The zinc finger transcription factor PLAGL2 enhances stem cell fate and activates expression of ASCL2 in intestinal epithelial cells

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Plasmodium falciparum translational machinery condones polyadenosine repeats

Plasmodium falciparum is a causative agent of human malaria. Sixty percent of mRNAs from its extremely AT-rich (81%) genome harbor long polyadenosine (polyA) runs within their ORFs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate polyA runs cause ribosome stalling and frameshifting, triggering mRNA surveillance pathways and attenuating protein synthesis. Here, we show that P. falciparum is an exception to this rule. We demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately translated in P. falciparum cells. We show that polyA runs do not elicit any response from No Go Decay (NGD) or result in the production of frameshifted proteins. This is in stark contrast to what we observe in human cells or T. thermophila, an organism with similar AT-content. Finally, using stalling reporters we show that Plasmodium cells evolved not to have a fully functional NGD pathway

Gold nanoparticle-enhanced X-ray microtomography of the rodent reveals region-specific cerebrospinal fluid circulation in the brain

Cerebrospinal fluid (CSF) is essential for the development and function of the central nervous system (CNS). However, the brain and its interstitium have largely been thought of as a single entity through which CSF circulates, and it is not known whether specific cell populations within the CNS preferentially interact with the CSF. Here, we develop a technique for CSF tracking, gold nanoparticle-enhanced X-ray microtomography, to achieve micrometer-scale resolution visualization of CSF circulation patterns during development. Using this method and subsequent histological analysis in rodents, we identify previously uncharacterized CSF pathways from the subarachnoid space (particularly the basal cisterns) that mediate CSF-parenchymal interactions involving 24 functional-anatomic cell groupings in the brain and spinal cord. CSF distribution to these areas is largely restricted to early development and is altered in posthemorrhagic hydrocephalus. Our study also presents particle size-dependent CSF circulation patterns through the CNS including interaction between neurons and small CSF tracers, but not large CSF tracers. These findings have implications for understanding the biological basis of normal brain development and the pathogenesis of a broad range of disease states, including hydrocephalus

Elevations of intracellular calcium reflect normal voltage-dependent behavior, and not constitutive activity, of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

In smooth muscle, the gating of dihydropyridine-sensitive Ca2+ channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for Ca2+ influx and determining the bulk average cytoplasmic Ca2+ concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca2+ signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (−70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca2+ current (ICa) and evoked a rise in [Ca2+] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca2+] increase ([Ca2+]c) was approximately uniform, whereas that of the subplasma membrane space ([Ca2+]PM) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active Ca2+ channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca2+ channels were not normally constitutively active. The repetitive localized [Ca2+]PM rises (“persistent Ca2+ sparklets”) that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca2+ channels regulate the bulk average [Ca2+]c. A dihydropyridine blocker of Ca2+ channels, nimodipine, which blocked ICa and accompanying [Ca2+]c rise, reduced neither the resting bulk average [Ca2+]c (at −70 mV) nor the rise in [Ca2+]c, which accompanied an increased electrochemical driving force on the ion by hyperpolarization (−130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent Ca2+ channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca2+]c regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca2+]

Integrative neural behavior in a mammalian nervous system revealed using calcium imaging

Historically, research on nervous systems has been hindered by the complexity of such systems, and the inability to record activity in multiple neurons at the same time. As such, our current understanding of the way a nervous system functions has been the result of electrophysiological studies performed on single, or small groups of neurons, as well as research on relatively simple invertebrate models. In the work presented in this dissertation, we have used imaging techniques to explore the functions of several classes of neurons and auxiliary cells in the enteric nervous system of the murine large bowel during a compound motor behavior, the colonic migrating motor complex (CMMC). Using low-powered Ca2+ imaging in whole tissue preparations, we explored the relationship between the longitudinal and circular muscle layers of the colon, showing a high degree of synchronization between the two layers during the CMMC. This synchronization is further shown to be a function of enteric innervation, as addition of nicotinic and muscarinic antagonists disrupted coordinated Ca2+ waves in the two muscle layers. Using a mouse model for Hirschsprung’s disease (aganglionosis), we have shown that CMMCs do not occur in mice that lack enteric innervation, and Ca 2+ waves that persist are unsynchronized, and do not propagate sufficient distances to generate pellet propulsion. The myenteric plexus of the colon contains 13 different classes of enteric neurons, including sensory neurons, ascending and descending interneurons, and both excitatory and inhibitory motor neurons. The use of Ca2+ imaging as a technique to study the activity of enteric neurons provided little information regarding the class of neurons being observed. This dissertation details a number of techniques which we have developed to facilitate the identification of these enteric neurons using post hoc staining, and have subsequently used to identify inhibitory motor neurons, sensory neurons, types of interneurons and different classes of interstitial cells in the murine colon. Our work has identified AH/Dogiel Type II neurons, which stained intensely with the mitochondrial marker, Mitotracker, as the first responders in the generation of the CMMC in response to mucosal stimulations. Responses in these cells were abolished in the presence of 5-HT3 receptor antagonists, and when the mucosa was removed, suggesting that 5-HT release from enterochromaffin cells in the mucosa activates AH/Dogiel Type II neurons to generate a CMMC. Furthermore, we have shown that during the CMMC, there is an increase in Ca 2+ transient frequency in neurons that tested negative for NOS, and were presumably excitatory motor neurons, as well as a marked decrease in the activity of NOS+ve neurons, which were likely inhibitory motor neurons. Recent studies have identified a colonic occult reflex, which is activated by colonic elongation, leading to the activation of mechanosensitive descending inhibitory interneurons, which inhibit the activation of the peristaltic reflex. This occult reflex is thought to underlie slow transit constipation, and as the CMMC in the murine large bowel is responsible for fecal pellet propulsion, we sought to establish the effects of colonic elongation on the CMMC. Our findings show that colonic elongation led to a nitric oxide-mediated reduction in the amplitude of migrating complexes. During such elongation, enteric neurons, including AH/Dogiel Type II neurons exhibited reduced amplitude and frequency of spontaneous Ca2+ transients. Ca2+ imaging experiments of the myenteric plexus region during the CMMC revealed an increase in the Ca2+ transient frequency of ICC-MY, which, in the colon, are believed to underlie synchronization of the longitudinal and circular muscle layers. Using high-power imaging, we observed both excitatory and inhibitory nerve varicosities in close apposition to ICC-MY, which activated or inactivated the cells, respectively. Pharmacological studies revealed that ICC-MY have both muscarinic and NK1 receptors, which are likely the targets of ACh and TK release from excitatory motor neurons. Collectively these findings offer insight into the neuronal mechanism that underlies the generation and propagation of the colonic migrating motor complex in the murine large intestine. From a larger perspective, studies described in this dissertation represent an important step in our understanding of how neural networks in a mammalian nervous system functions to generate a complex rhythmic motor behavior

