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Item Author Correction: Moderate Nrf2 Activation by Genetic Disruption of Keap1 Has Sex-Specific Effects on Bone Mass in Mice(Springer Nature, 2021-05-10) Yin, Yukun; Corry, Kylie A.; Loughran, John P.; Li, Jiliang; Biology, School of ScienceCorrection to: Scientific Reports https://doi.org/10.1038/s41598-019-57185-1, published online 15 January 2020Item The Genius of the Zebrafish Model: Insights on Development and Disease(MDPI, 2021-05) Marrs, James A.; Sarmah, Swapnalee; Biology, School of ScienceItem S-Palmitoylation of the sodium channel Nav1.6 regulates its activity and neuronal excitability(Elsevier, 2020-05) Pan, Yanling; Xiao, Yucheng; Pei, Zifan; Cummins, Theodore R.; Biology, School of ScienceS-Palmitoylation is a reversible post-translational lipid modification that dynamically regulates protein functions. Voltage-gated sodium channels are subjected to S-palmitoylation and exhibit altered functions in different S-palmitoylation states. Our aim was to investigate whether and how S-palmitoylation regulates Nav1.6 channel function and to identify S-palmitoylation sites that can potentially be pharmacologically targeted. Acyl-biotin exchange assay showed that Nav1.6 is modified by S-palmitoylation in the mouse brain and in a Nav1.6 stable HEK 293 cell line. Using whole-cell voltage clamp, we discovered that enhancing S-palmitoylation with palmitic acid increases Nav1.6 current, whereas blocking S-palmitoylation with 2-bromopalmitate reduces Nav1.6 current and shifts the steady-state inactivation in the hyperpolarizing direction. Three S-palmitoylation sites (Cys1169, Cys1170, and Cys1978) were identified. These sites differentially modulate distinct Nav1.6 properties. Interestingly, Cys1978 is exclusive to Nav1.6 among all Nav isoforms and is evolutionally conserved in Nav1.6 among most species. Cys1978S-palmitoylation regulates current amplitude uniquely in Nav1.6. Furthermore, we showed that eliminating S-palmitoylation at specific sites alters Nav1.6-mediated excitability in dorsal root ganglion neurons. Therefore, our study reveals S-palmitoylation as a potential isoform-specific mechanism to modulate Nav activity and neuronal excitability in physiological and diseased conditions.Item Longevity of Crown Margin Repairs Using Glass Ionomer Cement: A Retrospective Study(Allen Press, 2021-05) Watson, JI; Patel, JS; Ramya, MB; Capin, O; Diefenderfer, KE; Thyvalikakath, TP; Cook, NB; Biology, School of ScienceObjective: The objective of this study was to determine the survival time of crown margin repairs (CMRs) with glass ionomer and resin-modified glass ionomer cements on permanent teeth using electronic dental record (EDR) data. Methods: We queried a database of EDR (axiUm; Exan Group, Coquitlam, BC, Canada) in the Indiana University School of Dentistry (IUSD), Indianapolis, IN, USA, for records of patients who underwent CMRs of permanent teeth at the Graduate Operative Dentistry Clinic. Two examiners developed guidelines for reviewing the records and manually reviewed the clinical notes of patient records to confirm for CMRs. Only records that were confirmed with the presence of CMRs were retained in the final dataset for survival analysis. Survival time was calculated by Kaplan-Meier statistics, and a Cox proportional hazards model was performed to assess the influence of age, gender, and tooth type on survival time (a<0.05). Results: A total of 214 teeth (115 patients) with CMR were evaluated. Patient average age was 69.4 ± 11.7 years old. Posterior teeth accounted for 78.5% (n=168) of teeth treated. CMRs using glass ionomer cements had a 5-year survival rate of 62.9% and an annual failure rate (AFR) of 8.9%. Cox proportional-hazards model revealed that none of the factors examined (age, gender, tooth type) affected time to failure. Conclusion: The results indicate the potential of CMRs for extending the functional life of crowns with defective margins, thus reducing provider and patient burden of replacing an indirect restoration. We recommend future studies with a larger population who received CMR to extend the generalizability of our findings and to determine the influence of factors such as caries risk and severity of defects on survival time.Item Skeletal Dynamics of Down Syndrome: A Developing Perspective(Elsevier, 2020-04) LaCombe, Jonathan M.; Roper, Randall J.; Biology, School of ScienceIndividuals with Down syndrome (DS) display distinctive skeletal morphology compared to the general population, but disparate descriptions, methodologies, analyses, and populations sampled have led to diverging conclusions about this unique skeletal phenotype. As individuals with DS are living longer, they may be at a higher risk of aging disorders such as osteoporosis and increased fracture risk. Sexual dimorphism has been suggested between males and females with DS in which males, not females, experience an earlier decline in bone mineral density (BMD). Unfortunately, studies focusing on skeletal health related to Trisomy 21 (T21) are few in number