Faculty Bibliography

Epilepsy is a chronic condition characterized by unpredictable and recurrent spontaneous seizures. In a previous study, we reported that pharmacological inhibition of calpain prevented epileptogenesis in the rat pilocarpine model. In this study, we demonstrate that transgenic overexpression of calpastatin, the endogenous inhibitor of calpain, reduces calpain activation and lessens seizure burden in the mouse intrahippocampal kainate model. Blockade of calpain activation was evidenced by a reduction in the generation of spectrin breakdown products, a hallmark of calpain activation. CAST overexpression was associated with a significant reduction in seizure burden, further supporting the idea that blocking calpain overactivation prevents epilepsy. Moreover, a reduction in seizure burden was accompanied by a decrease in inflammatory markers but not cell death. Together, these observations corroborate the role of calpain overactivation in epileptogenesis and provide further support for the use of calpain inhibitors as a viable strategy to prevent epilepsy. PLAIN LANGUAGE SUMMARY: The mechanisms by which brain alterations lead to spontaneous seizures are not well understood. Acquired epilepsy often follows brain trauma. After a brain injury, the activation of the protease calpain has been associated with the development of spontaneous seizures. Our observations indicate that transgenic overexpression of calpastatin, an endogenous inhibitor of calpain, impacts epileptogenesis and reduces seizure burden. This suggests that inhibiting calpain could be a viable strategy to prevent epilepsy.

Auxins are a group of phytohormones that control plant growth and development. Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as herbicides. Phenoxyacetic acid derivatives are a widely used group of auxin herbicides in agriculture and research. Despite their prevalence, the identity of the transporters required for distribution of these herbicides in plants is both poorly understood and the subject of controversial debate. Here we show that PIN-FORMED auxin transporters transport a range of phenoxyacetic acid herbicides across the membrane. We go on to characterize the molecular determinants of substrate specificity using a variety of different substrates as well as protein mutagenesis to probe the binding site. Finally, we present cryogenic electron microscopy structures of Arabidopsis thaliana PIN8 bound to either 2,4-dichlorophenoxyacetic acid or 4-chlorophenoxyacetic acid. These structures represent five key states from the transport cycle, allowing us to describe conformational changes associated with the transport cycle. Overall, our results reveal that phenoxyacetic acid herbicides use the same export machinery as endogenous auxins and exemplify how transporter binding sites undergo transformations that dictate substrate specificity. These results provide a foundation for future development of novel synthetic auxins and for precision breeding of herbicide-resistant crop plants.

BACKGROUND:Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy associated with a high risk of ventricular arrhythmia (VA). Several animal models have been used to postulate a therapeutic role of the inhibition of the ryanodine 2 receptor via the use of flecainide for this disease. Clinical data describing its use are scarce, however, especially in patients without implantable cardioverter-defibrillators or with left ventricular (LV) involvement. OBJECTIVES/OBJECTIVE:This study sought to report safety and effectiveness long-term, multicenter data on the impact of flecainide therapy on arrhythmic outcomes in patients with a definite diagnosis of ARVC. METHODS:Patients with definite ARVC receiving flecainide at 12 academic institutions were enrolled in the study. Baseline was defined as the time of flecainide initiation. Premature ventricular complex burdens, nonsustained ventricular tachycardia (NSVT) rates, and sustained VA yearly/rates were collected and compared while on and off flecainide. Side effects and flecainide discontinuation were tracked. Analyses were performed in the overall cohort as well as stratifying for genotype (gene positive vs negative; plakohpillin-2 [PKP-2] vs non PKP-2) and for LV involvement. RESULTS:; LV ejection fraction 55.9 ± 7.3%; right ventricular ejection fraction 44.5 ± 10.5% at baseline) were enrolled, with 66 patients (34.6%) showing LV involvement. The median dose of flecainide was 200 mg/d [150-200 mg/d], with 166 patients (86.9%) also taking a beta-blocker. The median follow-up time on flecainide was 4.2 years [1.9-6.3 years]. Flecainide was well tolerated, with a low (7.9%) discontinuation rate. After flecainide initiation, a significant reduction in the 24-hour premature ventricular complex burden and in the rate of nonsustained ventricular tachycardia was observed (2,190 vs 418; P < 0.001; 35.1% vs 21.5%; P = 0.003). For patients with prior VA events, a significant reduction in the amount of VA episodes/y (1.1 [0.4-1.6] episodes/y vs 0 [0-0.3] episodes/y; P < 0.001) was observed. These safety and effectiveness findings were consistent across genotype subgroups, as well as in patients with and without LV involvement. CONCLUSIONS:Flecainide use had a favorable safety profile and was associated with an observed to a significant reduction in arrhythmic burden in patients with ARVC, irrespective of the underlying genotype or LV involvement.

Like many organisms, the roundworm Caenorhabditis elegans incorporates an assessment of environmental quality into its reproductive strategy. C. elegans hermaphrodites release fertilized eggs into food-rich environments but retain them in the absence of food. Here, we report the discovery of a neural circuit required for the modulation of reproductive behavior by food sensing. A mutation that electrically silences the AVK interneurons uncouples egg laying from detection of environmental food cues. We find that AVK activity inhibits egg laying, and AVKs themselves are inhibited by dopamine released from food-sensing neurons. AVKs express a large number of structurally and functionally diverse neuropeptides. Coordination of food-sensing and reproductive behavior requires a subset of AVK neuropeptides that converge on a small ensemble of premotor neurons that coexpress their cognate receptors. Modulation of C. elegans reproductive behavior, therefore, requires a cascade of neuromodulatory signals that uses disinhibition and combinatorial neuropeptide signals to activate reproductive behavior when food is sensed.

