Faculty Bibliography

The three mammalian RAS genes (HRAS, NRAS and KRAS) encode four proteins that play central roles in cancer biology. Among them, KRAS is mutated more frequently in human cancer than any other oncogene. The pre-mRNA of KRAS is alternatively spliced to give rise to two products, KRAS4A and KRAS4B, which differ in the membrane targeting sequences at their respective C-termini. Notably, both KRAS4A and KRAS4B are oncogenic when KRAS is constitutively activated by mutation in exon 2 or 3. Whereas KRAS4B is the most studied oncoprotein, KRAS4A is understudied and until recently considered relatively unimportant. Emerging work has confirmed expression of KRAS4A in cancer and found non-overlapping functions of the splice variants. The most clearly demonstrated of these is direct regulation of hexokinase 1 by KRAS4A, suggesting that the metabolic vulnerabilities of KRAS-mutant tumors may be determined in part by the relative expression of the splice variants. The aim of this review is to address the most relevant characteristics and differential functions of the KRAS splice variants as they relate to cancer onset and progression.

Troxerutin (TXR) is a phytochemical reported to possess anti-inflammatory and hepatoprotective effects. In this study, we aimed to exploit the antiarthritic properties of TXR using an adjuvant-induced arthritic (AIA) rat model. AIA-induced rats showed the highest arthritis score at the disease onset and by oral administration of TXR (50, 100, and 200 mg/kg body weight), reduced to basal level in a dose-dependent manner. Isobaric tags for relative and absolute quantitative (iTRAQ) proteomics tool were employed to identify deregulated joint homogenate proteins in AIA and TXR-treated rats to decipher the probable mechanism of TXR action in arthritis. iTRAQ analysis identified a set of 434 proteins with 65 deregulated proteins (log₂ case/control≥1.5) in AIA. Expressions of a set of important proteins (AAT, T-kininogen, vimentin, desmin, and nucleophosmin) that could classify AIA from the healthy ones were validated using Western blot analysis. The Western blot data corroborated proteomics findings. In silico protein-protein interaction study of tissue-proteome revealed that complement component 9 (C9), the major building blocks of the membrane attack complex (MAC) responsible for sterile inflammation, get perturbed in AIA. Our dosimetry study suggests that a TXR dose of 200 mg/kg body weight for 15 days is sufficient to bring the arthritis score to basal levels in AIA rats. We have shown the importance of TXR as an antiarthritic agent in the AIA model and after additional investigation, its arthritic ameliorating properties could be exploited for clinical usability.

As obligate intracellular parasites with reduced genomes, microsporidia must infect host cells in order to replicate and cause disease. They can initiate infection by utilizing a harpoon-like invasion organelle called the polar tube (PT). The PT is both visually and functionally a striking organelle and is a characteristic feature of the microsporidian phylum. Outside the host, microsporidia exist as transmissible, single-celled spores. Inside each spore, the PT is arranged as a tight coil. Upon germination, the PT undergoes a large conformational change into a long, linear tube and acts as a tunnel for the delivery of infectious cargo from the spore to a host cell. The firing process is extremely rapid, occurring on a millisecond timescale, and the emergent tube may be as long as 20 times the size of the spore body. In this chapter, we discuss what is known about the structure of the PT, the mechanics of the PT firing process, and how it enables movement of material from the spore body.

Wildtype or mutant proteins expressed beyond the capacity of a cell's protein folding system could be detrimental to general cellular function and survival. In response to misfolded protein overload in the endoplasmic reticulum (ER), eukaryotic cells activate the Unfolded Protein Response (UPR) that helps cells restore protein homeostasis in the endoplasmic reticulum (ER). As part of the UPR, cells attenuate general mRNA translation and activate transcription factors that induce stress-responsive gene expression.UPR signaling draws research interest in part because conditions that cause chronic protein misfolding in the ER or those that impair UPR signaling underlie several diseases including neurodegeneration, diabetes, and cancers. Model organisms are frequently employed in the field as the UPR pathways are generally well-conserved throughout phyla. Here, we introduce experimental procedures to detect UPR in Drosophila melanogaster.

Brain-derived Neurotrophic Factor (BDNF) binds to the TrkB tyrosine kinase receptor, which dictates the sensitivity of neurons to BDNF. A unique feature of TrkB is the ability to be activated by small molecules in a process called transactivation. Here we report that the brain neuropeptide oxytocin increases BDNF TrkB activity in primary cortical neurons and in the mammalian neocortex during postnatal development. Oxytocin produces its effects through a G protein-coupled receptor (GPCR), however, the receptor signaling events that account for its actions have not been fully defined. We find oxytocin rapidly transactivates TrkB receptors in bath application of acute brain slices of 2-week-old mice and in primary cortical culture by increasing TrkB receptor tyrosine phosphorylation. The effects of oxytocin signaling could be distinguished from the related vasopressin receptor. The transactivation of TrkB receptors by oxytocin enhances the clustering of gephyrin, a scaffold protein responsible to coordinate inhibitory responses. Because oxytocin displays pro-social functions in maternal care, cognition, and social attachment, it is currently a focus of therapeutic strategies in autism spectrum disorders. Interestingly, oxytocin and BDNF are both implicated in the pathophysiology of depression, schizophrenia, anxiety, and cognition. These results imply that oxytocin may rely upon crosstalk with BDNF signaling to facilitate its actions through receptor transactivation.

