Type I interferon causes thrombotic microangiopathy by a dose-dependent toxic effect on the microvasculature.

Many drugs have been reported to cause thrombotic microangiopathy (TMA), yet evidence supporting a direct association is often weak. In particular, TMA has been reported in association with recombinant type I interferon (IFN) therapies, with recent concern regarding the use of IFN in multiple sclerosis patients. However, a causal association has yet to be demonstrated. Here, we adopt a combined clinical and experimental approach to provide evidence of such an association between type I IFN and TMA. We show that the clinical phenotype of cases referred to a national center is uniformly consistent with a direct dose-dependent drug-induced TMA. We then show that dose-dependent microvascular disease is seen in a transgenic mouse model of IFN toxicity. This includes specific microvascular pathological changes seen in patient biopsies and is dependent on transcriptional activation of the IFN response through the type I interferon α/ß receptor (IFNAR). Together our clinical and experimental findings provide evidence of a causal link between type I IFN and TMA. As such, recombinant type I IFN therapies should be stopped at the earliest stage in patients who develop this complication, with implications for risk mitigation.

Subcellular Chemical Imaging of Antibiotics in Single Bacteria Using C60-Secondary Ion Mass Spectrometry.

The inherent difficulty of discovering new and effective antibacterials and the rapid development of resistance particularly in Gram-negative bacteria, illustrates the urgent need for new methods that enable rational drug design. Here we report the development of 3D imaging cluster Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) as a label-free approach to chemically map small molecules in aggregated and single Escherichia coli cells, with ∼300 nm spatial resolution and high chemical sensitivity. The feasibility of quantitative analysis was explored, and a nonlinear relationship between treatment dose and signal for tetracycline and ampicillin, two clinically used antibacterials, was observed. The methodology was further validated by the observation of reduction in tetracycline accumulation in an E. coli strain expressing the tetracycline-specific efflux pump (TetA) compared to the isogenic control. This study serves as a proof-of-concept for a new strategy for chemical imaging at the nanoscale and has the potential to aid discovery of new antibacterials.

Nonviral delivery of self-amplifying RNA vaccines.

Despite more than two decades of research and development on nucleic acid vaccines, there is still no commercial product for human use. Taking advantage of the recent innovations in systemic delivery of short interfering RNA (siRNA) using lipid nanoparticles (LNPs), we developed a self-amplifying RNA vaccine. Here we show that nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA. This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors. Given the many positive attributes of nucleic acid vaccines, our results suggest that a comprehensive evaluation of nonviral technologies to deliver self-amplifying RNA vaccines is warranted.

Structure-kinetic relationship of carbapenem antibacterials permeating through E. coli OmpC porin.

The emergence of Gram-negative "superbugs" exhibiting resistance to known antibacterials poses a major public health concern. Low molecular weight Gram-negative antibacterials are believed to penetrate the outer bacterial membrane (OM) through porin channels. Therefore, intracellular exposure needed to drive antibacterial target occupancy should depend critically on the translocation rates through these proteins and avoidance of efflux pumps. We used electrophysiology to study the structure-translocation kinetics relationships of a set of carbapenem antibacterials through purified porin OmpC reconstituted in phospholipid bilayers. We also studied the relative susceptibility of OmpC+ and OmpC- E. coli to these compounds as an orthogonal test of translocation. Carbapenems exhibit good efficacy in OmpC-expressing E. coli cells compared with other known antibacterials. Ertapenem, which contains an additional acidic group compared to other analogs, exhibits the fastest entry into OmpC (k(on) ≈ 2 × 10(4) M(-1) s(-1)). Zwitterionic compounds with highly polar groups attached to the penem-2 ring, including panipenem, imipenem and doripenem exhibit faster k(on) (>10(4) M(-1) s(-1)), while meropenem and biapenem with fewer exposed polar groups exhibit slower k(on) (∼5 × 10(3) M(-1) s(-1)). Tebipenem pivoxil and razupenem exhibit ∼13-fold slower k(on) (∼1.5 × 10(3) M(-1) s(-1)) than ertapenem. Overall, our results suggest that (a) OmpC serves as an important route of entry of these antibacterials into E. coli cells; and (b) that the structure-kinetic relationships of carbapenem translocation are governed by H-bond acceptor/donor composition (in accordance with our previous findings that the enthalpic cost of transferring water from the constriction zone to bulk solvent increases in the presence of exposed nonpolar groups).

