Comparative Respiratory Tract Pathology of Emerging Viral Infections

Increases in the number of infectious disease outbreaks affecting humans have been reported nearly every decade since the 1940’s. Many of these outbreaks have been caused by emerging zoonotic viruses. It has recently been estimated that there are at least 320,000 viruses in mammals that have yet to be discovered, some of which may affect humans. As humans continue to encroach upon previously isolated areas and have greater contact with wildlife, it is likely that zoonotic viruses will continue to emerge. Since many of these emerging viruses cause significant disease or death in humans it is vital that we study their pathology and pathogenesis in order to detect patterns in their mechanisms of disease, which could then be used to develop broad spectrum antivirals or vaccines. As such, we focused our studies on two recently emerged viruses, Nipah virus and the Middle East respiratory syndrome coronavirus (MERS-CoV), which have caused significant respiratory disease and death in humans

Examination of Novel Cardiac Mechanisms Influencing Mitochondrial Proteomes during Diabetes Mellitus

Cardiac complications, including diabetic cardiomyopathy, are the leading cause of morbidity and mortality within diabetes mellitus. Mitochondrial dysfunction has been suggested as an underlying factor in the initiation and progression of the pathology. Cardiac mitochondria are characterized as two spatially distinct subpopulations within the myocyte including mitochondria located beneath the sarcolemma, termed subsarcolemmal mitochondria (SSM), and those located between myofibrils, termed interfibrillar mitochondria (IFM). Mitochondrial subpopulations have been shown to respond differently to physiological and pathological stimuli with the IFM being the most impacted in a type 1 diabetic setting. Proteomic evaluations within various models of diabetes have highlighted dynamic alterations of the mitochondrial proteome as a consequence of the pathology. To date, no studies have identified how the proteomes of mitochondrial subpopulations are differentially impacted during a type 1 diabetic insult. Further, the mechanisms involved in diabetes-driven mitochondrial proteomic alterations remain limited. Therefore, the goal of the present studies was to determine whether subpopulation-specific proteomes were altered with type 1 diabetes mellitus. Further, we sought to identify mechanisms involved in mitochondrial proteomic dysregulation prevalent within diabetic cardiomyopathy. Type 1 diabetes mellitus was induced in 8 week old mice with multiple low dose (50mg/kg) injections of streptozotocin (STZ) administered for 5 days. Five weeks post hyperglycemic onset, hearts were excised and mitochondrial subpopulations were isolated. Proteomic analyses revealed that the proteome of diabetic IFM was significantly dysregulated compared to control with no changes within diabetic SSM compared to control. Further, nuclear-encoded mitochondrial protein import was significantly decreased in the diabetic IFM, which correlated with decreased abundance of essential protein import constituent mitochondrial heat shock protein 70 (MtHsp70). Because greater than 99% of proteins are of nuclear encoded origin and must be imported into the mitochondria, decrements to the import process may prove to be a novel mechanism of dysfunction within the diabetic IFM. The inner mitochondrial membrane (IMM), the location of essential mitochondrial complexes including import translocases of the inner membrane (TIM), has been shown to be particularly prone to diabetes induced oxidative damage. Reduction in oxidative damage within various pathologies has been shown to have beneficial effects upon mitochondrial functionality. Therefore, we overexpressed antioxidant mitochondrial phospholipid hydroperoxide glutathione peroxidase (mPHGPx) to assess its impact upon the mitochondrial import process and proteomic makeup during a diabetic insult. Remarkably, nuclear-encoded mitochondrial protein import was corrected within the diabetic mPHGPx IFM, which correlated with restitution of a large proportion of mitochondrial proteins negatively impacted by diabetes mellitus including those involved in oxidative phosphorylation, the tricarboxylic acid cycle, fatty acid oxidation, and mitochondrial protein import. MPHGPx is capable of scavenging mitochondrial lipid hydroperoxides specifically in mitochondrial membranes and hydrogen peroxide to a lesser extent. Overexpression of the antioxidant preserved or enhanced the protein content of essential mitochondrial import constituents translocase of the outer membrane 20 (Tom20), Tim23, Tim50, and MtHsp70 in mPHGPX diabetic IFM. Therefore, we believe proteomic correction within mPHGPx diabetic IFM may be a consequence of preservation of mitochondrial protein import machinery. These findings support the rationale for the use of mPHGPx as a mitochondrial-targeted therapeutic capable of protection in the diabetic heart. Additional mechanisms of mitochondrial proteomic dysregulation within diabetes mellitus may exist including alterations to the transcriptional/translational regulators, microRNAs (miRNAs). Therefore, we performed a broad scale miRNA analysis on control and STZ-treated mouse hearts 5 weeks post diabetic onset to determine the effect of diabetes mellitus on miRNA modulation. Twenty nine miRNAs were shown to be dysregulated within the diabetic heart including miRNA-141 (miR-141), which was enhanced by 5 fold. miRNA targeting analyses (targetscan.org) revealed miR-141 likely to regulated Slc25a3, the mitochondrial inorganic phosphate carrier. Slc25a3 is essential for ATP production as it acts as conduit for inorganic phosphate to pass from the cytoplasm into the matrix, providing phosphate to fuel the ATP synthase. IFM Slc25a3 protein content was decreased in the diabetic IFM, which correlated with decreased ATP synthesis rates. Similarly, overexpression of miR-141 decreased Slc25a3 protein content and ATP synthase activity within HEK293 cells. These findings show, for the first time, miRNA modulation within diabetes mellitus has a direct impact upon mitochondrial proteomic makeup as well as mitochondrial functionality. Further, miR-141 ablation may provide a protective benefit to mitochondria and subsequent cardiac function during a type 1 diabetic insult. Taken together, the studies highlighted above prove that mitochondrial proteomic dysregulation prevalent within diabetes mellitus is a complicated process involving spatially distinct mitochondrial subpopulations and multiple mechanisms of action. Targeted therapeutics aimed at the correction of one or more of these mechanisms may provide cardiac benefit from diabetic induced dysfunction via preservation of the mitochondrial proteome

