infarction are also mentioned.
Next Generation Sequencing and its Applications in Medical Research - Frances...Sri Ambati
The so-called “next-generation” sequencing (NGS) technologies allows us, in a short time and in parallel, to sequence massive amounts of DNA, overcoming the limitations of the original Sanger sequencing methods used to sequence the first human genome. NGS technologies have had an enormous impact on biomedical research within a short time frame. This talk will give an overview of these applications with specific examples from Mendelian genomics and cancer research. #h2ony
Differentiation of neural_cells_in_human_embryonic_stemHoney Cheng
The document discusses neural differentiation from human and mouse embryonic stem cells. It outlines inducing neural differentiation from hESCs through various stages, including rosette formation and differentiation into neural precursor cells, neurons, astrocytes, and oligodendrocytes. Methods of identifying differentiated cell types using immunocytochemistry and markers are also presented. The document also briefly discusses the linkage between neural cells and retinal cells during embryonic development.
The document discusses neural differentiation from human and mouse embryonic stem cells. It outlines inducing neural differentiation from hESCs through various stages, including rosette formation and differentiation into neural precursor cells, neurons, astrocytes, and oligodendrocytes. Methods for identifying and characterizing the differentiated neural cell types are also presented, such as immunocytochemical staining and electrophysiological recording. The linkage between neural cells and retinal cells is briefly discussed.
1) Noncoding RNAs (ncRNAs), such as microRNAs and long noncoding RNAs, play important physiological roles that have been revealed by analyzing mutant mice lacking these ncRNAs.
2) One such ncRNA is NEAT1, which is required for the formation of nuclear paraspeckles. Mice lacking NEAT1 are infertile due to a failure of corpus luteum development and progesterone production.
3) Another ncRNA is 4.5SH RNA, which is essential for mouse embryonic development. Mice completely lacking the 4.5SH RNA cluster are embryonic lethal.
Human genetic variation and its contribution to complex traitsgroovescience
This document summarizes a meeting discussing the human genome and human genetic variation.
The key points are:
1) In June 2000, the first draft of the human genome was announced, with fully sequenced publications in 2001. This marked a major milestone in understanding the human genome.
2) Since then, over 11 million SNPs and thousands of structural variants have been discovered through projects like HapMap and large sequencing studies. However, the majority of rare variants are still unknown.
3) Genome-wide association studies since 2006 have identified over 300 genetic loci associated with over 80 human traits and diseases. However, determining the functional consequences and molecular mechanisms remains a major challenge.
This document summarizes research on the subcellular distribution of the RNA-splicing factor NeuN/Fox-3 in Alzheimer's disease. The study found that in control brains, Fox-3 was largely nuclear, but in Alzheimer's brains it exhibited increased cytoplasmic localization. This may be due to alternative splicing of Fox-3 isoforms with different nuclear localization signals. The results suggest that stress factors in Alzheimer's disease may affect the subcellular distribution of Fox-3 and disrupt its RNA-splicing activity, contributing to disease pathogenesis.
This document describes a study investigating the mechanisms underlying X chromosome counting in female embryonic stem cells (ESCs). The researchers conducted an RNA interference screen targeting proteins involved in chromosome structure to identify those necessary for the unique organization of X-linked genes observed in XX ESCs, which is a signature of counting. Knockdown of the loading factor Nipbl for the cohesin complex decreased doublet signals and increased singlet signals without changing cell morphology. Knockdown of Smc2, a subunit of the condensin complex, had no effect. In total, 15 targets were identified that may affect counting.
The document discusses various techniques used for nucleic acid hybridization, including Southern blotting, Northern blotting, dot blot hybridization, and in situ hybridization. Southern blotting involves separating DNA fragments by size, transferring them to a membrane, and using a labeled probe to detect complementary DNA sequences. It can be used to detect mutations. Northern blotting is similar but detects RNA. Dot blot hybridization spots DNA/RNA samples directly onto a membrane. In situ hybridization detects nucleic acids within intact cells using labeled probes. Microarrays allow simultaneous screening of thousands of genes using hybridization on an array.
How Does Inflammation in the Adipose Tissue Contribute to DiabetesKiana Khosravian
This document summarizes research into how inflammation in adipose tissue contributes to diabetes. Mouse models were used where certain inflammatory signaling genes were conditionally knocked out in adipose tissue. Histological analysis found that obese knockout mice had similar numbers of fat cells as obese wildtype mice, but a higher level of apoptotic cells in fat tissue. The results suggest the knocked out gene affects cell viability and survival in fat tissue. Conditional gene knockout was achieved using the Cre-Lox system to selectively delete genes in adipose tissue.
"Cold Call Campaigns Success visually represent data and information related to the effectiveness of cold calling in sales and marketing strategies. These graphics use a combination of charts, graphs, and illustrations to convey key insights and statistics in a concise and engaging manner.
