bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of postnatal maturation on muscarinic acetylcholine receptor distribution in hypoglossal motorneurons
Authors: Koziol Sophia M. , White Monica M. , Banayat Tatum , Buhlman Lori M. , Revill Ann L.
Published: May 2022

Upper airway patency is decreased during rapid eye movement (REM) sleep due to loss of genioglossus (primary tongue protruder) tongue muscle tone. Hypoglossal motoneurons (XII MNs), which innervate the genioglossus muscle, receive less excitatory noradrenergic drive, and may be inhibited by activation of muscarinic acetylcholine receptors, during REM sleep. Indeed, preliminary data from intact adult rats during natural sleep implicated an inhibitory effect of muscarinic acetylcholine receptors on hypoglossal motoneurons. However, data using the rhythmic slice preparation from neonatal mice indicate that muscarine has a net excitatory effect on inspiratory burst amplitude at hypoglossal motoneurons. Our project examines the distribution of muscarinic acetylcholine receptors at XII MNs, and how that changes across postnatal maturation. We hypothesizes there would be an increase in inhibitory (M2) and decrease in excitatory (M1, M3, M5) muscarinic receptors in XII MNs with postnatal maturation. We performed double‐labeled immunofluorescence experiments on perfused 20μm transverse brainstem slices across six postnatal age groups under identical conditions: P0‐P2, P3‐P5, P6‐P10, P11‐P13, P14‐P17, and adult against muscarinic targets M1, M2, M3, M5. Slices were co‐labeled with choline acetyltransferase (ChAT) and 4’,6‐diamidino‐2‐phenylindole (DAPI). Images of the hypoglossal motor nucleus (HMN) were collected using a confocal microscope. ImageJ was used to determine the average HMN muscarine receptor subtype intensity across postnatal maturation. Preliminary data (n = 1) indicate that M1 receptors showed a consistent decrease in labeling intensity in the HMN across development (P5=100%, P13=80%, P17=67%, adult = 40%), whereas M3 (n = 1) (P4= 90%, P9= 73%, P13=89%, P17= 100%, adult= 41%) showed a transitory increases in expression before a final decrease into adulthood. M5 (n = 1 or 2) expression shows a bimodal distribution of labeling intensity (P0‐2= 100%, P4‐6= 58‐99%, P7‐10 = 52‐70%, P11‐13= 35‐58 %, P14‐17 = 27 % adult= 17‐37 %). In contrast, M2 receptor expression intensity remained relatively high with small fluctuations into adulthood (n=2), (P0‐2 = 92‐94 %, P4‐6 = 100%, P7‐10 = 92‐95 %, P11‐13 = 78‐107 %, P14‐17 = 88‐107 %, adult = 73‐77 %). These data partly support our hypothesis that there would be a decrease in expression intensity of excitatory M1, M3, and M5 receptor subtypes into adulthood. Contrary to our hypothesis, we observed minimal change in the expression intensity of inhibitory M2 receptors. We speculate that the decrease in labeling intensity of excitatory muscarinic receptor subtypes may support the observed shift of muscarinic modulation from excitation to inhibition with postnatal maturation.
High‐throughput CRISPR for Generating Therapeutic Nanobodies
Authors: Rowe Jacob B. , Lee Kyutae , Isom Daniel
Published: May 2022

Engineered antibodies have had a widespread impact throughout biomedical sciences, from their use as fundamental research tools to their employment as therapeutics. Closely related nanobodies, derived from heavy‐chain only antibodies, have begun gaining much attention as alternatives with improved pharmacological properties. Containing only the antigen recognition components, nanobodies maintain the exceptional specificity of conventional antibodies but their simplistic design leads to increased stability and decreased toxicity. In recent years, nanobodies have proven especially useful for studying G protein‐coupled receptors (GPCRs), a class of membrane receptors responsible for activating intracellular proteins (e.g., Gα proteins) and initiating subsequent signaling pathways. Most GPCR‐targeting nanobodies to date have been designed as crystallographic chaperones for stabilizing the GPCR‐Gα interaction for in‐depth structural insights. However, harnessing the intrinsic specificity provided by nanobodies for therapeutic purposes has been limited to few receptors. In this study, we present a unique platform for discovering functionally selective nanobodies for a broad range of GPCRs. Using a yeast model proven invaluable for discovering GPCR ligands and structure‐stabilizing nanobodies, we engineered a system for rapidly identifying nanobody‐based therapeutic candidates. In this system, each cell uniquely contains 1) a tethered nanobody displayed outside the cell membrane, 2) a human GPCR, 3) one of ten Gα proteins for characterizing GPCR‐Gα interactions, and 4) a fluorescent reporter induced upon GPCR/Gα activation. By combining all components into a single cell, we can directly access the therapeutic potential of each nanobody‐GPCR combination by monitoring the (in)activity of relevant signaling pathways inspired from this interaction. To date, we have completed the initial engineering stages of this platform and showcased its utility through pharmacological activation of various GPCRs (e.g., SSTR5, AGTR1, CXCR4, and C5aR) via tethered peptide/protein agonists. Next, we will use high‐throughput CRISPR to generate an extensive nanobody library for rapidly identifying and functionally quantifying GPCR‐targeting nanobodies. Using this approach for a large variety of GPCRs, this platform will provide a vast collection of nanobody‐based therapeutic candidates, and with minimal modifications may be expanded for identifying therapeutics for other classes of membrane receptors.
Localization of Orphan GPCRs within the Liver
Authors: Kurtz Ryan , Shepard Blythe D.
Published: May 2022

