bluuebunny/crossref_metadata_2025 huggingface dataset:
Meprin β Regulates Protein Kinase A Mediated TGF‐β Signaling in Ischemia Reperfusion‐Induced Kidney Injury
Authors: Ahmed Faihaa , Abousaad Shaymaa , Abouzeid Ayman , Ongeri Elimelda
Published: May 2022

Ischemia/reperfusion (IR) is a leading cause of acute kidney injury (AKI) and is associated with high morbidity and mortality rates. The underlying mechanisms involved in IR‐induced AKI are not fully understood. Meprins, zinc metalloproteases that are abundantly expressed in the brush border membranes of proximal kidney tubules, have been implicated in the pathology of IR. Meprins are capable to proteolytically processing the catalytic subunit of protein kinase A (PKA‐C) and several mediators of inflammation. The protein kinase A (PKA) signaling pathway plays an important role in renal fibrosis, promoting the production of transforming growth factor‐β (TGF‐β) and/or TGF‐β‐dependent molecules. The TGF‐β signaling is involved in wound healing and tissue repair of injured kidneys. Activation of Smad2 and Smad3 is the major downstream event of the TGF‐β signaling pathway. The objective of the current study was to determine how meprin β expression impacts PKA C‐mediated TGF‐β signaling in IR‐induced kidney injury. To this end, we used surgical procedures for to achieve unilateral IR with contralateral nephrectomy in wild‐type (WT) and meprin β knockout (βKO) mice. Meprin βKO mice are deficient in the meprin B (β‐β) and the heterodimeric isoform of meprin A (α‐β). The mice were sacrificed at 96 h post‐IR and kidney tissues obtained for analysis. Real‐time PCR, and immunohistochemical staining, were used to evaluate the expression levels of PKA C and TGF‐β. Statistical analysis utilized 2‐way ANOVA. Real‐time PCR data showed significant increases in mRNA levels of PKA C only in βKO kidneys after IR (p< 0.05). In contrast, the mRNA levels for TGF‐β were higher in both WT and βKO at 96 h post‐IR (p< 0.01). Interestingly, the baseline mRNA levels for TGF‐β were higher in meprin βKO mice (p< 0.001). Immunohistochemical staining for PKA C and TGF‐β was quantified using Image J software. The data showed significant increases in the levels of PKA C proteins in select kidney tubules of both genotypes (p≤ 0.001) after IR. Similarly, staining intensity for TGF‐β were higher in select kidney tubules of both WT (p=0.01) and meprin β deficient mice at 96 h IR (p=0.001). Immunofluorescence counterstaining of kidney tissues with anti‐meprin β, anti‐PKA C and anti‐TGF‐β antibodies showed a positive correlation between meprin β expression and tubular PKA C and TGF‐β levels in kidneys subjected to IR. These findings suggest that meprin β regulates PKA C‐mediated TGF‐β signaling pathway in IR‐induced kidney injury and provides new insights on the mechanisms underlying meprin β modulation of the pathophysiology of IR‐induced renal injury.
Rotational dynamics of the MLL complex on nucleosome and its implication in heterogeneity of the epigenetic landscape
Authors: Dou Yali
Published: May 2022

The MLL family histone methyltransferases deposit mono‐, di‐, tri‐methylation of histone H3 lysine 4 (H3K4me). Epigenomic studies highlight the discrete distribution of H3K4me3 and H3K4me1 at gene promoters and distal enhancers, respectively. However, how this is achieved remains unclear. We have performed single particle cryo‐EM studies for the MLL1 core complex on the nucleosome core particles (NCP). We revealed a surprisingly dynamic nature of the MLL1 complex on the NCP. We show that DPY30 and the intrinsically disordered regions (IDRs) of ASH2L work together to restrict the rotational dynamics of the MLL1 complex, which is necessary for dramatic increase of processivity and activity of the MLL1 complex. The DPY30 and ASH2L‐IDR dependent regulation applies to all members of the MLL/SET1 family enzymes. We further show that DPY30 is causal for de novo establishment of H3K4me3 in cells and its preferred localization at gene promoters may be the primary reason for selective enrichment of H3K4me3. Our study provides a new paradigm of how discrete H3K4me state is regulated on chromatin.
MDH CURE Community Faculty Fellows: A new mentoring program supporting Biochemistry CUREs
Authors: Gentile Lisa , Provost Joseph , Bell Ellis , Springer Amy
Published: May 2022