Altered morphology and 3D architecture of brain vasculature in a mouse model for Alzheimer's disease.

Substantial evidence from epidemiological, pathological, and clinical reports suggests that vascular factors are critical in the pathogenesis of Alzheimer's disease (AD), and changes in blood flow are currently the most reliable indicators of the disease. We previously reported that older APP23 transgenic (tg) mice have significant blood flow alterations correlated with structural modifications of blood vessels. For the present study, our objective was to analyze the age-dependent morphological and architectural changes of the cerebral vasculature of APP23 tg mice. To visualize the 3D arrangement of the entire brain vasculature, we used vascular corrosion casts. Already at young ages, when typically parenchymal amyloid plaques are not yet present, APP23 tg mice had significant alterations, particularly of the microvasculature, often accompanied by small deposits attached to the vessels. In older animals, vasculature abruptly ended at amyloid plaques, resulting in holes. Often, small deposits were sitting near or at the end of truncated vessels. Between such holes, the surrounding vascular array appeared more dense and showed features typical for angiogenesis. We propose that small amyloid aggregates associated with the microvasculature lead to morphological and architectural alterations of the vasculature, resulting in altered local blood flow. The characteristic early onset of vascular alterations suggests that imaging blood flow and/or vasculature architecture could be used as a tool for early diagnosis of the disease and to monitor therapies.

Hierarchical imaging and computational analysis of three-dimensional vascular network architecture in the entire postnatal and adult mouse brain.

The formation of new blood vessels and the establishment of vascular networks are crucial during brain development, in the adult healthy brain, as well as in various diseases of the central nervous system. Here, we describe a step-by-step protocol for our recently developed method that enables hierarchical imaging and computational analysis of vascular networks in postnatal and adult mouse brains. The different stages of the procedure include resin-based vascular corrosion casting, scanning electron microscopy, synchrotron radiation and desktop microcomputed tomography imaging, and computational network analysis. Combining these methods enables detailed visualization and quantification of the 3D brain vasculature. Network features such as vascular volume fraction, branch point density, vessel diameter, length, tortuosity and directionality as well as extravascular distance can be obtained at any developmental stage from the early postnatal to the adult brain. This approach can be used to provide a detailed morphological atlas of the entire mouse brain vasculature at both the postnatal and the adult stage of development. Our protocol allows the characterization of brain vascular networks separately for capillaries and noncapillaries. The entire protocol, from mouse perfusion to vessel network analysis, takes ~10 d.

Hybrid FMT-CT imaging of amyloid-beta plaques in a murine Alzheimer's disease model.

The need to study molecular and functional parameters of Alzheimer's disease progression in animal models has led to the development of disease-specific fluorescent markers. However, curved optical interfaces and a highly heterogeneous internal structure make quantitative fluorescence imaging of the murine brain a particularly challenging tomographic problem. We investigated the integration of X-ray computed tomography (CT) information into a state-of-the-art fluorescence molecular tomography (FMT) scheme and establish that the dual-modality approach is essential for high fidelity reconstructions of distributed fluorescence within the murine brain, as compared to conventional fluorescence tomography. We employ this method in vivo using a fluorescent oxazine dye to quantify amyloid-beta plaque burden in transgenic APP23 mice modeling Alzheimer's disease. Multi-modal imaging allows for accurate signal localization and correlation of in vivo findings to ex vivo studies. The results point to FMT-CT as an essential tool for in vivo study of neurodegenerative disease in animal models and potentially humans.

Development and Optimization of a Higher-Throughput Bacterial Compound Accumulation Assay.