The infographics may include data on conversion rates, lead generation, call-to-sale ratios, and other metrics to showcase the impact of cold calling on business growth. They can also highlight best practices, tips, and strategies for optimizing cold call campaigns to improve success rates.
By presenting complex information in a visually appealing format, these infographics make it easier for viewers to understand and digest the content quickly. This makes them an effective tool for businesses looking to communicate the benefits of cold calling and its role in driving sales success.
Overall, infographics on Cold Call Campaigns Success serve as a valuable resource for sales professionals, marketers, and business owners seeking to enhance their cold calling strategies and achieve greater success in their campaigns.
2. Introduction
Figure 1. Lumbar region of the mouse spinal cord (Sengul et al., 2012).
• Spinal cord is divided into lamina (Caspary et al., 2003)
• Network of neurons connect in a translaminar fashion
(Johannssen et al., 2013)
• Cell types are classified using molecular markers (Dobrott
et al., 2019).
• Indirect immunofluorescence can determine
the localization of a protein in a cell (Donaldson, 2015)
Figure 2. Sensory neurons project onto specific laminae while motor
neurons connect directly to muscles (Caspary et al., 2003).
3. Hypothesis
Figure 3. The roles of LXR signaling are cell type specific (Courtney et al., 2016)
• If LXR is present in the same cell as marker of
known location, then that cell expresses LXR.
• If a cell is double-labeled, then it will produce a
yellow signal.
• If a cell does not contain LXR, then it will be
red.
• If LXR is expressed in a GFAP labeled astrocyte,
then there will be a yellow signal within the cell
or on its surface.
• If LXR is expressed in an Iba1 labeled microglial
cell, then a yellow signal will be observed on or
inside the cell.
• If LXR is expressed in a NeuN labeled neuron,
then a yellow signal will be observed.
• If LXR is expressed in a Olig2 labeled
oligodendrocyte, then a yellow signal will be
observed.
Figure 4. Comparison of direct and indirect immunofluorescence
(Abcam, 2021)
4. Materials and methods
1. Labeled a slide with the name of the protein marker and date
2. Collected data from paraffin sections of female spinal cord
• 11M wild-type
• 29M wild-type female
3. Deparaffinized the tissue, retrieved the antigen with PT module heated
bath
4. Made a well around the tissue with PAP pen
5. Utilized 0.5% Triton solution for LXR nuclear staining
6. Primary antibody solutions in donkey serum
• anti-Olig2 1:600
• anti-GFAP 1:1000
• anti-Iba 1:1000
• anti-NeuN 1:1000
• anti-LXRs 1:2000
• anti-LXRβ 1:2000
7. Secondary antibody solution 1:400 in PBST
• Anti-Rb Green 488 (LXRs)
• Anti-Gt Green 488 (LXRβ)
• Anti-Ms Red 594 (GFAP, NeuN)
• Anti-Rb Red 594 (Olig2, Iba1)
8. DAPI and coverslip, sealed with nail polish
9. Examined slides on Olympus FV3000 inverted confocal microscope
• 20X UCPLFLN20X N/A 0.7, WD 0.8-1.8 mm, w/Correction Collar
Product Name Species Brand & Product Number
anti-Olig2 rabbit Abcam ab-109186
anti-Iba1 rabbit Abcam ab-178846
anti-GFAP mouse Santa Cruz sc-65343
anti-NeuN mouse Millipore MAB377
anti-LXRbeta goat homemade
anti-LXRs rabbit LifeSpan Biosci. LS-B262
anti-goat 488 donkey
ThermoFisher Alexa Fluor
A11055
anti-mouse 594 donkey
ThermoFisher Alexa Fluor
A21203
anti-rabbit 594 donkey
ThermoFisher Alexa Fluor
A21207
DAPI mounting medium Santa Cruz sc-24941
5. Results
• NeuN stained cells showed some puncta with LXR
• GFAP stained cells had no LXR immunoreactivity
• Few or no Olig2 stained cells showed nuclear LXR staining
• Some Iba1+ cells showed nuclear LXR immunoreactivity
15. Abcam. (2021, January 21). Direct vs indirect immunofluorescence. https://www.abcam.com/secondary-antibodies/direct-vs-
indirect-immunofluorescence
Caspary, T., & Anderson, K. V. (2003). Patterning cell types in the dorsal spinal cord: what the mouse mutants say. Nat. Rev.
Neurosci., 4(4), 289-297. https://doi.org/10.1038/nrn1073
Courtney, R., & Landreth, G. E. (2016). LXR regulation of brain cholesterol: from development to disease. Trends Endocrinol.
Metab., 27(6), 404-414. https://dx.doi.org/10.1016%2Fj.tem.2016.03.018
Dobrott, C., Sathyamurthy, A., & Levin, A. J. (2019). Decoding cell type diversity within the spinal cord. Current Opinion in
Physiology, 8, 1-6. https://doi.org/10.1016/j.cophys.2018.11.006
Donaldson, J. G. (2015). Immunofluorescence staining. Current protocols in cell biology, 69(1), 4-
3. https://doi.org/10.1002/0471143030.cb0403s69
Johannssen, H. C., & Helmchen, F. (2013). Two-photon imaging of spinal cord cellular networks. Experimental Neurology, 242(),
18-26. https://doi.org/10.1016/j.expneurol.2012.07.014
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Works Cited
Editor's Notes
Good morning everyone, my name is Andrew and I am presenting my project on localizing proteins in the mouse spinal cord using immunofluorescence and the confocal microscope.
