Perivascular invasion of primary human glioblastoma cells in organotypic human brain slices: human cells migrating in human brain.

INTRODUCTION: Glioblastoma (GBM) is an aggressive primary brain cancer. Lack of effective therapy is related to its highly invasive nature. GBM invasion has been studied with reductionist systems that do not fully recapitulate the cytoarchitecture of the brain. We describe a human-derived brain organotypic model to study the migratory properties of GBM IDH-wild type ex vivo. METHODS: Non-tumor brain samples were obtained from patients undergoing surgery (n = 7). Organotypic brain slices were prepared, and green fluorescent protein (GFP)-labeled primary human GBM IDH-wild type cells (GBM276, GBM612, GBM965) were placed on the organotypic slice. Migration was evaluated via microscopy and immunohistochemistry. RESULTS: After placement, cells migrated towards blood vessels; initially migrating with limited directionality, sending processes in different directions, and increasing their speed upon contact with the vessel. Once merged, migration speed decreased and continued to decrease with time (p < 0.001). After perivascular localization, migration is limited along the blood vessels in both directions. The percentage of cells that contact blood vessels and then continue to migrate along the vessel was 92.5% (- 3.9/ + 2.9)% while the percentage of cells that migrate along the blood vessel and leave was 7.5% (- 2.9/ + 3.9) (95% CI, Clopper-Pearson (exact); n = 256 cells from six organotypic cultures); these percentages are significantly different from the random (50%) null hypothesis (z = 13.6; p < 10-7). Further, cells increase their speed in response to a decrease in oxygen tension from atmospheric normoxia (20% O2) to anoxia (1% O2) (p = 0.033). CONCLUSION: Human organotypic models can accurately study cell migration ex vivo. GBM IDH-wild type cells migrate toward the perivascular space in blood vessels and their migratory parameters change once they contact vascular structures and under hypoxic conditions. This model allows the evaluation of GBM invasion, considering the human brain microenvironment when cells are removed from their native niche after surgery.

NIH Funding for Surgeon-Scientists in the US: What Is the Current Status?

BACKGROUND: Recent literature suggests that the future of surgeon-scientists in the US has been threatened for the past several decades. However, we documented an overall increase in NIH funding for surgeon-scientists, as well as the number of NIH-funded surgeons, from 2010 to 2020. STUDY DESIGN: NIH-funded principal investigators (PIs) were identified for June 2010 and June 2020 using the NIH internal data platform iSearch Grants (version 2.4). Biographical sketches were searched for key terms to identify surgeon-scientists. Grant research types and total grant costs were collected. American Association of Medical Colleges data were used to determine total surgeon and physician populations. Bivariate chi-square analyses were performed using population totals and were corroborated using z-tests of population proportions using JMP (version 13.0.0). A 2-tailed p value <0.05 was considered significant. RESULTS: In June of 2020, a total of 1,031 surgeon-scientists held $872,456,710 in NIH funding. The percentage of funded surgeons significantly increased from 2010 (0.5%) to 2020 (0.7%) (p < 0.05), and the percentage of funded other physicians significantly decreased from 2.2% in 2010 to 1.6% in 2020 (p < 0.05). All surgeons sustained R grant funding at both time points (58% in 2020 and 60% in 2010), and specifically maintained basic science-focused R grants (73% in 2020 and 78% in 2010). CONCLUSIONS: Our study found surgeon-scientists are increasing in number and NIH funding and are becoming more diverse in their research efforts, while maintaining a focus on basic science.

Influenza Hemagglutinin Modulates Phosphatidylinositol 4,5-Bisphosphate Membrane Clustering.

The lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms nanoscopic clusters in cell plasma membranes; however, the processes determining PIP2 mobility and thus its spatial patterns are not fully understood. Using super-resolution imaging of living cells, we find that PIP2 is tightly colocalized with and modulated by overexpression of the influenza viral protein hemagglutinin (HA). Within and near clusters, HA and PIP2 follow a similar spatial dependence, which can be described by an HA-dependent potential gradient; PIP2 molecules move as if they are attracted to the center of clusters by a radial force of 0.079 ± 0.002 pN in HAb2 cells. The measured clustering and dynamics of PIP2 are inconsistent with the unmodified forms of the raft, tether, and fence models. Rather, we found that the spatial PIP2 distributions and how they change in time are explained via a novel, to our knowledge, dynamic mechanism: a radial gradient of PIP2 binding sites that are themselves mobile. This model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.