The Gram-negative bacterial permeability barrier, coupled with efflux, raises formidable challenges to antibiotic drug discovery. The absence of efficient assays to determine compound penetration into the cell and impact of efflux makes the process resource-intensive, small-scale, and lacking much success. Here, we present BacPK: a label-free, solid phase extraction-mass spectrometry (SPE-MS)-based assay that measures total cellular compound accumulation in Escherichia coli. The BacPK assay is a 96-well accumulation assay that takes advantage of 9 s/sample SPE-MS throughput. This enables the analysis of each compound in a four-point dose-response in isogenic strain pairs along with a no-cell control and 16-point external standard curve, all in triplicate. To validate the assay, differences in accumulation were examined for tetracycline (Tet) and two analogs, confirming that close analogs can differ greatly in accumulation. Tet cellular accumulation was also compared for isogenic strains exhibiting Tet resistance due to the expression of an efflux pump (TetA) or ribosomal protection protein (TetM), confirming only TetA affected cellular Tet accumulation. Finally, using a diverse set of antibacterial compounds, we confirmed the assay's ability to quantify differences in accumulation for isogenic strain pairs with efflux or permeability alterations that are consistent with differences in susceptibility seen for the compounds.

Enabling drug discovery and development through single-cell imaging.

INTRODUCTION: Single-cell imaging-based assays are an area of active and growing investment in drug discovery and development. This approach offers researchers the capability to interrogate rare subpopulations of cells with minimal sample consumption and multiplexed readouts. Recent technological advances in the optical interrogation and manipulation of single cells have substantially increased the throughput and sensitivity of these assays. Areas covered: In this review, the authors focus on three classes of single-cell imaging-based analyses: single-cell microscopy combined with microfluidics, mass spectrometric imaging for subcellular compound localization, and imaging mass cytometry (IMC). They provide an overview of each technology and recent examples of their utility in advancing drug discovery, based on the potential for scalability, multiplexing, and capability to generate definitive data on cellular heterogeneity and target engagement. Expert opinion: Understanding target engagement and heterogeneity at the single-cell level will enable the development of safer and more effective therapies, particularly for new modalities like CAR-T cell therapies and gene editing approaches (AAV, CRISPR). Successful adoption of new single-cell imaging-based approaches in drug discovery will require tandem investment in advanced computational analysis and bioinformatic approaches, due to the complexity and multivariate nature of single-cell imaging data.

Functional consequences of hippocampal neuronal ectopia in the apolipoprotein E receptor-2 knockout mouse.

Little is known about the impact ectopically located neurons have on the functional connectivity of local circuits. The ApoER2 knockout mouse has subtle cytoarchitectural disruptions, altered prepulse inhibition, and memory abnormalities. We evaluated this mouse mutant as a model to study the role ectopic neurons play in the manifestation of symptoms associated with brain diseases. We found that ectopic CA1 pyramidal and inhibitory neurons in the ApoER2 knockout hippocampus are organized into two distinct stratum pyramidale layers. In vitro analyses found that ApoER2 is not required for neurons to reach maturity in regard to dendritic arborization and synaptic structure density, and electrophysiological testing determined that neurons in both strata pyramidale are integrated into the hippocampal network. However, the presence of these two layers alters the spatiotemporal pattern of hippocampal activity, which may explain why ApoER2 knockout mice have selective cognitive dysfunctions that are revealed only under challenging conditions.

(56)Fe-particle radiation reduces neuronal output and attenuates lipopolysaccharide-induced inhibition of long-term potentiation in the mouse hippocampus.