The mouse central nervous system consists of the brain and spinal cord. The other nerves in the body are the peripheral nervous system. The spinal cord is divided into 34 segments, consisting of cervical, thoracic, lumbar and coccygeal sections. Each segment sections each containing nerves that innervate different muscles. For example, the nerves in the L1 region of the spinal cord FIG1 appear to be involved with the flexing and extending of muscles. The peripheral nervous system sends signals to the spinal cord from specialized cells. These cells send projections through the dorsal root ganglia into the different lamina of the spinal cord. In FIG2 we see how these nerves feed into the dorsal horn of the spinal cord. Networks of neurons spread throughout the lamina in a translaminar fashion through axonal projections. The motor neurons on the other hand project out of the ventral spinal cord onto muscles. Different cell types can be identified using molecular markers. A method called indirect immunofluorescence can be used to determine if a protein is located within a cell.
This method of indirect immunofluorescence uses multiple antibodies to label an antigen. When the location of one protein is known a second marker may be added to the tissue in order to localize the unknown protein. By labeling the cells with LXR and markers for astrocytes, microglia, neurons, and oligodendrocytes the aim of this experiment was to determine which cells in the mouse spinal cord are highly IR for LXR.
Ablation of LXRs, notably LXR beta results in a neurodegenerative phenotype in knockout mice. This ALS-like loss of motor neurons is combined with accumulation of lipids according a 2005 article by Gustafsson etal. Although dysregulation of lipid homeostasis is assoicated with serveral ND diseases, there is no known mechanism for the death of MN. LXR controls the efflux of cholesterol in cells and regulates AQP4, and TFs like ABCA1. Astrocytes produce cholesterol which is used by neurons to maintain synapses. LXR is activated by the cholesterol metabolite 24-OHC. Microglia and astrocytes produce and lipidate apoE.
For this experiment, it was hypothesized that if nuclear LXR were present within a cell containing a known marker, then it would generate a yellow signal on the fluorescence microscope. Astrocytes were labeled with glial fibrillary acidic protein, microglia with Iba1, neurons with NeuN, and oligodendrocytes with Olig2.
The mouse spinal cord sections that were used came from an eleven month-old wild type female and a 29 M WT female. The sections were made from paraffin blocks containing a small piece of tissue from the L1 portion of the spinal cord. Xylene and alcohol were used to deparaffinize and rehydrate the tissue. A warm citrate buffer bath inside of a PT module was used to unmask the antigens. Using a wax pen a well was made around the section. Some triton was added to the section in order to permeablizie the nuclear membrane. If this is not done then the LXR antibody will not reach the cell nucleus. After the triton was washed off a solution of primary antibody was added to the tissue and allowed to incubate for an hour. This solution was washed off and the secondary antibody was added. The secondary antibody contains the fluorophore that illuminated the target antigen. LXR was labeled in green and the known markers Iba1 etc were labeled in Red. The last step is to add a drop of DAPI and a coverslip. Then the slide was sealed with nail polish and examined on the Olympus FV3000 inverted confocal microscope using a 20X objective which is equipped with correction collar. Our lab does not normally use #1.5 coverslips so the correction collar was adjusted in order to visualize the tissue sections at this magnification. The notch on the correction collar is shown in the picture. The table shows the different products I used to stain the tissue, what species they were made in, and the manufacturer.
After examination of this tissue, it was concluded that the NeuN+ neurons showed some puncta in the cytoplasm, GFAP+ cells had no LXR IR, Olig2+ cells had little or no LXR, and Iba1+ cells were highly IR. In the future, I would like to utilize DAPI that does not contain mounting media and #1.5 coverslips in order to examine the staining at higher magnification. Furthermore, I plan to examine the male mice, compare the expression of LXR to the female, and stain some brain tissue as a positive control.
This micrograph shows a NeuN stained neuron.
Although there is some green anti-LXR it does not appear in the nucleus, however, there are some puncta in the cytoplasm.
This micrograph shows some red GFAP+ astrocytes in the spinal cord
The LXR antibody does not appear in the nucleus or the axonal processes.
This is the Olig2 staining
There may be one yellow cell here but the nuclear DAPI staining does not appear to overlap with this area. This could a dead cell or some non-specific staining.
This is an Iba1+ microglia. Here you can see how the DAPI, red and green stains overlap indicating that the Iba1 antibody and LXR antibody are within the same cell.
This is another Iba1+ microglia showing similar results.
This an Iba1 immunostaining. It shows some of the cellular structures that branch out from the cell's main body.