and often too underpowered to answer questions about skeletal development, resultant osteoporosis, and sexual dimorphism, especially in stages of bone accrual. Further confounding the field are the varied methods of bone imaging, analysis, and data interpretation. This review takes a critical look at the current knowledge of DS skeletal phenotypes, both from human and mouse studies, and presents knowledge gaps that need to be addressed, differences in research methodologies and analyses that affect the interpretation of results, and proposes guidelines for overcoming obstacles to understand skeletal traits associated with DS. By examining our current knowledge of bone in individuals with T21, a trajectory for future studies may be established to provide meaningful solutions for understanding the development of and improving skeletal structures in individuals with and without DS.Item Pregnancy facilitates maternal liver regeneration after partial hepatectomy(American Physiological Society, 2020-04-01) Lee, Joonyong; Garcia, Veronica; Nambiar, Shashank Manohar; Jiang, Huaizhou; Dai, Guoli; Biology, School of ScienceLiver resection induces robust liver regrowth or regeneration to compensate for the lost tissue mass. In a clinical setting, pregnant women may need liver resection without terminating pregnancy in some cases. However, how pregnancy affects maternal liver regeneration remains elusive. We performed 70% partial hepatectomy (PH) in nonpregnant mice and gestation day 14 mice, and histologically and molecularly compared their liver regrowth during the next 4 days. We found that compared with the nonpregnant state, pregnancy altered the molecular programs driving hepatocyte replication, indicated by enhanced activities of epidermal growth factor receptor and STAT5A, reduced activities of cMet and p70S6K, decreased production of IL-6, TNFα, and hepatocyte growth factor, suppressed cyclin D1 expression, increased cyclin A1 expression, and early activated cyclin A2 expression. As a result, pregnancy allowed the remnant hepatocytes to enter the cell cycle at least 12 h earlier, increased hepatic fat accumulation, and enhanced hepatocyte mitosis. Consequently, pregnancy ameliorated maternal liver regeneration following PH. In addition, a report showed that maternal liver regrowth after PH is driven mainly by hepatocyte hypertrophy rather than hyperplasia during the second half of gestation in young adult mice. In contrast, we demonstrate that maternal liver relies mainly on hepatocyte hyperplasia instead of hypertrophy to restore the lost mass after PH. Overall, we demonstrate that pregnancy facilitates maternal liver regeneration likely via triggering an early onset of hepatocyte replication, accumulating excessive liver fat, and promoting hepatocyte mitosis. The results from our current studies enable us to gain more insights into how maternal liver regeneration progresses during gestation.NEW & NOTEWORTHY We demonstrate that pregnancy may generate positive effects on maternal liver regeneration following partial hepatectomy, which are manifested by early entry of the cell cycle of remnant hepatocytes, increased hepatic fat accumulation, enhanced hepatocyte mitosis, and overall accelerated liver regrowth.Item Whole exome sequencing and co-expression analysis identify an SCN1A variant that modifies pathogenicity in a family with Genetic Epilepsy and Febrile Seizures Plus (GEFS+)(Wiley, 2022) Hammer, Michael F.; Pan, Yanling; Cumbay, Medhane; Pendziwiat, Manuela; Afawi, Zaid; Goldberg-Stern, Hadassah; Johnstone, Laurel; Helbig, Ingo; Cummins, Theodore R.; Biology, School of ScienceObjective Family members carrying the same SCN1A variant often exhibit differences in the clinical severity of epilepsy. This variable expressivity suggests that other factors aside from the primary sodium channel variant influence the clinical manifestation. However, identifying such factors has proven challenging in humans. Methods We perform whole exome sequencing in a large family in which an SCN1A variant (p.K1372E) is segregating that is associated with a broad spectrum of phenotypes ranging from lack of epilepsy, to febrile seizures and absence seizures, to Dravet Syndrome. We assessed the hypothesis that the severity of SCN1A-related phenotype was affected by alternate alleles at a modifier locus (or loci). Results One of our top candidates identified by WES was a second variant in the SCN1A gene (p.L375S) that was exclusively shared by unaffected carriers of K1372E allele. To test the hypothesized that L375S nullifies the loss-of-function effect of K1372E, we transiently expressed Nav1.1 carrying the two variants in HEK293T cells and compared their biophysical properties with the wild-type (WT) variant, and then co-expressed WT with K1372E or L375S with K1372E in equal quantity and tested the functional consequence. The data demonstrated that co-expression of the L375S and K1372E alleles reversed the loss-of-function property brought by the K1372E variant, while WT-K1372E co-expression remained partial loss-of-function. Significance