A comparison of residual nitrite and nitrate, lipid oxidation, cut-surface color, and sensory and visual characteristics for nitrite-added and no-nitrite- or -nitrate-added Canadian-style bacon

The objective of this research was to compare residual nitrite, residual nitrate, lipid oxidation, and sensory and visual characteristics of conventionally cured Canadian-style bacon containing sodium nitrite to no-nitrite- or -nitrate-added Canadian-style bacon (naturally cured) during 7 or 12 weeks of storage. Three treatments were used for the first and second experiments: control, natural cure (NC) using celery powder with a nitrate reducing culture, and natural cure with cherry powder (NCCP) with a nitrate reducing culture; all of the pork loins used for these treatments were mechanically injected with brines. Six treatments were evaluated in a third experiment: control, control with ascorbate (NA), natural cure with preformed nitrite in celery powder (NCEL), NC, NCCP, and natural cure with lemon powder (NCLP); all treatments in the third experiment were processed by grinding the pork loins, then mixing with the ingredients. All natural cure treatments included celery powder and starter culture as ingredients and finished products were found to contain both residual nitrite and nitrate after incubation of the products for nitrate conversion. The control had significantly more (P\u3c0.05) residual nitrite than NC and NCCP in the first experiment whereas in the second and third experiments the control and NC treatments had significantly more residual nitrite than NCCP. The control had significantly less residual nitrate (P\u3c0.05) than the naturally cured treatments in the first experiment, but significantly more (P\u3c0.05) in the second experiment. NCLP had significantly less residual nitrate (P\u3c0.05) than NCCP in the third experiment, but no other treatments were significantly different. Significant differences between TBARS values were observed only in the third experiment where NC and NCLP had significantly higher levels of TBARS (P\u3c0.05) than all other treatments. The control was significantly darker and more red (P\u3c0.05) than NC and NCCP in the first experiment, while in the second experiment it had similar lightness and redness to NCCP when evaluated by a Hunter Lab instrument. No significant differences (P\u3e0.05) between the treatments for lightness or redness were found in the third experiment. Results from sensory panelists indicated that the control had more cured meat color intensity and was more tender, but contained more off-flavor (P\u3c0.05) than NC and NCCP. According to the panel scores, NC and NCCP had significantly greater cured meat flavor intensity and overall were rated as having better flavor acceptability (P\u3c0.05) than the control, although these results were not consistent with chemical analyses. These results demonstrate that natural curing processes can be successfully utilized for both injected and ground and then formed Canadian-style bacon

Human immunodeficiency virus infection of the human thymus and disruption of the thymic microenvironment in the SCID-hu mouse.