Exposure to space radiation consisting of high-energy charged (56)Fe particles represents a significant health risk for astronauts. (56)Fe-particle radiation affects the synaptic plasticity of the hippocampus and alters its response to the experimental immunological stressor lipopolysaccharide (LPS). We previously showed in mice that 1 month after exposure to (56)Fe-particle radiation, the LPS-induced inhibition of hippocampal long-term potentiation (LTP) was significantly attenuated, resulting in seemingly normal LTP. In the current study, we investigated this phenomenon further at longer times postirradiation. We exposed mice to accelerated iron particles ((56)Fe; 600 MeV/nucleon; 1, 2, 4 Gy; brain only), and 1, 3, 6 or 12 months postirradiation we administered LPS. Four hours after the intraperitoneal LPS injection, we prepared hippocampal slices to measure synaptic excitability and plasticity between CA3-CA1 neurons. In unexposed mice, we confirmed that LPS inhibited LTP at all times. However, in mice exposed to 2 Gy, the LPS-induced LTP inhibition was attenuated and reversed to control values. Such reversal was evident at 1 and 3 months but not 6 and 12 months postirradiation. In addition, at 6 and 12 months postirradiation, we observed inhibition of population spike (PS) amplitudes at 4 Gy that correlated with decrements in dendritic potentials, suggesting synaptic damage. Our data show that (56)Fe-particle radiation affects the response of the hippocampus to an immunological stressor and that the alterations progress over time.

Advances and challenges in bacterial compound accumulation assays for drug discovery.

The identification of potent in vitro inhibitors of essential bacterial targets is relatively straightforward, however vanishingly few of these molecules have Gram-negative antibacterial potency and spectrum because of a failure to accumulate inside the bacteria. The Gram-negative bacterial cell envelope provides a formidable barrier to entry and couples with efflux pumps to prevent compound accumulation. Assays to measure the cellular permeation, efflux and accumulation of compounds in bacteria continue to be innovated and refined to guide drug discovery. Important advances in the label-free detection of compounds associated with or passing through bacteria rely on mass spectrometry This technique holds the promise of bacterial subcellular resolution and the throughput needed to test libraries of compounds to evaluate structure-accumulation relationships.

Effects of lipopolysaccharide on 56Fe-particle radiation-induced impairment of synaptic plasticity in the mouse hippocampus.

Space radiation, including high-mass, high-Z, high-energy particles (HZE; e.g. (56)Fe), represents a significant health risk for astronauts, and the central nervous system (CNS) may be a vulnerable target. HZE-particle radiation may directly affect neuronal function, or during immunological challenge, it may alter immune system-to-CNS communication. To test these hypotheses, we exposed mice to accelerated iron particles ((56)Fe; 600 MeV/nucleon; 1, 2, 4 Gy; brain only) and 1 month later prepared hippocampal slices to measure the effects of radiation on neurotransmission and synaptic plasticity in CA1 neurons. In a model of immune system-to-CNS communication, these electrophysiological parameters were measured in irradiated mice additionally challenged with the peripheral immunological stressor lipopolysaccharide (LPS) injected intraperitoneally 4 h before the slice preparation. Exposure to (56)Fe particles alone increased dendritic excitability and inhibited plasticity. In control mice (0 Gy), LPS treatment also inhibited synaptic plasticity. Paradoxically, in mice exposed to 2 Gy, the LPS treatment restored synaptic plasticity to levels similar to those found in controls (0 Gy, no LPS). Our results indicate that HZE-particle radiation alters normal electrophysiological properties of the CNS and the hippocampal response to LPS.

Hippocampal dendritic arbor growth in vitro: regulation by Reelin-Disabled-1 signaling.

The cytoplasmic adaptor protein Disabled-1 (Dab1), which is a key component of the Reelin-signaling pathway, has been suggested to be required for neuronal dendritic development. However, only data from studies on immature cultures [< or = 6 days in vitro (DIV)] and cytoarchitectural analyses of mutant mice have been used to formulate this hypothesis. Therefore, to determine if Reelin-Dab1 signaling is specifically required for neurons to develop mature dendrites in respect to length and complexity, we analyzed dendritic development in mature cultures derived from Dab1 knockout (ko) embryos. No significant differences in dendritic length or complexity between Dab1 ko and wt cultures were found at 20 DIV. An examination of dendritic development in maturing cultures found significant differences in dendritic length between mutant and wt cultures at 4 DIV, but detected no differences in complexity. In addition, by 7 DIV, all measures were statistically the same between cultures. Therefore, although Reelin-Dab1 signaling promotes hippocampal dendrite development, Dab1 is not required for neurons to reach maturity with respect to dendritic length and complexity. Furthermore, analyses of 4 DIV cultures derived from Dab1 heterozygotes or mice that express only the natural splice form of Dab1 (p45) found that Dab1(p45/-) hemizygote, but not Dab1(p45/p45) and Dab1 heterozygote cultures had significantly shorter dendrites than those in wt cultures. Thus, a substantial attenuation of the Reelin-Dab1 signal is required before dendrite elongation is significantly decreased at 4 DIV. Moreover, experiments that incorporated a Reelin-neutralizing antibody support the hypothesis that the role(s) Reelin-signaling plays in dendritic maturation is different than the one it has in neuronal positioning.