G protein‐coupled receptors (GPCRs) are the largest gene family in the genome and are involved in almost every aspect of physiology ranging from hormone receptors to eyesight. They also happen to represent the largest class of “druggable” molecules; in fact, 20‐30% of all FDA‐approved drugs on the market target GPCRs. Despite this, the vast majority of GPCRs remain underappreciated with incomplete expression patterns and characterization. These receptors, termed GPRs, have been linked to metabolic pathways and are known to respond to naturally produced metabolites. We reasoned that the liver, the largest metabolic organ in the body, is primed to take advantage of some of these GPRs in order to regulate its internal environment. To identify these receptors, we performed a large‐scale screen for over 300 GPCRs on whole murine liver using TaqMan qPCR array cards. This screen identified a total of 57 GPRs – with 7 of the them deemed to be “high expressing” with ΔCt values comparable to the more well‐characterized hepatic GPCRs (i.e. glucagon receptor). We confirmed expression of each of these GPRs (GPRc5c, GPR125, GPR146, GPR137, GPR116, GPR108, GPR17) via gene‐specific PCR from multiple murine liver samples.

In order to characterize these understudied receptors within the liver, we must first understand where they are localized and what activates them. Given the dearth of reliable antibodies, we began by examining expression using RNAscope, a powerful in situ technique that specifically localizes mRNA sequences. GPR108, which has previously been linked to regulation of Toll‐Like Receptor signaling, was found within hepatocytes. GPR146, a known C‐peptide receptor which has been shown to impact blood cholesterol levels, was highly expressed in hepatocytes, and weakly localized to cholangioctyes and vessels. GPR109a, a highly studied receptor known to respond to short chain fatty acids and a potential therapeutic target for liver disease, was found to be primarily expressed in cholangiocytes, which notably play a key role in modification of bile. Finally, GPR125, an atypical adhesion GPCR, exhibited weak expression in hepatocytes but rather strong staining in select cholangiocytes and the liver vasculature. This was confirmed using both immunohistochemistry and immunofluorescence microscopy where we noted basolateral staining along the small cholangiocytes and hepatocytes. Given its localization and function as an adhesion receptor, GPR125 is well‐positioned within the liver to respond to, and to regulate, extracellular matrix proteins and their composition in both health and disease. Efforts are currently underway to deorphanize these receptors using the PRESTO‐Tango assay and to better understand their roles in the pathogenesis of metabolic diseases in the liver.
Metabolic Alterations in Aorta Induces Brain Cholesterolemia, Memory Loss and Cognitive Dysfunction in Diabetic Mice
Authors: Aluganti Narasimhulu Chandrakala , Singla Dinender
Published: May 2022

Aim

Diabetic hyperlipidemia and associated cognitive dysfunction are of major concern and is essential to understand the mechanism and pathophysiology of diabetic brains, which remains unknown. In this study, we investigated whether hypercholesterolemia induces apoptosis and cognitive dysfunction, in diabetic apolipoprotein E (APOE) and paraoxonase 1 (PON1) double knockout (DKO) mice brains.

Hypothesis

We hypothesize that hypercholesterolemia induces apoptosis that involves cognitive dysfunction and furthers to Alzheimer’s.

Methods

ApoE‐PON1 DKO mice (6±2 months) were divided into control and STZ groups. At Day‐28, brain function, blood glucose, and serum lipids were measured followed by subjecting animals to Glucose Tolerance Test. Echocardiography was performed, and mice aortas were isolated. Brain‐parameters were examined using RT‐PCR, immunohistochemistry, and histologically.

Results

We observed a significant (p<0.05) increase in blood glucose levels, serum lipids, increased aortic lesions, as well as increased cholesterol loading and apoptotic markers in brains of diabetic mice as compared to control. A significant reduction in reverse cholesterol transport, and HDL‐associated proteins were observed. A significant (p<0.05) decrease in brain weight, size, and brain function were observed in diabetic mice. Furthermore, a significant (p<0.05) increase in PTEN and decrease in AKT levels were observed in diabetic mice. In addition, a significant (p<0.05) increase in Alzheimer’s specific markers were also observed.

Conclusion

In conclusion, our data suggest that hyperlipidemia induces apoptosis, that leads to neuronal cell death, memory‐loss, and cognitive dysfunction, which furthers Alzheimer’s in diabetic ApoE and PON1 DKO mice. Further therapeutic approaches require to be developed to target hyperlipidemia in diabetic brains.
Engineering Prophylactic Biosynthesis of an Antituberculosis Antibiotic
Authors: Robbins Laurel , Najibi Zarina , Conrad William
Published: May 2022

Tuberculosis is a deadly respiratory disease caused by the bacterium Mycobacterium tuberculosis which does not currently have a fully effective vaccine. The goal of this project is to develop an effective prophylaxis for tuberculosis using mammalian cells to biosynthesize the anti‐tuberculosis antibiotic, d‐cycloserine (dCS). We took two approaches to activate this biosynthetic pathway in mammalian cells. Initially, Chinese Hamster Ovarian cancer cells (CHO) were transfected with plasmids encoding all six dCS biosynthesis enzymes (dcsA‐G) and isolated total protein was tested for synthetic capability. The expression of the genes detected by fluorescence microscopy was low and the in‐vitro dCS biosynthesis from lysates was not detected by HPLC. Currently, individual enzymes are being optimized, starting with dcsE, which converts L‐serine to O‐acetyl‐L‐serine. To focus on dcsE, human lung epithelial cancer cells (A549) were transfected with a higher concentration of DNA. High dcsE expression was detected by fluorescence microscopy and a Western Blot was used to detect the presence of our GFP tagged dcsE protein. The Western Blot indicated that GFP dimerized and there is no dcsE tagged with GFP.
Loss of Rbm12, a Genetic Risk Factor for Psychosis, Leads to Hyperactivated GPCR/cAMP Signaling Pathway
Authors: Semesta Khairunnisa M. , Garces Angelica , Tsvetanova Nikoleta G.
Published: May 2022

