It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.

It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.

INTRODUCTION: Cortical spreading depolarization (CSD) represents a pathomechanistic target for neuroprotective therapy given the association with lesion development and poor outcomes following traumatic brain injury (TBI).METHODS: Experiments were conducted using 9-week-old Sprague Dawley rats. Daily neurobehavioral scores were recorded by trained, blinded observers. Using an established weight-drop model, animals received either single moderate (modTBI; n = 23) or four daily mild (rmTBI; n = 30) head impacts. Sham animals received brief anesthetic without TBI (n = 40). Animals underwent cranial window surgery and CSDs were electrically triggered by cortical stimulation; electrophysiological, imaging and blood-flow were monitored using intravital microscopy. Ketamine (100uM topical and 25 mg/kg IP, respectively) and memantine (10 mg/kg IP) were tested in vivo. Subsequently, a RCT was conducted (N=31) using memantine (10 mg/kg) or saline (2.5 cc/kg). Primary outcome: proportion of animals resilient to rmTBI (neurobehavioral scores ≥ 6 out of 12).RESULTS: In modTBI, and rmTBI animal models, intraperitoneal ketamine and memantine reduced the proportion of electrically triggered CSDs by 79-88%, and 42-73%, respectively. The average relative amplitude of change in ECoG were reduced by 70-75%, and 51-60%, respectively. Memantine significantly reduced spreading depression and cortical hypoperfusion following CSD. RCT animals in the memantine group were more likely resilient to injury (93% vs. 56%; p = 0.023) and had higher mean neurological scores (9.27 (SD 3.08) vs. 5.56 (SD 3.05)), p < 0.001) compared to saline.CONCLUSIONS: The NMDA-receptor antagonist memantine supresses CSDs and reduces pathological cerebral hypoperfusion in vivo following CSDs. In a rmTBI RCT, memantine prevented neurological decline.

The following are abstracts from the research competition at the 14th annual Canadian Undergraduate Conference on Healthcare. The conference was entitled "The Future of Healthcare: A Multidisciplinary Approach," held on November 9th-11th, 2018 at Queen's University. Abstracts are grouped under the categories of oral and poster presentations, with sub-categories based on the general field in which the abstract is found. For more information about the conference, please go to https://www.cucoh.com/.

Traumatic brain injury (TBI) involves an acute injury (primary damage), which may evolve in the hours to days after impact (secondary damage). Seizures and cortical spreading depolarization (CSD) are metabolically demanding processes that may worsen secondary brain injury. Metabolic stress has been associated with mitochondrial dysfunction, including impaired calcium homeostasis, reduced ATP production, and elevated ROS production. However, the association between mitochondrial impairment and vascular function after TBI is poorly understood. Here, we explored this association using a rodent closed head injury model. CSD resulted in neurobehavioral decline after TBI. Craniotomy was performed to elicit CSD via electrical stimulation or to induce seizures via 4-aminopyridine application. We measured vascular dysfunction following CSDs and seizures in TBI animals using laser doppler flowmetry. We observed a more profound reduction in local cortical blood flow in TBI animals compared to healthy controls. Following TBI, CSD resulted in mitochondrial dysfunction and pathological signs of increased oxidative stress adjacent to the vasculature. We explored these findings further using electron microscopy and found that TBI and CSDs resulted in vascular morphological changes and mitochondrial cristae damage in astrocytes, pericytes and endothelial cells. Overall, we provide evidence that CSDs induce mitochondrial dysfunction, impaired cortical blood flow, and neurobehavioral deficits in the setting of TBI.