Brett J. Hilton

Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers.

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. “Fluorescent timer” protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of “fluorescent timer” protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. “Fluorescent timer” protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.

Roles for Endoplasmic Reticulum–Associated Degradation and the Novel Endoplasmic Reticulum Stress Response Gene Derlin-3 in the Ischemic Heart

Rationale: Stresses, such as ischemia, impair folding of nascent proteins in the rough endoplasmic reticulum (ER), activating the unfolded protein response, which restores efficient ER protein folding, thus leading to protection from stress. In part, the unfolded protein response alleviates ER stress and cell death by increasing the degradation of terminally misfolded ER proteins via ER-associated degradation (ERAD). ERAD is increased by the ER stress modulator, activating transcription factor (ATF)6, which can induce genes that encode components of the ERAD machinery. Objective: Recently, it was shown that the mouse heart is protected from ischemic damage by ATF6; however, ERAD has not been studied in the cardiac context. A recent microarray study showed that the Derlin-3 (Derl3) gene, which encodes an important component of the ERAD machinery, is robustly induced by ATF6 in the mouse heart. Methods and Results: In the present study, activated ATF6 induced Derl3 in cultured cardiomyocytes, and in the heart, in vivo. Simulated ischemia (sI), which activates ER stress, induced Derl3 in cultured myocytes, and in an in vivo mouse model of myocardial infarction, Derl3 was also induced. Derl3 overexpression enhanced ERAD and protected cardiomyocytes from simulated ischemia–induced cell death, whereas dominant-negative Derl3 decreased ERAD and increased simulated ischemia–induced cardiomyocyte death. Conclusions: This study describes a potentially protective role for Derl3 in the heart, and is the first to investigate the functional consequences of enhancing ERAD in the cardiac context.

Evolution of TNF-induced apoptosis reveals 550 My of functional conservation

Significance The TNF receptor-ligand superfamily is a central mediator of apoptosis or programmed cell death. Here we show that TNF-induced apoptosis has been functionally maintained for more than half a billion years of evolution. In response to human TNFα, coral cells underwent the classical stages of apoptosis including cellular blebbing, caspase activation, and eventual cell death. Next, the reciprocal experiment showed that coral TNF kills human cells through direct interaction with the death receptor pathway. In addition, corals were found to possess more putative TNF receptors than any organism previously described, including humans. This work provides important insight into the general evolution of apoptosis and demonstrates remarkable conservation of the TNF apoptotic response. The Precambrian explosion led to the rapid appearance of most major animal phyla alive today. It has been argued that the complexity of life has steadily increased since that event. Here we challenge this hypothesis through the characterization of apoptosis in reef-building corals, representatives of some of the earliest animals. Bioinformatic analysis reveals that all of the major components of the death receptor pathway are present in coral with high-predicted structural conservation with Homo sapiens. The TNF receptor-ligand superfamilies (TNFRSF/TNFSF) are central mediators of the death receptor pathway, and the predicted proteome of Acropora digitifera contains more putative coral TNFRSF members than any organism described thus far, including humans. This high abundance of TNFRSF members, as well as the predicted structural conservation of other death receptor signaling proteins, led us to wonder what would happen if corals were exposed to a member of the human TNFSF (HuTNFα). HuTNFα was found to bind directly to coral cells, increase caspase activity, cause apoptotic blebbing and cell death, and finally induce coral bleaching. Next, immortalized human T cells (Jurkats) expressing a functional death receptor pathway (WT) and a corresponding Fas-associated death domain protein (FADD) KO cell line were exposed to a coral TNFSF member (AdTNF1) identified and purified here. AdTNF1 treatment resulted in significantly higher cell death (P < 0.0001) in WT Jurkats compared with the corresponding FADD KO, demonstrating that coral AdTNF1 activates the H. sapiens death receptor pathway. Taken together, these data show remarkable conservation of the TNF-induced apoptotic response representing 550 My of functional conservation.

Bacterial induction of Snail1 contributes to blood-brain barrier disruption

Bacterial meningitis is a serious infection of the CNS that results when blood-borne bacteria are able to cross the blood-brain barrier (BBB). Group B Streptococcus (GBS) is the leading cause of neonatal meningitis; however, the molecular mechanisms that regulate bacterial BBB disruption and penetration are not well understood. Here, we found that infection of human brain microvascular endothelial cells (hBMECs) with GBS and other meningeal pathogens results in the induction of host transcriptional repressor Snail1, which impedes expression of tight junction genes. Moreover, GBS infection also induced Snail1 expression in murine and zebrafish models. Tight junction components ZO-1, claudin 5, and occludin were decreased at both the transcript and protein levels in hBMECs following GBS infection, and this repression was dependent on Snail1 induction. Bacteria-independent Snail1 expression was sufficient to facilitate tight junction disruption, promoting BBB permeability to allow bacterial passage. GBS induction of Snail1 expression was dependent on the ERK1/2/MAPK signaling cascade and bacterial cell wall components. Finally, overexpression of a dominant-negative Snail1 homolog in zebrafish elevated transcription of tight junction protein-encoding genes and increased zebrafish survival in response to GBS challenge. Taken together, our data support a Snail1-dependent mechanism of BBB disruption and penetration by meningeal pathogens.