G‐protein coupled receptors (GPCRs) are pivotal to neuronal functions such as neurotransmission and memory formation, and aberrant GPCR signaling has been implicated in complex neuropsychiatric disorders. Our ability to develop efficient therapeutic strategies therefore hinges on better understanding of the molecular factors that govern GPCR activity. Upon activation, GPCRs modulate a myriad of downstream second messengers and molecules that generate cellular responses, including the transcription of cAMP‐dependent target genes. Through a genome‐wide CRISPR‐based screen with a fluorescent cAMP transcriptional reporter, we identified RNA‐binding motif 12 (Rbm12) as a novel potent repressor of the GPCR/cAMP pathway. While Rbm12 is a genetic risk factor for familial psychosis, its precise cellular functions are unknown. We hypothesize that the loss of Rbm12’s regulation of the GPCR/cAMP pathway contributes to the development of psychosis. To investigate this intriguing possibility, we performed CRISPR‐based gene editing to show that Rbm12 deficiency leads to increased cAMP accumulation and hyperactivation of multiple cAMP‐dependent target genes downstream of several key neurotransmitter GPCRs. The signaling hyperactivation caused by Rbm12 loss is similarly conserved in human induced pluripotent stem cell‐derived neurons. Furthermore, we demonstrate that disease‐linked truncating mutations in Rbm12 (c.2377G>T and c.2532delT) fail to rescue the signaling hyperactivation due to possible loss‐of‐function and protein instability, respectively. Together, these experiments provide critical insights into this uncharacterized gene. By uncovering the novel regulatory role of a neuropsychiatric disease risk gene in GPCR signaling, we expand our understanding of the molecular basis of complex neurobiological disorders and enable the identification of novel druggable targets.
CRISPR/Cas9 with Homology‐Directed Repair Effectively Silences the Human Topoisomerase IIα Intron‐19 5′ Splice Site and Results in Etoposide Resistance in Human Leukemia K562 Cells
Authors: Hernandez Victor A. , Carvajal‐Moreno Jessika , Wang Xinyi , Pietrzak Maciej , Yalowich Jack , Elton Terry
Published: May 2022

DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/170 a target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2α/170. Our laboratory recently demonstrated that reduced levels of TOP2α/170 in acquired resistance to etoposide is associated with alternative RNA processing of the TOP2α gene and intron retention. Intron 19 retention in an etoposide‐resistant human leukemia K562 subline, K/VP.5, resulted not only in decreased TOP2α/170 but also translation and overexpression of a C‐terminal truncated 90‐kDa isoform of TOP2α, TOP2α/90. We found that this isoform heterodimerized with TOP2α/170 and was a determinant of acquired resistance to etoposide. Splice site analysis revealed that the exon 19/intron 19 boundary of the TOP2α gene is suboptimal, providing a potential explanation for intron 19 retention. To further test the hypothesis that intron 19 retention is a determinant of acquired drug resistance, CRISPR/Cas9 with homology‐directed repair (HDR) was used to silence the intron 19 5′ splice site of the TOP2α gene in parental K562 cells (etoposide‐sensitive) by making two nucleotide changes to the splice site (GAG//GTAAAC→GAA//CTAAAC) thereby forcing intron 19 retention by abrogating spliceosome function. Gene‐edited clones were identified by quantitative polymerase chain reaction (qPCR) and verified by Sanger sequencing. Clones with TOP2α edited alleles contained decreased TOP2α/170 mRNA/protein resulting in resistance to etoposide as assessed by reduced etoposide‐induced DNA damage (γH2AX, Comet assays) and growth inhibition. Forced expression of TOP2α/90 in the gene‐edited K562 cells further decreased etoposide‐induced DNA damage in support of a dominant negative role for this truncated isoform. Together results support the role of alternative TOP2α mRNA splicing as a determinant of resistance to TOP2α‐targeting agents.
Mitochondrial Fission is Essential to Maintain Cristae Morphology and Bioenergetics
Authors: Robertson Gabriella , Patel Mira , Riffle Stellan , Marshall Andrea , Beasley Heather , Garza‐Lopez Edgar , Hinton Antentor O. , Stoll Maria , Mears Jason , Gama Vivian
Published: May 2022

Mitochondria and peroxisomes are both dynamic signaling organelles that constantly undergo fission. While mitochondrial fission and fusion are known to coordinate cellular metabolism, proliferation, and apoptosis, the physiological relevance of peroxisome dynamics and the implications for cell fate are not fully understood. DRP1 (dynamin‐related protein 1) is an essential GTPase that executes both mitochondrial and peroxisomal fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L, present with encephalopathy due to mitochondrial and peroxisomal elongation (EMPF). EMPF is a devastating neurodevelopmental disease with no effective treatment. To interrogate the molecular mechanisms by which DRP1 mutations cause developmental defects, we are using patient‐derived fibroblasts and iPSC‐derived models from patients with mutations in different domains of DRP1 who present with clinically disparate conditions. Using super resolution imaging, we find that patient cells, in addition to displaying elongated mitochondrial and peroxisomal morphology, present with aberrant cristae structure. Given the direct link between cristae morphology and oxidative phosphorylation efficiency, we explored the impact of these mutations on cellular energy production. Patient cells display a lower coupling efficiency of the electron transport chain, increased proton leak, and Complex III deficiency. In addition to these metabolic abnormalities, mitochondrial hyperfusion results in hyperpolarized mitochondrial membrane potential. Intriguingly, human fibroblasts are capable of cellular reprogramming into iPSCs and appear to display peroxisome‐mediated mitochondrial adaptations that could help sustain these cell fate transitions. Understanding the mechanism by which DRP1 mutations cause cellular dysfunction will give insight into the role of mitochondrial and peroxisome dynamics in neurodevelopment.
Humanized ApoE4 Female Mice Exhibit a More Robust Hypoxic Ventilatory Response Than Female E3 Mice
Authors: Mendenhall Laura E. , Taylor Chase E. , Johnson Lance A. , Alilian Warren J.
Published: May 2022