The Malate Dehydrogenase Cures Community Faculty Fellows (MCC‐FF) Program is part of an NSF RCN sponsored program focused on expanding and sustaining community among faculty teaching a course in the course undergraduate research experience (CURE) format using malate dehydrogenase (MDH). All faculty interested in adopting an MDH CURE are welcomed to become part of this supportive national group working together to provide research opportunities to their students within the context of the labs they teach. In addition, each faculty fellow will be part of a regional hub of local faculty teaching MDH CUREs. These regional hubs will host workshops, office hours, and expertise in MDH CUREs. Opportunities to teach well developed MDH CUREs or to design new MDH CUREs exist, as do opportunities to collaborate on particular MDH labs with faculty at different institutions.

Starting in Summer 2022, we will be forming the first of four, two‐year MCC‐FF cohorts providing individually mentored training. The program will provide two years of individually mentored experience to the faculty members who want to teach labs in a CURE format using MDH. During the first summer they will participate in virtual and hands‐on professional development (a one day virtual workshop, a 2 day hands‐on workshop, and a flexibly scheduled week long period of time where faculty fellows have access to their mentors as they adapt techniques/protocols to their home institutions). In the second year, these faculty fellows will teach their first MCC CURE course and develop mentorship skills help mentor subsequent cohorts of faculty fellows. Up to nine faculty per cohort will be selected for additional financial support including funds to purchase minor equipment/instrumentation. The selection of these faculty will give preference to faculty from under‐resourced institutions and/or those who work with large populations of students from backgrounds traditionally underrepresented in higher education. Faculty fellows will be selected from different types of institutions (community colleges, MSIs, PUIs, regional comprehensives, research intensives), teaching different types of courses (introductory, advanced, interdisciplinary, remote/online), at different places in their careers (new faculty members, experienced faculty members interested in changing pedagogical approaches). For faculty who teach with a postdoctoral fellow, graduate TA, or UG TA, these students will also be affiliated with the cohort and have access to training/support. Expectations of cohort membership are that faculty will be working to get an MDH CURE up and running as part of their teaching load within 2 years, will be an active part of their local network, and in the future will become a mentor to new faculty fellows.
Identifying Genes with Consistently Noisy Expression in S. cerevisiae
Authors: DeStefanis Thomas , Palenchar Peter
Published: May 2022

Variability in gene expression is important for creating differences in populations of genetically identical organisms, helps populations survive changes in the environment, and allows infective yeasts to adapt to a host’s immune status more easily. This variability can be due to intrinsic stochastic differences between identical cells grown under the same conditions. Small extrinsic differences in environmental conditions can also result in variation in gene expression between genetically identical cells. We sought to leverage existing S. cerevisiae expression data to identify genes with consistently high variability in their expression levels. Methods used to extract expression data can create technical variation, which may make it difficult to identify actual biologically relevant results. The presence of cell walls in many organisms, including yeast, has limited the amount of single‐cell gene expression data. Because of this, our analyses were conducted on Affymetrix microarray and RNA‐seq expression datasets, comprised of non‐single cell studies done under 240 unique growth conditions. We identified 22 genes with consistently high variation in their expression in S. cerevisiae. Gene Ontology analysis using goseq indicated a significant biological relatedness between the genes, finding that 10 out of the 22 genes are involved in ribosome biogenesis. These results suggest that ribosome biogenesis genes have evolved to have high variability in their expression, and the results are not due to technical variation. Transcription factors that regulate these 22 genes were identified to better understand if they played a role in variation. Analyses found 15 transcription factors that are used significantly more by the genes with high expression variation in comparison to the genome. These transcription factors emphasize the biological relatedness of the genes and may play a role in the variation itself. These findings offer the first step in characterizing genes with high variation in their expression, helping to gain a greater understanding of the importance of variability in gene expression.
Teres Intermedius: An Accessory Rotator Cuff Muscle Discovered During Routine Cadaveric Dissection
Authors: Padden Preston A. , McCullough Megan , Davis Jillian S.
Published: May 2022