Rounding precedes rupture and breakdown of vacuolar membranes minutes before malaria parasite egress from erythrocytes.

Because Plasmodium falciparum replicates inside of a parasitophorous vacuole (PV) within a human erythrocyte, parasite egress requires the rupture of two limiting membranes. Parasite Ca2+ , kinases, and proteases contribute to efficient egress; their coordination in space and time is not known. Here, the kinetics of parasite egress were linked to specific steps with specific compartment markers, using live-cell microscopy of parasites expressing PV-targeted fluorescent proteins, and specific egress inhibitors. Several minutes before egress, under control of parasite [Ca2+ ]i , the PV began rounding. Then after ~1.5 min, under control of PfPKG and SUB1, there was abrupt rupture of the PV membrane and release of vacuolar contents. Over the next ~6 min, there was progressive vacuolar membrane deterioration simultaneous with erythrocyte membrane distortion, lasting until the final minute of the egress programme when newly formed parasites mobilised and erythrocyte membranes permeabilised and then ruptured-a dramatic finale to the parasite cycle of replication.

Exploitation of a newly-identified entry pathway into the malaria parasite-infected erythrocyte to inhibit parasite egress.

While many parasites develop within host cells to avoid antibody responses and to utilize host cytoplasmic resources, elaborate egress processes have evolved to minimize the time between escaping and invading the next cell. In human erythrocytes, malaria parasites perforate their enclosing erythrocyte membrane shortly before egress. Here, we show that these pores clearly function as an entry pathway into infected erythrocytes for compounds that inhibit parasite egress. The natural glycosaminoglycan heparin surprisingly inhibited malaria parasite egress, trapping merozoites within infected erythrocytes. Labeled heparin neither bound to nor translocated through the intact erythrocyte membrane during parasite development, but fluxed into erythrocytes at the last minute of the parasite lifecycle. This short encounter was sufficient to significantly inhibit parasite egress and dispersion. Heparin blocks egress by interacting with both the surface of intra-erythrocytic merozoites and the inner aspect of erythrocyte membranes, preventing the rupture of infected erythrocytes but not parasitophorous vacuoles, and independently interfering with merozoite disaggregation. Since this action of heparin recapitulates that of neutralizing antibodies, membrane perforation presents a brief opportunity for a new strategy to inhibit parasite egress and replication.

Blast shockwaves propagate Ca(2+) activity via purinergic astrocyte networks in human central nervous system cells.

In a recent study of the pathophysiology of mild, blast-induced traumatic brain injury (bTBI) the exposure of dissociated, central nervous system (CNS) cells to simulated blast resulted in propagating waves of elevated intracellular Ca(2+). Here we show, in dissociated human CNS cultures, that these calcium waves primarily propagate through astrocyte-dependent, purinergic signaling pathways that are blocked by P2 antagonists. Human, compared to rat, astrocytes had an increased calcium response and prolonged calcium wave propagation kinetics, suggesting that in our model system rat CNS cells are less responsive to simulated blast. Furthermore, in response to simulated blast, human CNS cells have increased expressions of a reactive astrocyte marker, glial fibrillary acidic protein (GFAP) and a protease, matrix metallopeptidase 9 (MMP-9). The conjoint increased expression of GFAP and MMP-9 and a purinergic ATP (P2) receptor antagonist reduction in calcium response identifies both potential mechanisms for sustained changes in brain function following primary bTBI and therapeutic strategies targeting abnormal astrocyte activity.

Automated analysis of a diverse synapse population.