Infection with the human immunodeficiency virus (HIV) results in immunosuppression and depletion of circulating CD4+ T cells. Since the thymus is the primary organ in which T cells mature it is of interest to examine the effects of HIV infection in this tissue. HIV infection has been demonstrated in the thymuses of infected individuals and thymocytes have been previously demonstrated to be susceptible to HIV infection both in vivo, using the SCID-hu mouse, and in vitro. The present study sought to determine which subsets of thymocytes were infected in the SCID-hu mouse model and to evaluate HIV-related alterations in the thymic microenvironment. Using two different primary HIV isolates, infection was found in CD4+/CD8+ double positive thymocytes as well as in both the CD4+ and CD8+ single positive subsets of thymocytes. The kinetics of infection and resulting viral burden differed among the three thymocyte subsets and depended on which HIV isolate was used for infection. Thymic epithelial (TE) cells were also shown to endocytose virus and to often contain copious amounts of viral RNA in the cytoplasm by in situ hybridization, although productive infection of these cells could not be definitively shown. Furthermore, degenerating TE cells were observed even without detection of HIV in the degenerating cells. Two striking morphologic patterns of infection were seen, involving either predominantly thymocyte infection and depletion, or TE cell involvement with detectable cytoplasmic viral RNA and/or TE cell toxicity. Thus, a variety of cells in the human thymus is susceptible to HIV infection, and infection with HIV results in a marked disruption of the thymic microenvironment leading to depletion of thymocytes and degeneration of TE cells

Morphometric analyses of the visual pathways in macular degeneration

Introduction. Macular degeneration (MD) causes central visual field loss. When field defects occur in both eyes and overlap, parts of the visual pathways are no longer stimulated. Previous reports have shown that this affects the grey matter of the primary visual cortex, but possible effects on the preceding visual pathway structures have not been fully established. Method. In this multicentre study, we used high-resolution anatomical magnetic resonance imaging and voxel-based morphometry to investigate the visual pathway structures up to the primary visual cortex of patients with age-related macular degeneration (AMD) and juvenile macular degeneration (JMD). Results. Compared to age-matched healthy controls, in patients with JMD we found volumetric reductions in the optic nerves, the chiasm, the lateral geniculate bodies, the optic radiations and the visual cortex. In patients with AMD we found volumetric reductions in the lateral geniculate bodies, the optic radiations and the visual cortex. An unexpected finding was that AMD, but not JMD, was associated with a reduction in frontal white matter volume. Conclusion. MD is associated with degeneration of structures along the visual pathways. A reduction in frontal white matter volume only present in the AMD patients may constitute a neural correlate of previously reported association between AMD and mild cognitive impairment. Keywords: macular degeneration - visual pathway - visual field - voxel-based morphometryComment: appears in Cortex (2013

Tracking Down the Intuitiveness of Gesture Interaction in the Truck Domain

AbstractTouchless hand gesture control potentially leads to a safer, more comfortable and more intuitive Human Vehicle Interaction (HVI) if relevant ergonomic requirements are met. To achieve intuitive interaction and thus to favor user acceptance, the gesture interface should be conform to user expectation and enable the users to apply their prior knowledge. This particularly concerns the gestures used for input. The conducted experiment investigates which gestures subjects tend to use for various functions of a truck and how these gestures are affected by the subjects’ prior knowledge. In total, 17 potential functions were considered for this purpose. Within the experiment, 74 subjects performed gestures for each of these functions while being recorded on video. The video data shows a variety of gestures differing in hand pose, execution space, and palm orientation. Nevertheless, several interindividual similarities in gesturing can be observed, which made it possible to analyze the gestures in terms of the prior knowledge applied. The results show that gestures differ according to the sources of prior knowledge like culture and instincts. Depending on the function, the gestures observed within the experiment are based on gestures of quasi-direct manipulation, emblematic gestures, instinctive gestures, standardized gestures and gestures expressing the users’ mental model. However, the applicability of these gestures is limited by capabilities of gesture recognition and is depending on how the user interface will be designed

The Granzyme B ELISPOT assay: an alternative to the (51)Cr-release assay for monitoring cell-mediated cytotoxicity