The teres minor is a unipennate intrinsic shoulder muscle that arises on the lateral border of the scapula and runs obliquely to insert on the greater tubercle of the humerus. It stabilizes and contributes to external rotation of the glenohumeral joint. Teres major, while resembling the teres minor in origin and fiber direction, ultimately diverges from the teres minor as it passes anterior to the humerus and acts predominantly as a medial rotator and adductor of the shoulder. Described variants in the teres musculature are rare. We found a unilateral accessory muscle in the teres region of the right shoulder in an 81‐year‐old Caucasian male cadaver in a traditional anatomy lab. The accessory muscle arises from the lateral aspect of the scapula and travels parallel and posterior to the teres minor to insert onto the posterior head of the deltoid. The variant muscle is of similar size to the teres minor (also present), but its fibers are encapsulated in their own distinct fascial sheath, entirely separate from that of the anatomically normal teres muscles. We provide a detailed description of the accessory muscle and propose that similar variants may be clinically important due to their proximity to the anatomical quadrangular space, as compression of the structures traversing this area may result in pain, tenderness, paresthesia, and cyanosis in the shoulder region.
Role of IL‐5/IL‐5RA Axis in Eosinophilic Allergic Response in Canines with Dermatological Problems
Authors: Tavener Selena K. , Badri Dayakar V. , Panickar Kiran S.
Published: May 2022

The inflammatory response in canines with dermatological problems is likely mediated by multiple effector cells including lymphocytes, eosinophils, and mast cells. Several cytokines produced by Th2 lymphocytes including IL‐4 and IL‐31 are implicated in the pathogenesis of dermatological disorders and subsequent elevation of serum IgE and increased inflammation in the skin. Our study assessed the gene expression profile of inflammatory markers in blood samples collected from canines with dermatological problems to identify novel inflammatory markers that could lead to improved nutritional interventions. This study was approved by IACUC and the Animal Welfare Committee, Hills PNC. Gene expression was assessed in blood collected in PAXgene RNA tubes from canines with dermatological problems (Derm; n = 12; 4‐14yr) or controls (Con; n = 16; 4‐14yr), as diagnosed clinically by a veterinarian. Changes in a panel of 84 genes were analyzed using the RT2 ProfilerTM PCR Array for dog inflammatory cytokines and receptors. Blood was also analyzed for complete blood count (CBC) for circulating concentrations of leukocytes. There was a significant increase in the interleukin‐5 receptor alpha (IL‐5RA) level in derm dogs when compared to controls (1.71 fold, p<0.05). There was also an increase in C‐C chemokine receptor 3 (CCR3), IL‐4 and IL‐5 (all >1.25 fold, ns) in the derm dogs. IL‐5RA and CCR3 are receptors that are also expressed on eosinophils and IL‐4 is involved in the production of IL‐5. Given that IL‐5 plays a key role in the maturation and activation of eosinophils, and committed eosinophil precursors in the bone marrow express IL‐5RA and CCR3, our results indicate an important role of the IL‐5/IL‐5RA axis in dermatological disorders in canines. Consistent with this, there was a significant increase in circulating eosinophil levels in derm dogs (n=14; 0.35 x 103/μl) when compared to controls (0.28 x 103/μl; p<0.05). Further, increased levels of IL‐4 and IL‐5 are associated with increased serum IgE and subsequent allergic response. We report increased serum IgE levels in derm dogs (147.7 ± 52.9 µg/ml; n=14) when compared with healthy dogs (72.5 ± 49.5 µg/ml). In addition, other pro‐inflammatory proteins that were increased in derm dogs include CCR10 (1.67), CCL16 (1.34), CCR8 (1.35), CXCR5 (1.34), LTA (1.37), and BMP2 (1.44). Interestingly, derm dogs showed a decreased level of NAMPT, a putative cytokine that is hypothesized to enhance the maturation of B cell precursors (‐1.37 fold decrease). Taken together, our results indicate the important role of eosinophilic allergic response in dermatological disorders in dogs. Targeting the IL‐5/IL‐5RA signaling axis may be important as part of newer therapeutic approaches, including nutritional strategies, to reduce dermatitis in dogs.
FXR1 Decreases Blood Pressure by Regulating Vascular Contractility
Authors: St. Paul Amanda , Preston Kyle , Corbett Cali , Leigh Tani , Kelemen Sheri , Okune Rachael , Eguchi Satoru , Haines Dale , Autieri Michael
Published: May 2022