Technical considerations in longitudinal multispectral small animal molecular imaging.

In a previous study, we investigated physical methods to reduce whole-body, diet-related autofluorescence interference in several mouse strains through changes in animal diet. Measurements of mice with an in vivo multispectral imaging system over a 21-day period allowed for the quantification of concentration changes in multiple in vivo fluorophores. To be an effective instrument, a multispectral imaging system requires a priori spectral knowledge, the form and importance of which is not necessarily intuitive, particularly when noninvasive in vivo longitudinal imaging studies are performed. Using an optimized spectral library from a previous autofluorescence-reduction study as a model, we investigated two additional spectral definition techniques to illustrate the results of poor spectral definition in a longitudinal fluorescence imaging study. Here we systematically evaluate these results and show how poor spectral definition can lead to physiologically irrelevant results. This study concludes that the proper selection of robust spectra corresponding to each specific fluorescent molecular label of interest is of integral importance to enable effective use of multispectral imaging techniques in longitudinal fluorescence studies.

Assessing Antibiotic Permeability of Gram-Negative Bacteria via Nanofluidics.

While antibiotic resistance is increasing rapidly, drug discovery has proven to be extremely difficult. Antibiotic resistance transforms some bacterial infections into deadly medical conditions. A significant challenge in antibiotic discovery is designing potent molecules that enter Gram-negative bacteria and also avoid active efflux mechanisms. Critical analysis in rational drug design has been hindered by the lack of effective analytical tools to analyze the bacterial membrane permeability of small molecules. We design, fabricate, and characterize the nanofluidic device that actively loads more than 200 single bacterial cells in a nanochannel array. We demonstrate a gigaohm seal between the nanochannel walls and the loaded bacteria, restricting small molecule transport to only occur through the bacterial cell envelope. Quantitation of clindamycin translocation through wild-type and efflux-deficient (ΔtolC) Escherichia coli strains via nanofluidic-interfaced liquid chromatography mass spectrometry shows higher levels of translocation for wild-type E. coli than for an efflux-deficient strain. We believe that the assessment of compound permeability in Gram-negative bacteria via the nanofluidic analysis platform will be an impactful tool for compound permeation and efflux studies in bacteria to assist rational antibiotic design.

New polyurethane-based material for vascular corrosion casting with improved physical and imaging characteristics.

Vascular corrosion casting has been established as a method to reconstruct the three-dimensional (3D) structure of blood vessels of organs and tissues. After replacing the blood volume with a low viscosity resin, the surrounding tissue is removed to replicate the vascular architecture, typically using scanning electron microscopy (SEM). To date available casting resins have had significant limitations such as lack of viscosity, leading to insufficient perfusion of smallest capillaries in organs and tissues of smaller species, interaction with surrounding tissue or fragility of resulting casts. We have reported here about a new polyurethane-based casting resin (PU4ii) with superior physical and imaging characteristics. Low viscosity, timely polymerization, and minimal shrinking of PU4ii produces high quality casts, including the finest capillaries. These casts are highly elastic while retaining their original structure to facilitate postcasting tissue dissection and pruning of casts. SEM images illustrate the high reproduction quality, including endothelial cell imprints, features that allow one to discern arterial and veinal vessels. For quantitative analysis, casts from PU4ii can be imaged using micro-computed tomography to produce digital 3D reconstructions. The inherent fluorescence of PU4ii is sufficient to reproduce casts with or without tissue using confocal microscopy (CM). Because of the simplified casting procedure, the high reproducibility and the superior reproduction quality, a combination of vascular corrosion casting using PU4ii with advanced imaging technologies has great potential to support the description of vascular defects and drug effects in disease models using mutant mice.