Synapses of the mammalian central nervous system are highly diverse in function and molecular composition. Synapse diversity per se may be critical to brain function, since memory and homeostatic mechanisms are thought to be rooted primarily in activity-dependent plastic changes in specific subsets of individual synapses. Unfortunately, the measurement of synapse diversity has been restricted by the limitations of methods capable of measuring synapse properties at the level of individual synapses. Array tomography is a new high-resolution, high-throughput proteomic imaging method that has the potential to advance the measurement of unit-level synapse diversity across large and diverse synapse populations. Here we present an automated feature extraction and classification algorithm designed to quantify synapses from high-dimensional array tomographic data too voluminous for manual analysis. We demonstrate the use of this method to quantify laminar distributions of synapses in mouse somatosensory cortex and validate the classification process by detecting the presence of known but uncommon proteomic profiles. Such classification and quantification will be highly useful in identifying specific subpopulations of synapses exhibiting plasticity in response to perturbations from the environment or the sensory periphery.

Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers.

A lack of methods for measuring the protein compositions of individual synapses in situ has so far hindered the exploration and exploitation of synapse molecular diversity. Here, we describe the use of array tomography, a new high-resolution proteomic imaging method, to determine the composition of glutamate and GABA synapses in somatosensory cortex of Line-H-YFP Thy-1 transgenic mice. We find that virtually all synapses are recognized by antibodies to the presynaptic phosphoprotein synapsin I, while antibodies to 16 other synaptic proteins discriminate among 4 subtypes of glutamatergic synapses and GABAergic synapses. Cell-specific YFP expression in the YFP-H mouse line allows synapses to be assigned to specific presynaptic and postsynaptic partners and reveals that a subpopulation of spines on layer 5 pyramidal cells receives both VGluT1-subtype glutamatergic and GABAergic synaptic inputs. These results establish a means for the high-throughput acquisition of proteomic data from individual cortical synapses in situ.

Array tomography: rodent brain fixation and embedding.

Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. This protocol describes the fixation and processing required to prepare tissues for immunofluorescence array tomography.

Array tomography: production of arrays.

Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time consuming and require some practice to perfect. This protocol describes the sectioning of embedded tissues and the mounting of the serial arrays. The procedures require some familiarity with the techniques used for ultramicrotome sectioning for electron microscopy.

Array tomography: immunostaining and antibody elution.

Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. In this protocol, tissue arrays are prepared for imaging by tagging with primary antibodies against specific cellular targets, followed by labeling with fluorescent secondary antibodies. Alternatively, fluorescent proteins that have been introduced into the tissue before dissection can be used.

Array tomography: imaging stained arrays.

Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. In this protocol, tissue arrays are imaged using conventional wide-field fluorescence microscopy. Images can be captured manually or, with the appropriate software and hardware, the process can be automated.

Array tomography: semiautomated image alignment.

Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. Successful array tomography requires that the captured images be properly stacked and aligned, and the software to achieve these ends is freely available. This protocol describes the construction of volumetric image stacks from images of fluorescently labeled arrays for three-dimensional image visualization, analysis, and archiving.

Array tomography: high-resolution three-dimensional immunofluorescence.

Array tomography, which is described in this article, is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Although the fabrication procedures can be relatively difficult, the high resolution, depth invariance, and molecular discrimination offered by array tomography justify the effort involved. Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture.

Classical MHCI molecules regulate retinogeniculate refinement and limit ocular dominance plasticity.

Major histocompatibility complex class I (MHCI) genes were discovered unexpectedly in healthy CNS neurons in a screen for genes regulated by neural activity. In mice lacking just 2 of the 50+ MHCI genes H2-K(b) and H2-D(b), ocular dominance (OD) plasticity is enhanced. Mice lacking PirB, an MHCI receptor, have a similar phenotype. H2-K(b) and H2-D(b) are expressed not only in visual cortex, but also in lateral geniculate nucleus (LGN), where protein localization correlates strongly with synaptic markers and complement protein C1q. In K(b)D(b-/-) mice, developmental refinement of retinogeniculate projections is impaired, similar to C1q(-/-) mice. These phenotypes in K(b)D(b-/-) mice are strikingly similar to those in beta2 m(-/-)TAP1(-/-) mice, which lack cell surface expression of all MHCIs, implying that H2-K(b) and H2-D(b) can account for observed changes in synapse plasticity. H2-K(b) and H2-D(b) ligands, signaling via neuronal MHCI receptors, may enable activity-dependent remodeling of brain circuits during developmental critical periods.