These results support the hypothesis that L375S counteracts the loss-of-function effect of K1372E such that individuals carrying both alleles in trans do not present epilepsy-related symptoms. We demonstrate that monogenic epilepsies with wide expressivity can be modified by additional variants in the disease gene, providing a novel framework for gene-phenotype relationship in genetic epilepsies.Item Fluctuating Asymmetry and Sexual Dimorphism in Human Facial Morphology: A Multi-Variate Study(MDPI, 2021-02) Ekrami, Omid; Claes, Peter; Van Assche, Ellen; Shriver, Mark D.; Weinberg, Seth M.; Marazita, Mary L.; Walsh, Susan; Van Dongen, Stefan; Biology, School of ScienceBackground: Fluctuating asymmetry is often used as an indicator of developmental instability, and is proposed as a signal of genetic quality. The display of prominent masculine phenotypic features, which are a direct result of high androgen levels, is also believed to be a sign of genetic quality, as these hormones may act as immunosuppressants. Fluctuating asymmetry and masculinity are therefore expected to covary. However, there is lack of strong evidence in the literature regarding this hypothesis. Materials and methods: In this study, we examined a large dataset of high-density 3D facial scans of 1260 adults (630 males and 630 females). We mapped a high-density 3D facial mask onto the facial scans in order to obtain a high number of quasi-landmarks on the faces. Multi-dimensional measures of fluctuating asymmetry were extracted from the landmarks using Principal Component Analysis, and masculinity/femininity scores were obtained for each face using Partial Least Squares. The possible correlation between these two qualities was then examined using Pearson's coefficient and Canonical Correlation Analysis. Results: We found no correlation between fluctuating asymmetry and masculinity in men. However, a weak but significant correlation was found between average fluctuating asymmetry and masculinity in women, in which feminine faces had higher levels of fluctuating asymmetry on average. This correlation could possibly point to genetic quality as an underlying mechanism for both asymmetry and masculinity; however, it might also be driven by other fitness or life history traits, such as fertility. Conclusions: Our results question the idea that fluctuating asymmetry and masculinity should be (more strongly) correlated in men, which is in line with the recent literature. Future studies should possibly focus more on the evolutionary relevance of the observed correlation in women.Item Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures(Elsevier, 2021-02-02) Miyoshi, Takushi; Zhang, Qianli; Miyake, Takafumi; Watanabe, Shin; Ohnishi, Hiroe; Chen, Jiji; Vishwasrao, Harshad D.; Chakraborty, Oisorjo; Belyantseva, Inna A.; Perrin, Benjamin J.; Shroff, Hari; Friedman, Thomas B.; Omori, Koichi; Watanabe, Naoki; Biology, School of ScienceFast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin). Specific antibodies show fast dissociation with half-lives ranging from 0.98 to 2.2 s. Unexpectedly, fast-dissociating yet specific antibodies are not so rare. A combination of fluorescently labeled Fab probes synthesized from these antibodies and light-sheet microscopy, such as dual-view inverted selective plane illumination microscopy (diSPIM), reveal rapid turnover of espin within long-lived F-actin cores of inner-ear sensory hair cell stereocilia, demonstrating that fast-dissociating specific antibodies can identify novel biological phenomena.Item A Multivariate Approach to Determine the Dimensionality of Human Facial Asymmetry(MDPI, 2020-03) Ekrami, Omid; Claes, Peter; White, Julie D.; Weinberg, Seth M.; Marazita, Mary L.; Walsh, Susan; Shriver, Mark D.; Van Dongen, Stefan; Biology, School of ScienceMany studies have suggested that developmental instability (DI) could lead to asymmetric development, otherwise known as fluctuating asymmetry (FA). Several attempts to unravel the biological meaning of FA have been made, yet the main step in estimating FA is to remove the effects of directional asymmetry (DA), which is defined as the average bilateral asymmetry at the population level. Here, we demonstrate in a multivariate context that the conventional method of DA correction does not adequately compensate for the effects of DA in other dimensions of asymmetry. This appears to be due to the presence of between-individual variation along the DA dimension. Consequently, we propose to decompose asymmetry into its different orthogonal dimensions, where we introduce a new measure of asymmetry, namely fluctuating directional asymmetry (F-DA). This measure describes individual variation in the dimension of DA, and can be used to adequately correct the asymmetry measurements for the presence of DA. We provide evidence that this measure can be useful in disentangling the different dimensions of asymmetry, and further studies on this measure can provide valuable insight into the underlying biological processes leading to these different asymmetry dimensions.