Age-dependent cerebrovascular abnormalities and blood flow disturbances in APP23 mice modeling Alzheimer's disease.

Neuropathological changes associated with Alzheimer's disease (AD) such as amyloidplaques, cerebral amyloid angiopathy, and related pathologies are reproduced in APP23 transgenic mice overexpressing amyloid precursor protein (APP) with the Swedish mutation. Magnetic resonance angiography (MRA) was applied to probe, in vivo, the cerebral arterial hemodynamics of these mice. Flow voids were detected at the internal carotid artery of 11-month-old APP23 mice. At the age of 20 months, additional flow disturbances were observed in large arteries at the circle of Willis. Vascular corrosion casts obtained from the same mice revealed that vessel elimination, deformation, or both had taken place at the sites where flow voids were detected by MRA. The detailed three-dimensional architecture of the vasculature visible in the casts assisted the identification of smaller vessels most likely formed as substitution or anastomosis within the circle of Willis. Angiograms and corrosion casts from nontransgenic, age-matched mice manifested no major abnormalities in the cerebrovascular arterial flow pattern. Because no transgene overexpression has been found in the cerebrovasculature of APP23 mice and no deposits of amyloid-beta (Abeta) were observed in large arteries in the region of the circle of Willis, the present results suggest that soluble Abeta may exert deleterious effects on the vasculature. Our findings support the idea that cerebral circulatory abnormalities evolving progressively could contribute to AD pathogenesis. The study also shows the power of MRA to identify changes of vascular function in genetically engineered mice. MRA as a noninvasive technique could be applied to test new therapeutic concepts in animal models of AD and in humans.

Targeted disruption of RC3 reveals a calmodulin-based mechanism for regulating metaplasticity in the hippocampus.

We used homologous recombination in the mouse to knock-out RC3, a postsynaptic, calmodulin-binding PKC substrate. Mutant brains exhibited lower immunoreactivity to phospho-Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) but had the same synaptic density as wild type and did not exhibit a gross neuroanatomical phenotype. Basal excitatory synaptic transmission in CA1 was depressed, long-term potentiation (LTP) was enhanced, and the depressant effects of the metabotropic glutamate receptor (mGluR) agonist (RS)-3,5-dihydroxyphenylglycine was occluded compared with littermate controls. The frequency-response curve was displaced to the left, and long-term depression (LTD) could not be induced unless low-frequency stimuli were preceded by high-frequency tetani. Depotentiation was much more robust in the mutant, and only one stimulus was required to saturate LTD in primed mutant hippocampi, whereas multiple low-frequency stimuli were required in wild-type slices. Thus, ablation of RC3 appears to render the postsynaptic neuron hypersensitive to Ca(2+), decreasing its LTD and LTP thresholds and accentuating the effects of priming stimuli. We propose an mGluR-dependent CaM-based sliding threshold mechanism for metaplasticity that is governed by the phosphorylation states of RC3 and CaMKII.

Age-related effects of Ginkgo biloba extract on synaptic plasticity and excitability.

EGb 761 is a standardized extract from the Ginkgo biloba leaf and is purported to improve age-related memory impairment. The acute and chronic effect of EGb 761 on synaptic transmission and plasticity in hippocampal slices from young adult (8-12 weeks) and aged (18-24 months) C57Bl/6 mice was tested because hippocampal plasticity is believed to be a key component of memory. Acutely applied EGb 761 significantly increased neuronal excitability in slices from aged mice by reducing the population spike threshold and increased the early phase of long-term potentiation, though there was no effect in slices from young adults. In chronically treated mice fed for 30 days with an EGb 761-supplemented diet, EGb 761 significantly increased the population spike threshold and long-term potentiation in slices from aged animals, but had no effect on slices from young adults. The rapid effects of EGb 761 on plasticity indicate a direct interaction with the glutamatergic system and raise interesting implications with respect to a mechanism explaining its effect on cognitive enhancement in human subjects experiencing dementia.