Breathing is essential to life and can be easily disrupted by many adverse events. Disorders such as high cervical spinal cord injury (SCI) and sleep apnea can decrease quality of life and enhance the ill effects of comorbidities like Alzheimer’s disease (AD). Previous studies have shown an increase in cognitive impairments in subjects with both sleep apnea and AD. Apolipoprotein E (ApoE) is a protein found throughout the body and central nervous system (CNS) that is involved in the regulation and transport of cholesterol and lipids. Out of the three APOE human alleles (E2, E3, and E4), the E4 allele has been implicated in the early onset and higher risk of AD when compared to E2 or E3. Previous research in our lab has shown that the interaction between APOE genotype and sex has different effects on breathing motor plasticity following a cervical SCI in mice. However, it is unknown how baseline respiratory pattern and capability differs between uninjured individuals expressing either ApoE4 or E3, specifically under hypoxic conditions. Unpublished data from our lab has examined these differences in male mice, showing that E4 male mice have high variation in respiratory pattern, as well as a less robust response to hypoxia. Using ApoE as our independent variable, we measured respiratory parameters of female C57/BL6 ApoE3 and ApoE4 humanized mice using whole body plethysmography (WBP), hypothesizing that ApoE3 animals would display a greater baseline respiratory rate and have a more adaptive, heightened response to hypoxic exposure than those expressing ApoE4. Preliminary data comparing E3 female animals (n=8) and E4 female animals (n=8) showed differences across several key measurements. Interestingly, ApoE4 humanized female mice had a more robust response to hypoxic exposure that failed to persist following cessation of hypoxia. However, at baseline, E4 female animals showed no differences in respiratory pattern in contrast to our previously collected male ApoE4 data. These results indicate a critical role of the APOE genotype in the regulation of breathing and response to hypoxic conditions which differs between males and females. To further understand this distinction, future experiments will examine differences in levels of brainstem and spinal cord serotonin expression between ApoE3 and E4 males and females, as well as plasticity following injury in areas high in ApoE expression.
Lights, Camera, PAKtion: Design and Engineering of a Subcellular Targeting Optogenetic PAK1 Kinase and Sensor
Authors: Kinney William D. , Haar Lauren , Lawrence David S.
Published: May 2022

p21‐activated kinase 1 (PAK1) is a major ‘pathogenic kinase’ that directs cell motility, cytoskeletal remodeling and has been shown to function as a downstream regulator for various cancer signaling cascades. Such biological roles require a tight spatial and kinetic control of its localization and activity. Unfortunately, the spatiotemporal nature of these pathways renders the application of conventional tools inadequate for studying localized PAK1 induced cell behavior. Therefore, we have designed and engineered an optogenetic analog of PAK1 to address the difficulties in assessing PAK1 intracellular activity.

Optogenetics is a powerful light‐triggered technology that, via control of protein biochemical activity, allows for the spatial and temporal control over a variety of biological processes. We developed an engineering strategy to construct a genetically expressed, light‐responsive optogenetic analog of PAK1 (optoPAK1) and demonstrated that, upon illumination, optoPAK1 migrates to specified intracellular sites. OptoPAK1 was designed to function independently of endogenous biochemical regulation in a constitutively active manner with minimal activity in the dark state. We employed the improved light‐induced dimer (iLid) system as a means to recruit and photoactivate the protein analog at discrete subcellular domains. Furthermore, we designed intracellular spatially segregated reporters of optoPAK1 activity and preliminary data displayed that optoPAK1 phosphorylates these reporters in a light‐dependent fashion. Taken together, optoPAK1 and its corresponding biosensor offer the opportunity to appropriately study the relationship between PAK1 compartmentalized biochemical activity and cell behavior.
Physiological Changes in Response to Skeletal Muscle Specific High Expression of mu‐Crystallin Following High and Low‐Fat Diets
Authors: Noland Kaila , O'Neill Andrea , Pimparkar Ujwala , Aja Susan , Bloch Robert
Published: May 2022

Approximately 20 percent of the population expresses high levels of the thyroid hormone binding protein mu‐crystallin in skeletal muscle while the rest of the population expresses little to no mu‐crystallin. Since the thyroid hormones, T3 and T4, are potent regulators of metabolism and thermogenic regulation, our studies focus on the consequences of skeletal‐specific high expression (SM‐HE) of mu‐crystallin. We hypothesize that SM‐HE of mu‐crystallin influences cellular and metabolic changes in a diet‐ and sex‐ specific manner by influencing changes in gene expression.

To evaluate this, we generated transgenic mice (Crym‐tg) that over‐express mu‐crystallin in skeletal muscle, equivalent to human high expressors, which resulted in high levels of accumulation of T3 in muscle tissue. Respiratory exchange ratio (RER) in these mice indicated a small but significant increase in utilization of fat as an energy source.