Ketogenic Diet Improves Forelimb Motor Function after Spinal Cord Injury in Rodents

High fat, low carbohydrate ketogenic diets (KD) are validated non-pharmacological treatments for some forms of drug-resistant epilepsy. Ketones reduce neuronal excitation and promote neuroprotection. Here, we investigated the efficacy of KD as a treatment for acute cervical spinal cord injury (SCI) in rats. Starting 4 hours following C5 hemi-contusion injury animals were fed either a standard carbohydrate based diet or a KD formulation with lipid to carbohydrate plus protein ratio of 3:1. The forelimb functional recovery was evaluated for 14 weeks, followed by quantitative histopathology. Post-injury 3:1 KD treatment resulted in increased usage and range of motion of the affected forepaw. Furthermore, KD improved pellet retrieval with recovery of wrist and digit movements. Importantly, after returning to a standard diet after 12 weeks of KD treatment, the improved forelimb function remained stable. Histologically, the spinal cords of KD treated animals displayed smaller lesion areas and more grey matter sparing. In addition, KD treatment increased the number of glucose transporter-1 positive blood vessels in the lesion penumbra and monocarboxylate transporter-1 (MCT1) expression. Pharmacological inhibition of MCTs with 4-CIN (α-cyano-4-hydroxycinnamate) prevented the KD-induced neuroprotection after SCI, In conclusion, post-injury KD effectively promotes functional recovery and is neuroprotective after cervical SCI. These beneficial effects require the function of monocarboxylate transporters responsible for ketone uptake and link the observed neuroprotection directly to the function of ketones, which are known to exert neuroprotection by multiple mechanisms. Our data suggest that current clinical nutritional guidelines, which include relatively high carbohydrate contents, should be revisited.

An assay to monitor HIV-1 protease activity for the identification of novel inhibitors in T-cells.

The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR). The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP) gene only in the presence of an effective PR Inhibitor (PI). Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells.

Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry

The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent‐coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6‐month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell‐based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry‐based analysis.

Dorsolateral funiculus lesioning of the mouse cervical spinal cord at C4 but not at C6 results in sustained forelimb motor deficits.

Although upper extremity functional recovery is a high priority for spinal cord injured patients with cervical injuries, few injury models have been developed in mice with sustained deficits in forelimb motor function. Here, we characterize a dorsolateral funiculus (DLF) crush model in mice, which ablates the rubrospinal tract (RST) unilaterally and thus allows correlation of motor recovery to axonal regeneration in the assessment of molecular regeneration targets. We conducted unilateral DLF crush injuries at cervical levels C4 and C6 and assessed motor recovery in a battery of tests: the rearing test of forelimb asymmetry, the grooming test, staircase pellet reaching, a horizontal ladder task, and CatWalk gait analysis. All tasks revealed lesion effects on forepaw function when DLF crush was instigated at level C4, but deficits were generally only transient in mice with DLF crush performed at level C6. Anterograde tracing of the RST with biotinylated dextran amine revealed the tracts complete ablation. The characterization of a C4 DLF model in mice provides an important tool for assessing molecular regeneration targets to promote functional recovery after spinal cord injury.

A Novel Two-Tag System for Monitoring Transport and Cleavage through the Classical Secretory Pathway - Adaptation to HIV Envelope Processing.

The classical secretory pathway is essential for the transport of a host of proteins to the cell surface and/or extracellular matrix. While the pathway is well-established, many factors still remain to be elucidated. One of the most relevant biological processes that occur during transport involves the cleavage of pro-proteins by enzymes residing in the endoplasmic reticulum/Golgi/TransGolgi Network compartment. Teasing out the requirements involved in the classical secretory pathway and cleavage during transport would shed new light into mis-regulation leading to disease. Current methodologies fail to link transport and cleavage at the single cell level. Here, we describe a cell-based assay that relies on an engineered protein scaffold that can discriminate between transport to the cell surface, in the absence or presence of cleavage. Our novel two-tag system works in a robust and quantitative manner and distinguishes between cleaved and non-cleaved events based on cell surface expression of one or two epitope tags, respectively. Here, we have used the HIV-1 envelope as a substrate, which is cleaved during transport, as proof of principle. Importantly, this assay can be easily coupled to existing siRNA-based screens to identify novel regulators and effectors involved in transport and/or cleavage of cell surface proteins. In addition, unlike other in vivo based assays, the assay described here can also be easily adapted to drug discovery purposes.

Energetic differences between bacterioplankton trophic groups and coral reef resistance.

Coral reefs are among the most productive and diverse marine ecosystems on the Earth. They are also particularly sensitive to changing energetic requirements by different trophic levels. Microbialization specifically refers to the increase in the energetic metabolic demands of microbes relative to macrobes and is significantly correlated with increasing human influence on coral reefs. In this study, metabolic theory of ecology is used to quantify the relative contributions of two broad bacterioplankton groups, autotrophs and heterotrophs, to energy flux on 27 Pacific coral reef ecosystems experiencing human impact to varying degrees. The effective activation energy required for photosynthesis is lower than the average energy of activation for the biochemical reactions of the Krebs cycle, and changes in the proportional abundance of these two groups can greatly affect rates of energy and materials cycling. We show that reef-water communities with a higher proportional abundance of microbial autotrophs expend more metabolic energy per gram of microbial biomass. Increased energy and materials flux through fast energy channels (i.e. water-column associated microbial autotrophs) may dampen the detrimental effects of increased heterotrophic loads (e.g. coral disease) on coral reef systems experiencing anthropogenic disturbance.