BACKGROUND: The interferon-γ (IFN-γ) ELISPOT assay is one of the most useful techniques for immunological monitoring of cancer vaccine trials and has gained increased application as a measure of specific T cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN-γ secretion is not limited to only cytolytic cells. Granzyme B (GrB) is a key mediator of target cell death via the granule-mediated pathway. Therefore, the release of GrB by cytolytic lymphocytes upon effector-target interaction may be a more specific indicator of CTL and NK cytotoxic ability than IFN-γ secretion. METHODS: We assessed whether the GrB ELISPOT assay is a viable alternative to the (51)Cr-release and IFN-γ ELISPOT assays for measuring antigen-specific CTL cytotoxicity. Direct comparisons between the three assays were made using human CTL cell lines (αEN-EBV and αJY) and an in vitro stimulated anti-Flu matrix peptide (FMP)-specific CTL. RESULTS: When the GrB ELISPOT was directly compared to the IFN-γ ELISPOT and (51)Cr-release assays, excellent cross-correlation between all three assays was shown. However, measurable IFN-γ secretion in the ELISPOT assay was observed only after 1 hour of incubation and cytotoxicity assessed via the (51)Cr-release assay after 4 hours, whereas GrB secretion was detectable within 10 min of effector-target contact with significant secretion observed after 1 h. Titration studies demonstrated a strong correlation between the number of effector cells and GrB spots per well. Irrelevant targets or antigens did not induce significant GrB secretion. Additionally, GrB secretion was abrogated when CTL cultures were depleted of CD8+ cells. CONCLUSION: Our findings demonstrate that the GrB ELISPOT assay is a superior alternative to the (51)Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-γ ELISPOT assay, the GrB ELISPOT directly measures the release of a cytotolytic protein. Detection of low frequency tumor-specific CTL and their specific effector functions can provide valuable insight with regards to immunological responses

Retinotopic remapping of the visual system in deaf adults

Sound is a vital cue in helping hearing people orient their gaze and attention towards events outside their central line of sight, especially in the far periphery, where vision is poor. Without sound cues, deaf individuals must rely on vision as an ‘early warning system’ for peripheral events, and in fact numerous behavioural studies demonstrate that deaf adults have superior visual sensitivity, particularly to far peripheral stimuli. We asked whether an increased demand on peripheral vision throughout development might be reflected in early visual brain structures. Using functional magnetic resonance imaging (fMRI), we mapped visual field representations in 16 early, profoundly deaf adults and 16 hearing age-matched controls. To target the far periphery, we used wide-field retinotopic mapping stimuli to map visual field eccentricity out to 72°, well beyond conventional mapping studies. Deaf individuals exhibited a larger representation of the far peripheral visual field in both the primary visual cortex and the lateral geniculate nucleus of the thalamus. Importantly, this was not due to a total expansion of the visual map, as there was no difference between groups in overall size of either structure, but a smaller representation of the central visual field in the deaf group, suggesting a redistribution of neural resources. Here, we demonstrate for the first time that the demands placed on vision due to lifelong hearing loss can sculpt visual maps at the first level of inputs from the retina, increasing neural resources for processing stimuli in the far peripheral visual field

Evaluating the cytotoxicity of innate immune effector cells using the GrB ELISPOT assay

BACKGROUND: This study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the (51)Cr-release assay for measuring cytotoxic activity of innate immune effector cells. We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells. METHODS: We optimized the GrB ELISPOT assay using the human-derived TALL-104 cytotoxic cell line as effectors against K562 target cells. Titration studies were performed to assess whether the ELISPOT assay could accurately enumerate the number of GrB-secreting effector cells. TALL-104 were treated with various secretion inhibitors and utilized in the GrB ELISPOT to determine if GrB measured in the ELISPOT was due to degranulation of effector cells. Additionally, CD107a expression on effector cells after effector-target interaction was utilized to further confirm the mechanism of GrB release by TALL-104 and lymphokine-activated killer (LAK) cells. Direct comparisons between the GrB ELISPOT, the IFN-γ ELISPOT and the standard (51)Cr-release assays were made using human LAK cells. RESULTS: Titration studies demonstrated a strong correlation between the number of TALL-104 and LAK effector cells and the number of GrB spots per well. GrB secretion was detectable within 10 min of effector-target contact with optimal secretion observed at 3–4 h; in contrast, optimal IFN-γ secretion was not observed until 24 h. The protein secretion inhibitor, brefeldin A, did not inhibit the release of GrB but did abrogate IFN-γ production by TALL-104 cells. GrB secretion was abrogated by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester), which sequesters intracellular Ca(2+), thereby preventing degranulation. The number of effector cells expressing the degranulation associated glycoprotein CD107a increased after interaction with target cells and correlated with the stimulated release of GrB measured in the ELISPOT assay. CONCLUSIONS: Because of its high sensitivity and ability to estimate cytotoxic effector cell frequency, the GrB ELISPOT assay is a viable alternative to the (51)Cr-release assay to measure MHC non-restricted cytotoxic activity of innate immune cells. Compared to the IFN-γ ELISPOT assay, the GrB ELISPOT may be a more direct measure of cytotoxic cell activity. Because GrB is one of the primary effector molecules in natural killer (NK) cell-mediated killing, detection and enumeration of GrB secreting effector cells can provide valuable insight with regards to innate immunological responses