PURPOSE/OBJECTIVE:The purpose of this study is to examine the effect of age on outcomes following repair of acute displaced patella fractures Methods: 248 patients who sustained a displaced patella fracture and underwent open reduction and internal fixation were identified. Patients included underwent a similar operative protocol, were prescribed a standard post-operative protocol of therapy, and were seen at standard follow-up intervals. Patients were divided into groups of < 65 years old (young) and ≥ 65 years old (older). Statistical analysis was run to determine if there was a significant difference in range of knee motion and rate of major complications. RESULTS:Of the 248 patients, 149 were young and 99 were older. The mean age of the older group was 74.5 ± 6.7 and the mean age of the young group was 50 ± 12. Fracture pattern and BMI were similar the groups, however the older group had a higher average CCI (p<0.001). Additionally, the groups had similar length of follow up (p=0.693) and similar mean time to radiographic healing (p=0.533). Older patients had limited knee extension at 6 months (compared young patients (p=0.031). Finally, older patients had a higher rate of all complications compared to young patients. Two percent of older patients developed a fracture related infection (FRI), 4% developed a symptomatic nonunion and 11% were underwent re-operation including removal of hardware, total knee replacement, irrigation and debridement and manipulation under anesthesia. CONCLUSION/CONCLUSIONS:Complication rates following patella fracture fixation in older patients were higher than young patients, despite having similar injury patterns, surgical treatment and follow up. These findings can better inform treating physicians during surgical intervention of older patients with patella fractures.

Our brains integrate sensory, cognitive and internal state information with memories to extract behavioral relevance. Cortico-hippocampal interactions likely mediate this interplay, but underlying circuit mechanisms remain elusive. Unlike the entorhinal cortex-to-hippocampus pathway, we know little about the organization and function of the hippocampus-to-cortex feedback circuit. Here we report in mice, two functionally distinct parallel hippocampus-to-entorhinal cortex feedback pathways: the canonical disynaptic route via layer 5 and a novel monosynaptic input to layer 2/3. Circuit mapping reveals that hippocampal input predominantly drives excitation in layer 5 but feed-forward inhibition in layer 2/3. Upon repetitive pairing with cortical layer 1 inputs, hippocampal inputs undergo homosynaptic potentiation in layer 5, but induce heterosynaptic plasticity and spike output in layer 2/3. Behaviorally, hippocampal inputs to layer 5 and layer 2/3 support object memory encoding versus recall, respectively. Two-photon imaging during navigation reveals hippocampal suppression reduces spatially tuned cortical axonal activity. We present a model, where hippocampal feedback could iteratively shape ongoing cortical processing.

Microsporidia are single-celled intracellular parasites that cause opportunistic diseases in humans. Encephalitozoon intestinalis is a prevalent human-infecting species that invades the small intestine. Macrophages are potential reservoirs of infection, and dissemination to other organ systems is also observed. The macrophage response to infection and the developmental trajectory of the parasite are not well studied. Here we use single cell RNA sequencing to investigate transcriptional changes in both the parasite and the host during E. intestinalis infection of human macrophages in vitro. The parasite undergoes large transcriptional changes throughout the life cycle, providing a blueprint for parasite development. While a small population of infected macrophages mount a response, most remain transcriptionally unchanged, suggesting that the majority of parasites may avoid host detection. The stealthy microsporidian lifestyle likely allows these parasites to harness macrophages for replication. Together, our data provide insights into the host response in primary human macrophages and the E. intestinalis developmental program.

The continuity of a species depends on germ cells. Germ cells are different from all the other cell types of the body (somatic cells) as they are solely destined to develop into gametes (sperm or egg) to create the next generation. In this review, we will touch on 4 areas of embryonic germ cell development in Drosophila melanogaster: the assembly and function of germplasm, which houses the determinants for germ cell specification and fate and the mitochondria of the next generation; the process of pole cell formation, which will give rise to primordial germ cells (PGCs); the specification of pole cells toward the PGC fate; and finally, the migration of PGCs to the somatic gonadal precursors, where they, together with somatic gonadal precursors, form the embryonic testis and ovary.

Astrocytes are a highly abundant glial cell type and perform critical homeostatic functions in the central nervous system. Like neurons, astrocytes have many discrete heterogeneous subtypes. The subtype identity and functions are, at least in part, associated with their anatomical location and can be highly restricted to strategically important anatomical domains. Here, we report that astrocytes forming the glia limitans superficialis, the outermost border of the brain and spinal cord, are a highly specialized astrocyte subtype and can be identified by a single marker: myocilin (Myoc). We show that glia limitans superficialis astrocytes cover the entire brain and spinal cord surface, exhibit an atypical morphology, and are evolutionarily conserved from zebrafish, rodents, and non-human primates to humans. Identification of this highly specialized astrocyte subtype will advance our understanding of CNS homeostasis and potentially be targeted for therapeutic intervention to combat peripheral inflammatory effects on the CNS.