The majority of 20th century investigations into anesthetic effects on the nervous system have used electrophysiology. Yet some fundamental limitations to electrophysiologic recordings, including the ...invasiveness of the technique, the need to place (potentially several) electrodes in every site of interest, and the difficulty of selectively recording from individual cell types, have driven the development of alternative methods for detecting neuronal activation. Two such alternative methods with cellular scale resolution have matured in the last few decades and will be reviewed here: the transcription of immediate early genes, foremost c-fos, and the influx of calcium into neurons as reported by genetically encoded calcium indicators, foremost GCaMP6. Reporters of c-fos allow detection of transcriptional activation even in deep or distant nuclei, without requiring the accurate targeting of multiple electrodes at long distances. The temporal resolution of c-fos is limited due to its dependence upon the detection of transcriptional activation through immunohistochemical assays, though the development of RT-PCR probes has shifted the temporal resolution of the assay when tissues of interest can be isolated. GCaMP6 has several isoforms that trade-off temporal resolution for signal to noise, but the fastest are capable of resolving individual action potential events, provided the microscope used scans quickly enough. GCaMP6 expression can be selectively targeted to neuronal populations of interest, and potentially thousands of neurons can be captured within a single frame, allowing the neuron-by-neuron reporting of circuit dynamics on a scale that is difficult to capture with electrophysiology, as long as the populations are optically accessible.
Whilst the general presumption of the public is that general anaesthesia prevents awareness of any sensory stimuli, Lennertz and colleagues have shown in this issue of the British Journal of ...Anaesthesia that 11% of young adults were able to respond to auditory commands when neuromuscular blocking drugs were prevented from reaching one arm using the isolated forearm technique. This occurred with anaesthetic regimens that followed usual clinical practice in each of the 10 countries that enrolled patients, and it was significantly more common in women than in men. This high incidence demands attention. Further characterisation of the experience of these patients is essential to our understanding of the state of general anaesthesia.
Protein kinase C (PKC) isozymes have remained elusive cancer targets despite the unambiguous tumor promoting function of their potent ligands, phorbol esters, and the prevalence of their mutations. ...We analyzed 8% of PKC mutations identified in human cancers and found that, surprisingly, most were loss of function and none were activating. Loss-of-function mutations occurred in all PKC subgroups and impeded second-messenger binding, phosphorylation, or catalysis. Correction of a loss-of-function PKCβ mutation by CRISPR-mediated genome editing in a patient-derived colon cancer cell line suppressed anchorage-independent growth and reduced tumor growth in a xenograft model. Hemizygous deletion promoted anchorage-independent growth, revealing that PKCβ is haploinsufficient for tumor suppression. Several mutations were dominant negative, suppressing global PKC signaling output, and bioinformatic analysis suggested that PKC mutations cooperate with co-occurring mutations in cancer drivers. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapies should focus on restoring, not inhibiting, PKC activity.
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•Cancer-associated PKC mutations are LOF and can act in a dominant-negative manner•Correcting a heterozygous PKCβ LOF mutation reduces tumor volume•Hemizygous deletion shows PKC is haploinsufficient for tumor suppression•Therapeutic strategies should aim to restore PKC activity instead of inhibiting it
Cancer-associated kinase mutations have generally been characterized as oncogenic, but an analysis of PKC mutations reveals that the majority are loss of function, indicating a tumor-suppressive role for this kinase and a shift in therapeutic strategies targeting PKC.
WHAT WE ALREADY KNOW ABOUT THIS TOPICA decrease in frontoparietal functional connectivity has been demonstrated with multiple anesthetic agents, and this decrease has been proposed as a final common ...functional pathway to produce anesthesia.Two alternative measures of long-range cortical interaction are coherence and phase-amplitude coupling. Although phase-amplitude coupling within frontal cortex changes with propofol administration, the effects of propofol on phase-amplitude coupling between different cortical areas have not previously been reported.
WHAT THIS ARTICLE TELLS US THAT IS NEWUsing a previously published monkey electrocorticography data set, it was found that propofol induced coherent slow oscillations in visual and oculomotor networks made up of cortical areas with strong anatomic projections.Frontal eye field within-area phase-amplitude coupling increased.Contrary to expectations from previous functional connectivity studies, interareal phase-amplitude coupling also increased with propofol.
BACKGROUND:Frontoparietal functional connectivity decreases with multiple anesthetics using electrophysiology and functional imaging. This decrease has been proposed as a final common functional pathway to produce anesthesia. Two alternative measures of long-range cortical interaction are coherence and phase-amplitude coupling. Although phase-amplitude coupling within frontal cortex changes with propofol administration, the effects of propofol on phase-amplitude coupling between different cortical areas have not previously been reported. Based on phase-amplitude coupling observed within frontal lobe during the anesthetized period, it was hypothesized that between-lead phase-amplitude coupling analysis should decrease between frontal and parietal leads during propofol anesthesia.
METHODS:A published monkey electrocorticography data set (N = 2 animals) was used to test for interactions in the cortical oculomotor circuit, which is robustly interconnected in primates, and in the visual system during propofol anesthesia using coherence and interarea phase-amplitude coupling.