Introduction

Hypertension is a major risk factor for cardiovascular disease and stroke. Optimally treated hypertensive patients still have a 50% greater cardiovascular risk than untreated normotensive subjects. Despite current medication, half a million deaths in the United States include hypertension as a primary contributing cause in 2018 presenting a need for additional targets. Vascular smooth muscle cells (VSMCs) are the fulcrum of vascular disease, particularly hypertension. VSMCs play a critical role in vascular contractility and the regulation of blood pressure. Fragile X‐related protein (FXR1) is a muscle‐enhanced RNA binding protein and we previously found siRNA knock down of FXR1 increases inflammatory mRNA stability. Overexpression of FXR1 decreases inflammatory mRNA stability in VSMC. Little is known concerning FXR1 protein binding partners and its role in vascular disease. The specific aim of this study is to test the hypothesis that FXR1 regulates vascular contractility by RNA stability and protein interactions.

Results

We generated a novel, VSMC‐specific FXR1 conditional knock out mouse (FXR1VSMC/VSMC) in order to establish an in vivo role of FXR1 in vascular disease. Preliminary data indicates that these mice show decreased diastolic (P < 0.05) blood pressure at baseline compared to controls. Gene ontology of RNA immuno‐precipitation sequencing analysis in human VSMCs identified that FXR1 binds to mRNA that participate in VSMC contractility and regulation of blood pressure‐ related pathways. Although considered an RNA stability protein, mass‐spectrometry identified that FXR1 interacts with proteins related to contractile processes such as cell migration, adhesion and stress fiber formation. FXR1 depletion in human VSMCs decreases mRNA abundance of contractile machinery. Future studies will determine the effect of FXR1 depletion on mRNA stability of these transcripts. Additionally, siRNA knock down of FXR1 decreased VSMC adhesion, migration and collagen gel contraction corroborating in vivo observations.

Conclusion

These data are the first to suggest FXR1 regulates blood pressure and vascular contractility potentially by two mechanisms: mRNA stability and functional activity by protein‐protein interactions. The findings support FXR1 activity may represent a target for therapeutic invention to regulate blood pressure.
The miR‐200family regulates the epithelial stem cell niche during craniofacial and dental development
Authors: Amendt Brad A. , Leonard Riley J. , Su Dan , Eliason Steven L.
Published: May 2022

Introduction & objective‐

The murine lower incisor ectodermal organ contains a single epithelial stem cell niche that provides epithelial progenitor cells to the continuously growing rodent incisor. Calvaria sutures also contain stem cells that are regulated by specific factors. We have discovered that the microRNA (miR) ‐200 family is required for stem cell differentiation through targeting stem cell factors. We asked if the miR‐200 family regulated specific stem cell niches during development.

Materials & methods

‐ Our group used the plasmid‐based miR inhibitor system (PMIS) to knock down specific miR‐200 family members in mice and study their role during developmental processes. Our system inhibits one miR within a cluster to understand the role of that miR, which cannot be accomplished using gene targeting methods.