Integrative Neural Behavior in a Mammalian Nervous System Revealed Using Ca2+ Imaging

Historically, research on nervous systems has been hindered by the complexity of such systems, and the inability to record activity in multiple neurons at the same time. As such, our current understanding of the way a nervous system functions has been the result of electrophysiological studies performed on single, or small groups of neurons, as well as research on relatively simple invertebrate models. In the work presented in this dissertation, we have used imaging techniques to explore the functions of several classes of neurons and auxiliary cells in the enteric nervous system of the murine large bowel during a compound motor behavior, the colonic migrating motor complex (CMMC).Using low-powered Ca2+ imaging in whole tissue preparations, we explored the relationship between the longitudinal and circular muscle layers of the colon, showing a high degree of synchronization between the two layers during the CMMC. This synchronization is further shown to be a function of enteric innervation, as addition of nicotinic and muscarinic antagonists disrupted coordinated Ca2+ waves in the two muscle layers. Using a mouse model for Hirschsprung's disease (aganglionosis), we have shown that CMMCs do not occur in mice that lack enteric innervation, and Ca2+ waves that persist are unsynchronized, and do not propagate sufficient distances to generate pellet propulsion.The myenteric plexus of the colon contains 13 different classes of enteric neurons, including sensory neurons, ascending and descending interneurons, and both excitatory and inhibitory motor neurons. The use of Ca2+ imaging as a technique to study the activity of enteric neurons provided little information regarding the class of neurons being observed. This dissertation details a number of techniques which we have developed to facilitate the identification of these enteric neurons using post hoc staining, and have subsequently used to identify inhibitory motor neurons, sensory neurons, types of interneurons and different classes of interstitial cells in the murine colon.Our work has identified AH/Dogiel Type II neurons, which stained intensely with the mitochondrial marker, Mitotracker, as the first responders in the generation of the CMMC in response to mucosal stimulations. Responses in these cells were abolished in the presence of 5- HT3 receptor antagonists, and when the mucosa was removed, suggesting that 5-HT release from enterochromaffin cells in the mucosa activates AH/Dogiel Type II neurons to generate a CMMC. Furthermore, we have shown that during the CMMC, there is an increase in Ca2+ transient frequency in neurons that tested negative for NOS, and were presumably excitatory motor neurons, as well as a marked decrease in the activity of NOS+ve neurons, which were likely inhibitory motor neurons.Recent studies have identified a colonic occult reflex, which is activated by colonic elongation, leading to the activation of mechanosensitive descending inhibitory interneurons, which inhibit the activation of the peristaltic reflex. This occult reflex is thought to underlie slow transit constipation, and as the CMMC in the murine large bowel is responsible for fecal pellet propulsion, we sought to establish the effects of colonic elongation on the CMMC. Our findings show that colonic elongation led to a nitric oxide-mediated reduction in the amplitude of migrating complexes. During such elongation, enteric neurons, including AH/Dogiel Type II neurons exhibited reduced amplitude and frequency of spontaneous Ca2+ transients.Ca2+ imaging experiments of the myenteric plexus region during the CMMC revealed an increase in the Ca2+ transient frequency of ICC-MY, which, in the colon, are believed to underlie synchronization of the longitudinal and circular muscle layers. Using high-power imaging, we observed both excitatory and inhibitory nerve varicosities in close apposition to ICC-MY, which activated or inactivated the cells, respectively. Pharmacological studies revealed that ICC-MY have both muscarinic and NK1 receptors, which are likely the targets of ACh and TK release from excitatory motor neurons.Collectively these findings offer insight into the neuronal mechanism that underlies the generation and propagation of the colonic migrating motor complex in the murine large intestine. From a larger perspective, studies described in this dissertation represent an important step in our understanding of how neural networks in a mammalian nervous system functions to generate a complex rhythmic motor behavior