In high‐ and low‐fat diet studies, we found differences between Crym‐tg and controls as well as sex specific differences. Following a 16‐week exposure, Crym‐tg males on high‐fat diet and Crym‐tg females on low‐fat diet weighed more than controls whereas Crym‐tg males on low‐fat diet and Crym‐tg females on high‐fat diet weighed less than controls. These differences were consistent with the results of glucose tolerance tests, in which only the Crym‐tg mice that weighed more than controls showed a significant increase in sensitivity to glucose. We are currently evaluating these results to understand their basis in transcriptomic and proteomic data, which have already revealed increased expression of genes involved in fatty acid oxidation and decreased expression of genes involved in glycolytic metabolism (Kinney et al. (2021) Curr Res Physiol. 4:47‐59.) as well as some members of the insulin signaling pathway.
Cell type specificity on integrin α2 mediated functions in pancreatic cancer
Authors: Mia Md. S. , Sun Dali , Venkatachalem Sathish , Mathew Sijo
Published: May 2022

Background

Among the various cancers, pancreatic cancer has the third highest mortality rate per 100,000 population each year. In 2021, an estimated 60,430 people will be diagnosed with pancreatic cancer in the United States, with more than 48,220 deaths. Desmoplasia is a hallmark of pancreatic cancer. Cell surface receptor integrin promotes tumor progression by modulating the reciprocal signaling between the actin‐cytoskeleton and the desmoplastic tumor microenvironment. Integrin’s are heterodimeric cell surface receptors consisting of non‐covalently associated α and β subunits. Integrin β1 forms heterodimers with 12 different α subunits, and its expression is upregulated in pancreatic cancer. Integrin α2 is one of the α subunits that shows both pro and anti‐tumorigenic properties in cancer and belongs to the I domain group of integrin where the I domain in α subunit mediates the binding to matrix proteins. This study tested the hypothesis that in pancreatic cancer, upregulation of integrin β1 differentially regulates integrin α2 mediated cell functions in a cell‐specific manner.

Method

Two of the major pancreatic cancer types, Mia Paca‐2 and Panc‐1 cell lines were used in this study. The protein level of integrin β1 was downregulated using genetic approaches and confirmed by immunoblotting, immunocytochemistry, and qRT‐PCR. Cell spreading and adhesion assays were performed with WT cells and clones with lower integrin β1 expression. Cells were grown on the 3D matrix to see the ability of cells to proliferate in the presence of matrix proteins.

Results

Downregulation of integrin β1 in both Mia Paca‐2 and Panc‐1 cells decreased tumor growth in the collagen‐Matrigel 3D matrix significantly (n=10, p<0.0001). With the reduction of expression of integrin β1, there is a significant decrease in the expression of integrin α5 in Mia paca‐2 and Panc‐1 cell, which resulted in a significant decrease in cell adhesion (n=3; P<0.05) and spreading (n=40; p<0.05) on fibronectin and vitronectin. However, there were contrasting results in the case of integrin α2 expression and functions with integrin β1 downregulation. An increase in integrin α2 expression was recorded in Miapaca‐2 cells (n=3, p<0.05) with increased cell adhesion (n=3; p<0.05) and cell spreading (n=3; p<0.05) on collagen‐1 by the deletion of integrin β1. In the case of Panc‐1, there is reduced expression of integrin α2 that resulted in decreased cell adhesion (n=3; p<0.05) and cellular spreading (n=30; p<0.05) on collagen 1 with the knock‐down of integrin β1.

Conclusion

The results indicate that the tumor suppressor property of integrin α2 varies between different tumor types in pancreatic cancer. Further studies are required to understand the mechanism of the anti‐tumorigenic property of integrin α2 in pancreatic cancer.
UL25 Deletion in hCMV and Impacts on Viral Replication Cycles
Authors: Laskie Asa K.
Published: May 2022

Human Cytomegalovirus (hCMV) is a beta herpes virus that impacts a significant portion of the adult U.S. population (50‐94% seroprevalence, depending on the region). Furthermore, hCMV is the leading infectious cause of congenital defects, predominantly sensory neural hearing loss and various neurological disorders. hCMV is a problem for immunocompromised transplant recipients. A major challenge in dealing with hCMV infections, as well as herpes virus infections in general, is the ability of the virus to go into a latent state of replication, where it produces very little gene products, and avoids host immune responses. Understanding the parameters of latent infectious phases of herpesviruses is necessary if substantial progress is to be made in treating all herpes virus infections. Additionally, understanding the mechanisms of latency in one herpes virus will provide insight on the mechanisms of all herpes virus, since they are evolutionarily related. With this in mind, the purpose of our research is to understand the molecular mechanisms that transition herpesviruses from a latent phase of replication, to a more productive, lytic phase. Specifically, we are asking what the role of the hCMV tegument protein UL25 was in the latent phase of replication. UL25 is a protein known to impact chromatin structure, transcription activation, and p53 inhibition. To understand hCMVs role in latent replication, we are designing UL25 deficient recombinants of clinical hCMV strains. After recovering and characterizing the recombinant virus lacking UL25, we will measure expression of marker genes for latent infection in cells quiescently infected with either wild‐type or UL25 deficient hCMV strains to see if UL25 plays a significant role in the maintenance or establishment of latency using cell culture models of quiescent infections.
Does VEGF Expression Correlate with Lymphatic Metastasis in Breast Cancer?
Authors: Rukmini Ashita , Mathur Sandeep , Vuthaluru Seenu
Published: May 2022

Introduction

Primary tumor characteristics are increasingly used for prognostication, predicting lymph node metastasis and planning adjuvant treatment in breast cancer. Vascular Endothelial Growth factor (VEGF) has been postulated to stimulate lymph vessels development and growth in tumors. Few studies on cell lines have documented how the new lymphatics develop or tumor cells invade these lymphatics. However, in human breast cancer the literature is sparse. Hence, this study is done to see the expression of VEGF and its correlation with lymphatic invasion in breast cancer patients.

