Poster Day 2024: Abstracts: Basic Science

The study of the ENP5 mouse on basal cell hyperplasia and eosinophilic epithelial infiltration within the esophagus did not show significance in preventing EoE disease features.

These primer sets were shown to amplify predicted products from cell cultures and will provide an assay to identify MAP4K4 isozymes from cell lines and isolated tissues.

Dysregulation of kinase signaling is implicated in the uncontrolled growth and survival of cancer cells. Therefore, understanding the intricacies of these pathways is paramount for the development of targeted therapies.  Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a member of the germinal center kinase (GCK) family, has been implicated in signaling pathways associated with cancer from cell division, migration, invasion, and adhesion. The kinase domain from MAP4K4 is a well-conserved domain shared across multiple genes in the GCK family from humans and found in many organisms from slime molds, yeasts, nematodes, and mammals.

The purpose of this work was to amplify, sequence, and compare the kinase domain of MAP4K4 between normal and dysregulated epithelial cell cultures. Identification of ATP-binding domain(s) and comparison of sequence homologies will provide insight to kinase activity in cells.

Using the NIH Primer-BLAST tool, we designed multiple oligonucleotides to amplify the MAP4K4 kinase domain from dividing human breast cell cultures. The MAP4K4 kinase domain spans exons 2-11 of the single copy gene located at 2q11.2 in the human genome. These primers generated a 200 base pair product from cDNA generated from total RNA isolated from multiple human cell lines. These PCR products were subcloned, sequenced, and aligned to compare transcripts from normal and metastatic cells.

We found that MAP4K4 sequences were conserved between both normal and metastatic cells. These data suggest that kinase activity is controlled by increased regulatory domain input or by increased expression levels of the gene.

Metastatic breast cells require complex signaling to an array of regulatory proteins to control cell migration. The coordination of these signaling proteins is necessary to regulate the actin cytoskeleton and allow a metastatic cell to migrate from a primary tumor in response to growth factors. The microRNA miR-141-3p has been shown to regulate the expression of key signaling proteins responsible for cancer metastasis in several tissues, including the mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4). Here, we lay the groundwork to investigate the role of miR-141-3p on cell migration and invasion in established human breast cancer cell lines.

The purpose of this study was to compare mir141-3p and MAP4K4 RNA expression in metastatic breast cancer with normal breast cell cultures using quantitative PCR.

All quantitative PCR was performed from cDNA generated from RNA purified from normal (MCF-10A) and metastatic (MDA-MB-231) human breast cell cultures. We have found that consistently, MCF-10A cells express miR-141-3p at more than 30% that of MDA-MB-231 cells. Conversely, MAP4K4 gene expression is down-regulated in MCF-10A and increased in MDA-MB-231 cells.

These data suggest that mir141-3p down-regulates MAP4K4 expression in normal epithelial cells through a regulatory domain in the upstream untranslated region of the MAP4K4 gene. Reduced expression of mir141-3p in MDA-MB-231 cells results in increased MAP4KA expression and thus increased levels of metastatic activity. Future studies aim to develop a liquid biopsy to identify microRNAs in patients for early detection of breast cancer.

Dysregulation of kinase signaling is implicated in the uncontrolled growth and survival of cancer cells. Therefore, understanding the intricacies of these pathways is paramount for the development of targeted therapies.  Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a member of the germinal center kinase (GCK) family, has been implicated in signaling pathways associated with cancer from cell division, migration, invasion, and adhesion. The citron homology domain from MAP4K4 is a regulatory domain that contains multiple domains of unknown function that may contribute to kinase activity in metastatic cancer cells.

The purpose of this work was to amply, sequence, and compare the citron homology domain of MAP4K4 between normal and dysregulated breast cell cultures using a PCR-based screening tool developed by our research group.