Results‐

We inhibited the miR‐200 family in developing murine embryos using new technology, resulting in an expanded epithelial stem cell niche and lack of cell differentiation. Inhibition of individual miRs within the miR‐200 cluster resulted in differential developmental and cell morphology defects. miR‐200 inhibition increased the expression of dental epithelial stem cell markers, expanded the stem cell niche and decreased progenitor cell differentiation. RNA‐seq. identified miR‐200 regulatory pathways involved in cell differentiation and compartmentalization of the stem cell niche. The miR‐200 family regulates signaling pathways required for cell differentiation and cell cycle progression. New miR‐200targets demonstrate gene networks and pathways controlling cell differentiation and maintenance of the stem cell niche.

Conclusion‐

This is the first report demonstrating how the miR‐200 family is required for in vivo progenitor cell proliferation and differentiation.

Significance/implication‐

This new approach has identified several new scientific advancements in stem cell niche biology. 1) miR‐200 family members are required for in vivo epithelial stem cell differentiation and stem cell niche maintenance; 2) The miR‐200 family regulates genes required for generating the different dental epithelial cell lineages; 3) miR‐200expression may be a common mechanism for defining the boundaries of adult epithelial stem cell niches; 4) By inhibiting the miRs that become active when a progenitor cell becomes fated to differentiate, the in vivo cell differentiation programs are determined assuming their terminal fates; 5) Dental epithelial stem cells and suture stem cells are regulated by members of the miR‐200 family to yield the differentiated progeny and determine cell fate.
Arterial Demonstration Device Illustrates Core Physiological Concepts
Authors: Sweeney Terrence E.
Published: May 2022

We have created a hand‐held, interactive arterial demonstration device that simplifies the teaching of several core physiological concepts. The device consists of low breaking point one‐way spirometer valve coupled serially to a flow‐through latex balloon that is fitted distally with a flow restrictor. The spirometer valve models the aortic valve; the latex balloon models the elastic arteries; the restrictor valve models the peripheral resistance. The user blows vigorously and intermittently into the proximal end of the device, modeling the ventricle's stroke volume. Initially designed to clarify the role of elastic arteries in cardiovascular function, it became evident that the arterial demonstration device could be employed to explain and demonstrate a number of physiological principles, including the distinct structure and functions of arteries and veins, the pathophysiological impact of aortic valve dysfunction (using device versions that incorporate dysfunctional valves), and the Law of LaPlace. Further, the device can be used to demonstrate several core physiological concepts, including causality; flow down gradients; energy acquisition, transformation and use; and mass balance in elastic structures1. This extends the device's utility beyond cardiovascular principles, a foundational idea behind the pedagogical approach of incorporating Core Concepts of Physiology2 in physiological instruction. Several such approaches are described.

1 Modell, H., Adv. Physiol. Educ. 23: 101‐107, 2000.

2 Michaels, J et al. The core concepts of physiology: A new paradigm for teaching physiology.DOI 10.1007/978‐1‐4939‐6909‐8, Springer, 2017.
Treatment of Multidrug‐Resistant Bacterial Infections Using Quantum Dots
Authors: McCollum Colleen , Bertram John , Nagpal Prashant , Chatterjee Anushree
Published: May 2022

Multidrug‐resistant (MDR) bacterial infections remain a major threat to public health, despite strides made in antibiotic development in the past century. The rise of MDR infections has demonstrated the dire need for new treatment strategies against infectious disease. MDR bacteria are predicted to cause ten million deaths annually worldwide by 2050, more than all cancers combined, unless significant strides are made in treatment options. While pharmaceutical companies devote years to develop a single drug for a single ailment, bacteria are quick to adapt, and MDR bacterial strains appear within only a couple years of the release of a new antibiotic. Our antibiotic discovery pipelines must accommodate the necessary shift away from traditional small molecule therapies toward more adaptive drugs.