Materials & Methods

Formalin fixed paraffin embedded blocks of breast cancer specimens and controls were retrieved & 4u thick sections stained with H&E stains for morphological evaluation. Serial sections were cut on APES (3‐Triethoxysilylpropylamine) coated slides for performing immunohistochemistry using monoclonal antibodies against VEGF and DII40. The expression intensity of VEGF and its distribution in the tumor samples were observed and semi‐quantitatively analyzed. VEGF expression was interpreted as follows: negative (0): <5% staining; weak (+): 5–25% staining; positive (++): 26–50% staining; strongly positive (+++): >50% staining. Statistical analysis: samples with 0/+ staining were considered in low VEGF expression, while samples with ++/+++ expression were considered as high VEGF expression. For DII40 well demarcated vessels showing positivity in endothelial cells were counted per 10hpf after selecting hot spots at scanner view. For statistical analysis, 0 was considered as no expression and 1/10HPF was considered as high expression. Association between levels of VEGF and D II 40 was examined by the two‐way scatter plot and a linear regression analysis of DII 40 over VEGF. The association of positivity between the two markers was assessed using Chi Square test.

Results

High VEGF expression was seen 33 of the 43 evaluable patients (76.74%%; 95% CI 61.37%‐88.24%) and high DII 40 expression was seen in 30 of the 44 patients (68.18%; 95% CI 52.42%‐81.39%). Of the 33 patients with high VEGF expression, DII 40 was expressed in 26 of them (78.79%). There was a strong association between VEGF expression and DII 40 expression (p<0.01) (Figure 1). The variance explained by VEGF level in the variance of DII 40 as measured by the correlation (R2) was 0.1265 (p=0.02). When the expression of VEGF and D II 40 was compared with tumor characteristics, there was statistically significant correlation with lymphovascular invasion (LVI) but not with tumor size, type, grade, ER/PR status, Her2neu status or lymph node involvement.

Conclusion

VEGF expression is high in most tumors. There is significant association between LVI & lymph vessels detection as studied by D II 40 expression and VEGF expression. Further studies in clinical settings are essential before incorporating VEGF expression for prognostication and customized patient care.
Effect of Sympathetic Neurotransmitters on Muscle Spindle Afferent Sensitivity to Muscle Stretch in Adult Mice
Authors: Ortiz Serena , Gomez Teodomiro , Andrade Timothy , Harnisch Arthur , Salazar Alexandra , Valdespino Steven R. , Snyder Erika , Vallinayagam Maya , Sanchez Phylicia , Wilkinson Katherine A.
Published: May 2022

The primary sensory input for proprioception, or the sense of body position in space, comes from muscle spindle afferents, which are mechanoreceptors that relay information about changes in muscle length. The muscle spindle is also innervated by sympathetic neurons, but the role of this sympathetic innervation on muscle spindle function is not well understood. Here we test the hypothesis that muscle spindle afferents will exhibit decreased firing in response to muscle stretch following exposure to the sympathetic neurotransmitters norepinephrine and epinephrine. To test this hypothesis we used an ex vivo mouse muscle‐nerve preparation. We recorded muscle spindle afferent firing activity during ramp‐and‐hold stretch and sinusoidal vibration before and after the addition of norepinephrine, epinephrine, or adrenergic receptor agonists. We observed significantly decreased firing during the end of stretch after both norepinephrine (100 µm, n=6) and epinephrine (30 µm, n=6). To identify the adrenergic receptor(s) involved, we also tested two ɑ2 adrenergic receptor agonists, Clonidine and Dexmedetomidine, and the ɑ1 receptor agonist phenylephrine. Both ɑ2 receptor agonists caused a decrease in muscle spindle afferent firing (1 mM clonidine, n=8 decreased firing; 100 µm dexmedetomidine, n=3). Phenylephrine had no significant effect on muscle spindle afferent firing (100 µm n=2; 30 µm n=6). These results show direct effects of sympathetic neurotransmitters on muscle spindle afferent stretch sensitivity and further support the hypothesis that sympathetic innervation of the muscle spindle plays an important role in modulating muscle spindle afferent activity and therefore motor control. Future studies will investigate the location of the adrenergic receptor(s) mediating this effect as well as the mechanism of action.
Positive Allosteric Modulation of the Mu Opioid Receptor
Authors: Kochan Kelsey , Traynor John
Published: May 2022