RESULTS:Propofol induces coherent slow oscillations in visual and oculomotor networks made up of cortical areas with strong anatomic projections. Frontal eye field within-area phase-amplitude coupling increases with a time course consistent with a bolus response to intravenous propofol (modulation index increase of 12.6-fold). Contrary to the hypothesis, interareal phase-amplitude coupling also increases with propofol, with the largest increase in phase-amplitude coupling in frontal eye field low-frequency phase modulating lateral intraparietal area β-power (27-fold increase) and visual area 2 low-frequency phase altering visual area 1 β-power (19-fold increase).
CONCLUSIONS:Propofol anesthesia induces coherent oscillations and increases certain frontoparietal interactions in oculomotor cortices. Frontal eye field and lateral intraparietal area show increased coherence and phase-amplitude coupling. Visual areas 2 and 1, which have similar anatomic projection patterns, show similar increases in phase-amplitude coupling, suggesting higher order feedback increases in influence during propofol anesthesia relative to wakefulness. This suggests that functional connectivity between frontal and parietal areas is not uniformly decreased by anesthetics.
It is not clear how, after a large perturbation, the brain explores the vast space of potential neuronal activity states to recover those compatible with consciousness. Here, we analyze recovery from ...pharmacologically induced coma to show that neuronal activity en route to consciousness is confined to a low-dimensional subspace. In this subspace, neuronal activity forms discrete metastable states persistent on the scale of minutes. The network of transitions that links these metastable states is structured such that some states form hubs that connect groups of otherwise disconnected states. Although many paths through the network are possible, to ultimately enter the activity state compatible with consciousness, the brain must first pass through these hubs in an orderly fashion. This organization of metastable states, along with dramatic dimensionality reduction, significantly simplifies the task of sampling the parameter space to recover the state consistent with wakefulness on a physiologically relevant timescale.
Methods for studying oogenesis Hudson, Andrew M.; Cooley, Lynn
Methods,
06/2014, Letnik:
68, Številka:
1
Journal Article
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Drosophila oogenesis is an excellent system for the study of developmental cell biology. Active areas of research include stem cell maintenance, gamete development, pattern formation, cytoskeletal ...regulation, intercellular communication, intercellular transport, cell polarity, cell migration, cell death, morphogenesis, cell cycle control, and many more. The large size and relatively simple organization of egg chambers make them ideally suited for microscopy of both living and fixed whole mount tissue. A wide range of tools is available for oogenesis research. Newly available shRNA transgenic lines provide an alternative to classic loss-of-function F2 screens and clonal screens. Gene expression can be specifically controlled in either germline or somatic cells using the Gal4/UAS system. Protein trap lines provide fluorescent tags of proteins expressed at endogenous levels for live imaging and screening backgrounds. This review provides information on many available reagents and key methods for research in oogenesis.
The planning process has been, arguably, slow to adapt and adopt new technologies: It is perhaps only now that it is starting to move into a more digitally focused era. Yet, it is not the current ...thinking around the digital that is going to change planning; it is the emerging metaverse. It is a change on the near horizon that is there but is currently largely unseen in the urban planning profession. The metaverse is, at first sight, a mirror to the current world, a digital twin, but it is more than this: It is an inhabited mirror world where the physical dimensions and rules of time and space do not necessarily apply. Operating across scales, from the change of use of a building up to a local plan and onwards to the scale of future cities, these emerging metaverses will exist either directly within computational space or emerge into our physical space via augmented reality. With economic systems operating via blockchain technology and the ability to instigate aspects of planning law, interspaced with design fiction type scenarios, they represent a new tool kit for the urban planner, spatial, economic, and social. We explore these emerging spaces, taking a look at their origins and how the use of game engines have allowed participation and design to become part of the workflow of these 3D spaces. Via a series of examples, we look at the current state of the art, explore the short term future, and speculate on digital planning using these incoming metaverses 10 years from now.
In tissue engineering, an unresolved challenge is how to build complex 3D scaffolds in order to recreate the structure and function of human tissues and organs. Additive manufacturing techniques, ...such as 3D bioprinting, have the potential to build biological material with unprecedented spatial control; however, printing soft biological materials in air often results in poor fidelity. Freeform Reversible Embedding of Suspended Hydrogels (FRESH) is an embedded printing approach that solves this problem by extruding bioinks within a yield-stress support bath that holds the bioinks in place until cured. In this Perspective, we discuss the challenges of 3D printing soft and liquid-like bioinks and the emergence for FRESH and related embedded printing techniques as a solution. This includes the development of FRESH and embedded 3D printing within the bioprinting field and the rapid growth in adoption, as well as the advantages of FRESH printing for biofabrication and the new research results this has enabled. Specific focus is on the customizability of the FRESH printing technique where the chemical composition of the yield-stress support bath and aqueous phase crosslinker can all be tailored for printing a wide range of bioinks in complex 3D structures. Finally, we look ahead at the future of FRESH printing, discussing both the challenges and the opportunities that we see as the biofabrication field develops.
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