Polymorphisms in the Apolipoprotein L1 (APOL1) gene are common in ancestrally African populations, and associate with kidney injury and cardiovascular disease. These risk variants (RV) provide an advantage in resisting Trypanosoma brucei, the causal agent of African trypanosomiasis, and are largely absent from non-African genomes. Clinical associations between the APOL1 high risk genotype (HRG) and disease are stronger in those with comorbid infectious or immune disease. To understand the interaction between cytokine exposure and APOL1 cytotoxicity, we established human umbilical vein endothelial cell (HUVEC) cultures representing each APOL1 genotype. Untreated HUVECs were compared to IFNÉ£-exposed; and APOL1 expression, mitochondrial function, lysosome integrity, and autophagic flux were measured. IFNÉ£ increased median APOL1 expression across all genotypes 22.1 (8.3 to 29.8) fold (p=0.02). Compared to zero risk variant-carrying HUVECs (0RV), HUVECs carrying 2 risk variant copies (2RV) showed both depressed baseline and maximum mitochondrial oxygen consumption (p<0.01), and impaired mitochondrial networking on MitoTracker assays. These cells also demonstrated a contracted lysosomal compartment, and an accumulation of autophagosomes suggesting a defect in autophagic flux. Upon blocking autophagy with non-selective lysosome inhibitor, hydroxychloroquine, autophagosome accumulation between 0RV HUVECs and untreated 2RV HUVECs was similar, implicating lysosomal dysfunction in the HRG-associated autophagy defect. Compared to 0RV and 2RV HUVECs, HUVECs carrying 1 risk variant copy (1RV) demonstrated intermediate mitochondrial respiration and autophagic flux phenotypes, which were exacerbated with IFNÉ£ exposure. Taken together, our data reveal that IFNÉ£ induces APOL1 expression, and that each additional RV associates with mitochondrial dysfunction and autophagy inhibition. IFNÉ£ amplifies this phenotype even in 1RV HUVECs, representing the first description of APOL1 pathobiology in variant heterozygous cell cultures.

Previous studies revealed an abundance of functional Connexin43 (Cx43) hemichannels consequent to loss of plakophilin-2 (PKP2) expression in adult murine hearts. The increased Cx43-mediated membrane permeability is likely responsible for excess entry of calcium into the cells, leading to an arrhythmogenic/cardiomyopathic phenotype. The latter has translational implications to the molecular mechanisms of inheritable arrhythmogenic right ventricular cardiomyopathy (ARVC). Despite functional evidence, visualization of these "orphan" (i.e., non-paired in a gap junction configuration) Cx43 hemichannels remains lacking. Immuno-electron microscopy (IEM) remains an extremely powerful tool to localize, with nanometric resolution, a protein within its native structural landscape. Yet, challenges for IEM are to preserve the antigenicity of the molecular target and to provide access for antibodies to reach their target, while maintaining the cellular/tissue ultrastructure. Fixation is important for maintaining cell structure, but strong fixation and vigorous dehydration (as it is routine for EM) can alter protein structure, thus impairing antigen-antibody binding. Here, we implemented a method to combine pre-embedding immunolabeling (pre-embedding) with serial block-face scanning electron microscopy (SBF-SEM). We utilized a murine model of cardiomyocyte-specific, Tamoxifen (TAM) activated knockout of PKP2. Adult hearts were harvested 14 days post-TAM, at this time hearts present a phenotype of concealed ARVC (i.e., an arrhythmogenic phenotype but no overt structural disease). Thick (200 µm) vibratome slices were immunolabelled for Cx43 and treated with nanogold or FluoroNanogold, coupled with a silver enhancement. Left or right ventricular free walls were dissected and three-dimensional (3D) localization of Cx43 in cardiac muscle was performed using SBF-SEM. Reconstructed images allowed us to visualize the entire length of gap junction plaques, seen as two parallel, closely packed strings of Cx43-immunoreactive beads at the intercalated disc. In contrast, in PKP2-deficient hearts we observed bulging of the intercellular space, and entire areas where only one of the two strings could be observed, indicating the presence of orphan Cx43. We conclude that pre-embedding and SBF-SEM allowed visualization of cardiac Cx43 plaques in their native environment, providing for the first time a visual complement of functional data indicating the presence of orphan Cx43 hemichannels resulting from loss of desmosomal integrity in the heart.

OBJECTIVE/UNASSIGNED:The present study aimed to enhance understanding of continuity and stability of positive parenting of infants, across age and different settings in women with a history of depression who are at elevated risk for postpartum depression. DESIGN/UNASSIGNED:= 103) with a history of major depression and their infants were observed during 5-min play and feeding interactions when their infants were 3, 6, and 12 months of age. Summary scores representing mothers' positive parenting were computed separately for each age and context based on ratings of five parenting behaviors. Mothers' depressive symptom levels were assessed at each infant age. RESULTS/UNASSIGNED:Continuity (consistency of level) and stability (consistency of rank order) were assessed across age and context at both the group and individual level. Across-age analyses revealed continuity in the play context and discontinuity in the feeding context, albeit only at the group level, as well as weak to moderate stability. Across-context analyses revealed higher positive parenting scores in play than feeding at all time points as well as weak to moderate stability. Variations in positive parenting across age and context were independent of mothers' postpartum depressive symptom levels. CONCLUSIONS/UNASSIGNED:Findings based on normative samples may not generalize to women with a history of depression, who may benefit from interventions aimed at enhancing their positive parenting over the course of infancy, regardless of postpartum depressive symptom level. Results also underscore the importance of assessing parenting at multiple age points and across varying contexts.