Opioid therapeutics, such as morphine, that act at the mu‐opioid receptor (MOR) are the clinical standard for patients struggling to manage symptoms associated with pain. It is widely understood that although opioids are effective at treating pain, their use leads to the development of severe adverse effects, such as constipation, addiction, and respiratory depression. Thus, there is a clear need for a safer alternative to manage pain. One such alternative is to enhance the effects of the body’s endogenous opioid system by positive allosteric modulation (PAM) of MOR. A known PAM, BMS‐986122, enhances MOR agonist potency in cellular models and MOR agonist mediated antinociception in vivo. In addition, this PAM is active alone in a variety of mouse pain assays by promoting the activity of endogenous opioid peptides. Moreover, at an effective antinociceptive dose, BMS‐986122 alone produces less severe adverse effects than morphine as determined by measures of constipation, respiratory depression, and conditioned place preference. However, we do not yet know how the overall pharmacology of opioids is affected by PAMs or if all opioid drugs are equally sensitive to PAM modulation. Here we compare the ability of BMS‐986122 to enhance the action of three structurally diverse opioid drugs, morphine, methadone, and fentanyl, in an acute pain assay and in an assay examining respiratory depression using CD‐1 male and female mice. We show that BMS‐986122 increases the antinociceptive effects of the opioid therapeutics in the warm water tail withdrawal assay without promoting the ability of the drugs to lower blood oxygen levels or heart rate. Future work will assess the effects of BMS‐986122 in additional acute and chronic pain models and with a more diverse group of opioids. If additional experiments support the concept that BMS‐986122 enhances MOR‐mediated antinociception but not MOR‐mediated respiratory depression, this will validate the development of MOR‐PAMs as standalone pain medications and support the use of PAMs as opioid‐sparing drugs for the effective management of pain.
Parkinson's Disease
Authors: Bai Kathleen , Cai Ziming , Cherif Ibrahim , Compaore Faltas C. , Fujikawa Ayaka , Hao Delilah , Jia Rachel , Koziakova Sabina C. , Liu Lenore , Qiu Elaine , Ramirez Victoria , Christensen Anna , Zhang Josh , Greig James , MacGillivray Brendan C.
Published: May 2022

Parkinson’s disease (PD) is a neurodegenerative disease that involves a progressive loss of dopaminergic neurons within the substantia nigra. Patients with PD experience a loss of motor function over time. In normal patients, muscle contractions are driven by action potentials through the binding of the neurotransmitter dopamine. Disruptions to dopaminergic activity would thus result in less efficient contractions. Patients with Parkinson’s can experience akinesia, tremors and other motor dysfunction.

Glucocerebrosidase (GCase), specifically β‐GCase, is a lysosomal enzyme involved in sphingolipid metabolism. β‐GCase falls under the hydrolase classification, allowing it to catabolize glucosylceramide (GlcCer) into glucose and ceramide. β‐GCase consists of 497 amino acid glycoproteins within its four domains. The glucocerebrosidase gene (GBA) mutations are often associated with Gaucher disease. However, β‐GCase synthesized from mutated GBA1 is often studied as a biomarker for PD susceptibility. In a healthy cell with the non‐mutated GBA gene, the GBA gene would be transcribed into mRNA and then transported out to the rough Endoplasmic Reticulum, where GCase is then synthesized. Lysosomal integral membrane protein‐2 (LIMP2) is a protein that transports GCase through the Golgi Apparatus and transfers the β‐GCase into a late endosome. When the late endosome fuses with a lysosome, where β‐GCase can hydrolyze its substrates. Mutated β‐GCase may affect this autophagy pathway.

This research will explore how mutated β‐GCase may disturb lysosomal functions, which would consequently lead to the aggregation of alpha‐synuclein (ASN), a protein that regulates synaptic vesicle trafficking and subsequently releases dopamine. Therefore, the detection and accumulation of mutated lysosomal enzymes, such as β‐GCase, in the cerebrospinal fluid might serve as biomarkers for Parkinson’s Disease.
Treatment with rapamycin reduces progressive proteinuria while inducing hyperglycemia in obese Dahl salt‐sensitive leptin‐receptor mutant rats prior to puberty
Authors: Brown Andrea K. , Ekperikpe Ubong S. , Cornelius Denise C. , Williams Jan M.
Published: May 2022

Prepubertal obesity (PPO) is now considered a risk factor for the development of proteinuria and renal injury in children. Recently, we observed the early development of renal injury in the obese SSLepRmutant strain is associated with elevations in GFR and glomerular injury prior to puberty. However, the mechanisms involved remain unclear. The mammalian target of rapamycin (mTOR) signaling pathway has been shown to contribute to various forms of renal disease. In the current study, we examined whether treatment with the mTOR inhibitor, rapamycin, reduces the early progression of renal injury in SSLepRmutant rats during PPO. Four week‐old SS and SSLepRmutant rats were treated with either vehicle (saline) or rapamycin (1.5 mg/kg/day, ip) until rats reached 8 weeks of age. At baseline, body weight was significantly higher in vehicle‐treated SSLepRmutant rats compared to SS rats and remained elevated over the course of the study (309±13 vs. 219±27 g, respectively). Treatment with rapamycin significantly reduced body weight in SSLepRmutant rats compared to vehicle SSLepRmutant rats (206±9 g). At baseline, non‐fasting blood glucose levels were similar in both SS and SSLepRmutant rats and were within normal physiological range (≤120 mg/dl). Interestingly, chronic treatment with rapamycin markedly increased blood glucose levels in SSLepRmutant rats vs. vehicle SSLepRmutant rats (438±8 vs. 123±5 mg/dl, respectively) but had no effects in SS rats. At the end of the study, plasma insulin levels were significantly elevated in vehicle‐treated SSLepRmutant rats compared to the values measured in SS rats (6±1 vs. 1±0.6 ng/dl, respectively), and rapamycin treatment only reduced plasma insulin in SSLepRmutant (2±0.7 ng/dl, respectively). At baseline, proteinuria was significantly higher in SSLepRmutant rats compared to SS rats (159±34 and 17±7 mg/day, respectively) and remained elevated throughout the study (558±79 and 73±34 mg/day, respectively). By the end of the study, rapamycin significantly reduced proteinuria in SSLepRmutant rats (225±67 mg/day). We observed a significant increase in creatinine clearance (CCr) in SSLepRmutant rats vs SS rats indicating increased GFR (4.0±0.6 vs. 0.9±0.1 ml/min, respectively). Chronic treatment with rapamycin significantly decreased CCr in SSLepRmutant rats (2.2±0.9 ml/min) while not affecting SS rats. Overall, these data suggest that treatment with rapamycin is effective in reducing progressive proteinuria via reducing GFR while reducing plasma insulin levels and contributing to hyperglycemia in obese SSLepRmutant rats.
Novel PKC signaling in Alzheimer’s Disease
Authors: Gauron M. C. , Lorden Gema , Prokopenko Dmitry , Tanzi Rudolph , Newton Alexandra
Published: May 2022