Using the NIH Primer-BLAST tool, we designed multiple oligonucleotides to amplify the citron homology domain from MAP4K4 by PCR. The citron homology domain is C-terminal of the MAP4K4 kinase domain (exons 25-33) and includes an interdomain with three varying regions of interest: 1) a charged arg/lys domain, 2) a proline-rich domain, and 3) a ser/thr region. Each of these domains have potential for regulatory and structural functions.

We found that our primer oligos amplified the citron homology domain. Sequence information was acquired and aligned with known MAP4K4 homologues to gain insight on domain function and metastatic activity.

Nucleotide sequences from both normal and metastatic cells were compared from multiple cell lines and identified three potential binding sides for autoregulation of the MAP4K4 kinase domain. Variations in exon splicing or mutations found in metastatic cells could explain increased metastatic activity observed in cell cultures.

This case study investigates the risks of high folic acid supplementation in expecting mothers, with a focus on those with the methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism. While folic acid is vital for DNA synthesis and preventing neural tube defects, excessive intake may pose health risks, especially for those with the MTHFR variant. These individuals have reduced folic acid processing ability, leading to unmetabolized folic acid (UMFA) accumulation, which is linked to vitamin B12 deficiency, cognitive and psychiatric issues, and adverse pregnancy outcomes. The study highlights the need for careful monitoring of folic acid intake and explores alternative supplements like 5-MTHF or 5-FTHF that may mitigate these risks by bypassing MTHFR conversion. Personalized nutrition considering genetic variations is crucial for optimizing health and avoiding potential harm.

Hannah Berko OMS-II, Isabella Porter OMS-III, Benford Mafuvadze PhD, MVSc, BVSc   

Natural and synthetic progestins increase transcriptional expression of pri-miR-190 and pri-miR-199a-1 through processes directly dependent on the progesterone receptor. Both microRNAs tested can be potential biomarkers in progesterone receptor positive breast cancers.        

The early postnatal period is a valuable model for studying synaptic plasticity, as pyramidal neurons and interneurons adjust their numbers to balance levels of excitation and inhibition. A recent study found that NMDA receptors and calpain-1 work together to maintain neuronal survival during this time, but it did not specify which neuron types were protected. This study investigated the level of cell death in both excitatory and inhibitory neurons in wild-type (WT) and calpain-1KO (C1KO) mice at postnatal day 7. We hypothesized that during the critical period, calpain-1 plays an integral role in neuronal survival. In addition, Calpain-1 exists as a homeostatic mechanism that maintains the balance between excitation and inhibition in the tri-synaptic circuit of the hippocampus, which would result in a similar fraction of degenerating pyramidal and inhibitory neurons. This project used co-staining techniques to visualize and quantify excitatory and inhibitory neuronal apoptosis levels in the hippocampus of WT and C1KO mice. The results showed increased apoptosis of both excitatory and inhibitory neurons in C1KO mice, with a higher percentage of apoptotic inhibitory neurons. In the tri-synaptic circuit of the hippocampus, interneuron apoptosis was significantly increased in the CA1 and dentate gyrus regions. These findings suggest that calpain-1 serves as a homeostatic mediator of excitation and inhibition in mouse brains and highlights its distribution in the hippocampus. Overall, the study demonstrates that calpain-1 activity prevents neuronal apoptosis in the mouse hippocampus and offers insights into early synaptic plasticity.

A. Kuzema, B, M.O. Ibiwoye, MD, MS, DTM&H, MPH, PhD, J. Lyons, MD and J.D. Foster, PhD, HHRT

This project sought to find associated phenotypic abnormalities correlating to mutations within the Glucokinase Regulatory Protein (GKRP). GKRP will modulate the body’s reaction to different levels of glucose in the blood which has clinical implications of hyperglycemia or hypertriglyceridemia depending on the genotype of the GKRP. This project aimed to characterize different genetic variants of GKRP through analysis of its enzymatic activity and how it compared to the native GKRP.  

The Vmax values were decreased in both of the genetic mutations. This can lead to hyperglycemia and subsequent diabetes in patients with these genetic mutations. Some limitations to this project could be that the assay was not sensitive enough to change within the protein activity.