Here, we investigate new ways of combating MDR bacteria and apply those methods in vivo to optimize them for clinical use. We present a novel method for the treatment of infections caused by MDR bacteria: antibiotic photoactivated semiconductor nanoparticles called quantum dots (QDs). Photoactivated QDs kill bacteria by producing superoxide, which targets iron clusters in bacterial cells, and additionally have been shown to potentiate the activity of antibiotics against resistant bacteria. We have previously shown that this specific generation of superoxide allows for killing of bacterial cells without causing toxicity to surrounding mammalian cells in vitro. Indium phosphide (InP) QDs injected subcutaneously into mice caused no measured toxicity, measured through body weight, organ histology, and inflammation and oxidative stress markers in serum, to the host animal after six consecutive days of treatment. InP QDs are activated by near‐infrared (NIR) light, which was provided to the injection site using high‐intensity LEDs. We then used InP QDs to treat subcutaneous abscesses of MDR clinical isolate Escherichia coli in mice. As NIR light has high transmittance through skin and tissue, QDs injected under the skin were sufficiently activated for bacterial killing. We observed decreased bacterial count by QD dosages of 2 and 4 μM compared to PBS treatment. A 6000‐fold drop in abscess bacterial viability was observed in mice treated with 4 μM QDs compared to PBS control.

This novel approach to treatment of infectious disease could provide necessary alternatives to small‐molecule drugs. The QDs offer a new method of using superoxide to kill bacteria, including bacteria that have developed resistance to small‐molecule drugs. Our QDs could revolutionize last‐resort treatments of burn and wound infections, with the potential to be expanded to other infection types.
Cytotoxicity in Human Kidney HK‐2 Cells of the Flavoring Aldehydes Cinnamaldehyde and Vanillin
Authors: Cox Ashley , Brown Kathleen C. , Valentovic Monica A.
Published: May 2022

Vaping and e‐cigarette use has markedly increased in adults as well as middle and high school age children. Flavoring agents are not tightly regulated resulting in widely variable levels present in various commercial preparations. Cinnamaldehyde and vanillin are popular flavoring agents contained in e‐liquids. Unfortunately, the safety as well as the long term effects of these compounds is not well understood. While the lung is the primary site of contact for such flavoring agents, aldehydes absorbed following inhalation can distribute throughout the body to other tissue sites including the kidney. These flavoring compounds can then impact cell function in tissues such as the kidney. The purpose of this study was to test the hypothesis that cinnamaldehyde and vanillin may have cytotoxic effects in the kidney. In the present study, the effects of cinnamaldehyde or vanillin at final concentrations of 0‐100 uM or 0‐1000 uM, respectively, were evaluated using human renal proximal tubular epithelial cells (HK‐2) for 24‐ and 48‐hour time periods. Cells were obtained by ATCC and cultured according to vendor specifications. All experiments were a minimum of 4 independent experiments utilizing 4 different cell passages. Viability was determined by use of the MTT assay and conversion to formazan. Cell Countess was done to visualize percent live and dead cells. Western blots were performed to assess expression of oxidative stress as well as mitophagy‐related proteins LC3B‐II, PINK, and PARKIN. Vanillin was not cytotoxic between 0‐100 uM relative to vehicle control at 24 hours. Cinnamaldehyde was cytotoxic beginning at 25 uM when compared to vehicle control using the MTT assay. Expression of mitophagy‐related protein LC3B‐II increased significantly at higher concentrations of cinnamaldehyde (p<0.05). Oxidative stress was evaluated using Western blot for protein modifications. Cinnamaldehyde did not increase 4‐hydroxynonenal (4HNE) adducted proteins within a 24 hour period. Mitochondrial function was evaluated using a Seahorse analyzer following a 24 hour exposure. Mitochondrial oxygen consumption was diminished by cinnamaldehyde. These findings suggest that cinnamaldehyde was more cytotoxic to HK‐2 cells than vanillin. Cinnamaldehyde further altered mitochondrial function and increased LC3B‐II expression. Further studies are required to explore the specific mechanisms of cytotoxicity to the kidney by flavoring aldehydes.
Investigating the Role of CRAC in FSH Receptor Function and Structure
Authors: Lynn Tatyana , Cohen Brian
Published: May 2022