Protein kinase C (PKC) isozymes are abundantly expressed in the brain, where the enhanced activity of conventional isozymes is associated with neurodegenerative diseases such as Alzheimer’s Disease (AD) and spinocerebellar ataxia. Whether novel PKC isozymes contribute to neurodegeneration remains to be established. Here we identify a rare variant in the novel isozyme, PKCη, present exclusively in affected siblings in families with AD. This mutation, K65R, is present on the C2 domain, a module which participates in maintaining autoinhibition of the enzyme but is located on a solvent‐exposed face that is not predicted to be involved in interdomain interactions. Unsurprisingly, live‐cell imaging experiments using a genetically encoded biosensor CKAR (C kinase activity reporter), revealed that the K65R mutant, overexpressed in COS7 cells, had the same basal activity as wild‐type (WT) PKCη, consistent with intact autoinhibition. However, PKCη K65R displayed a 20% increase in agonist‐stimulated activity compared with WT. This increase did not result from improper folding or maturation of the protein: western blot analysis revealed that both the overexpressed WT and PKCη K65R proteins were properly processed by priming phosphorylations at the activation loop (Thr513) and the two C‐terminal sites (Thr656 and Ser675). In contrast to the enhanced activity observed in cells, in vitro kinase assays of PKCη WT or K65R (using protein purified from insect cells) revealed that the mutation did not alter the kinetics of activation by phosphatidylserine or diacylglycerol, nor did it alter the Km for phosphorylation of peptide substrate. Furthermore, studies in HEK‐293T comparing the overexpressed PKCη WT to K65R revealed that the mutation did not alter protein stability. Specifically, cycloheximide treatment resulted in loss of PKCη with a similar half‐life of approximately 40 hours for both. To ensure overexpression did not saturate degradation machinery, we examined the turnover of endogenous PKCη in the microglial cell line BV‐2. We chose this line because analysis of the brain by cell type‐specific enhancer‐promotor interactome map revealed that PRKCH is expressed predominantly in microglia. Endogenous PKCη was readily detectable in this cell line and displayed the same turnover half time as that of overexpressed protein. In summary, our data suggest that K65R AD‐associated variant of PKCη confers higher kinase activity in a cellular environment without affecting the intrinsic activity of the pure protein or cellular stability of the enzyme, suggesting that post‐translational modifications or altered protein:protein interactions may be responsible for the enhanced cellular activity. Taken together with reports that PKCη RNA expression is increased in microglia from AD brain, our identification of an AD‐associated variant in PKCη with enhanced activity poises this novel PKC isozyme in a prime spot to regulate microglial‐mediated inflammation, a key contributor to the progression of AD.
Forty‐five Vasopressin‐Regulated Phosphoproteins Involved in Control of Collecting Duct Water Transport
Authors: Park Euijung , Yang Chin‐Rang , Raghuram Viswanathan , Kikuchi Hiroaki , Chen Lihe , Chou Chung‐Lin , Knepper Mark
Published: May 2022

Vasopressin regulates renal water excretion through actions to regulate aquaporin 2 trafficking and transcription. These actions are dependent on vasopressin‐induced phosphorylation changes in collecting duct cells. Although some phosphorylation targets are well known including aquaporin 2 itself, the full gamut of vasopressin‐induced phosphorylation changes is unexplored. Our prior studies using quantitative phosphoproteomics have identified phosphorylation changes in cultured mouse mpkCCD cells (PMID: 33346914) and native inner medullary collecting duct cells (PMID: 31313956). Here, we have curated a list of vasopressin‐regulated phosphoproteins based on a consensus of the two studies. This includes 51 phosphorylation sites in 45 proteins, archived at https://esbl.nhlbi.nih.gov/Databases/AVP‐Phos/. Among these sites are 23 consensus protein kinase A (PKA) sites that are increased in response to vasopressin, consistent with a central role for PKA in vasopressin signaling. Fifteen downregulated phosphorylation sites are associated with decreased activation of the MAP kinases, ERK1 and ERK2. The remaining 13 sites are likely to be phosphorylated by other kinases. Regulated phosphoproteins include several protein kinases, namely Mink1, Camkk2, Cdk18, Prkar2a, Erbb3, and Src, which play likely roles downstream from PKA. Gene Ontology term analysis reveals multiple vasopressin‐regulated phosphoproteins involved in known vasopressin‐regulated processes: “transmembrane transport” (Aqp2, Itpr1, Itpr2, Itpr3), “actin cytoskeleton organization” (Bin1, Myo9b, Pdlim5, Slc9a3r1, Specc1l), “regulation of GTPase activity” (Als2, Arfgef1, Ralgapa2), “exocytosis” (Exoc7, Stxbp5, Itsn1), “endocytosis” (Bin1, Itsn1), “cell cycle” (Sept9, Arhgef2, Specc1l, Src), “regulation of transcription” (Hdac4, Ctnnb1, Lrrfip1, Tsc22d4), “Wnt signaling pathway” (Ctnnb1, Slc9a3r1, Src). Overall, this study provides a list of vasopressin‐regulated phosphoproteins likely to be involved in vasopressin‐mediated regulation of collecting duct water transport, providing grist for future studies.