Human infertility is a complex disorder that can often be attributed to a dysfunction of the endocrine system. Follicle‐stimulating hormone (FSH) is one of many hormones that participate in a complex process in both women and men to regulate normal reproduction. The dysfunction of this hormone and its receptor are some of the many causes of infertility. FSH is secreted by the anterior pituitary and, in women, initiates a cascade of biological events that enable ovulation. FSH carries out its function by binding and activating specific receptors. The FSH receptor (FSHR) is a G protein‐coupled receptor (GPCR) that is located in the cell membrane of target cells in the ovaries and testes. GPCRs often interact with a variety of molecules beyond their ligands including, in some cases, cholesterol in the plasma membrane. The Cholesterol Recognition/Interaction Amino Acid Consensus Sequence (CRAC) is one such binding domain and is represented by the sequence LV‐XXXX‐Y‐XXXXX‐K/R. The human FSHR contains a sequence consistent with the CRAC motif in the first intracellular loop L‐TTLQ‐Y‐KLTVP‐R. We hypothesized that mutating this domain will negatively affect the signaling of the receptor upon ligand activation. To test this hypothesis tyrosine 375 was mutated to phenylalanine (Y375F) which should render the CRAC inactive. After establishing a cell line stably expressing the mutant receptor, activation of the p44/42 (ERK1/2) pathway was measured. Preliminary data suggest that signaling of the hFSHR‐Y375F mutant is decreased relative to the wild‐type receptor. As we learn more about this receptor, we continue to gain knowledge about conditions such as infertility. This information can facilitate the development of innovative treatments which can help many families who struggle to conceive.
Ser14‐Rpn6/PSMD11 Phosphorylation Mediates the Activation of 26S Proteasomes by cAMP and Protects against Cardiac Proteotoxic Stress in Mice
Authors: Yang Liuqing , Parajuli Nirmal , Wu Penglong , Liu Jinbao , Wang Xuejun
Published: May 2022

Background

Means to enhance proteasome (PSM) functioning are highly sought. In cultured cells, cAMP‐dependent protein kinase (PKA) was shown to activate 26S PSMs by phosphorylating Ser14 of Rpn6/PSMD11 (a key PSM subunit), but this remains to be established in animals. Hence, we sought to test the hypothesis that Ser14‐Rpn6 phosphorylation (pS14‐Rpn6) mediates the activation of PSMs by PKA and reduces proteotoxicity in vivo (Figure 1).

Methods and Results

We generated via CRSPR/Cas9 gene editing two mouse models, in which the codon for Ser14 in the Rpn6 gene was mutated to encode either Alanine (S14A) for blocking pS14‐Rpn6 or Aspartic acid (S14D) for mimicking this phosphorylation. Treatment with adenylate cyclase activator forskolin (5mg/kg, i.p.) increased cardiac pS14‐Rpn6 (2.52±0.08 vs. 1.0, p<0.001) and PSM activities (292.35±45.75 vs. 100, p<0.005) in wild type mice, but these were abolished by co‐treatment of PKA inhibitor H89 (15mg/kg). Forskolin did not show these effects in S14A mice, demonstrating that pS14‐Rpn6 is required for PKA to activate the PSM. Cross‐breeding transgenic GFPdgn (a proven substrate of the ubiquitin‐proteasome system [UPS]) into S14D mice revealed that S14D decreased cardiac GFPdgn proteins (0.81±0.08 vs. 1.0±0.08, p<0.05) without altering GFPdgn mRNA levels, indicating that S14D suffices to enhance UPS performance. Basal myocardial PSM activities were significantly higher in S14D mice compared to WT mice (1.52±0.38 vs. 1.0±0.16, p<0.005). To determine the effect of increasing pS14‐Rpn6 on proteotoxicity, we cross‐bred S14D into the CryAB‐R120G mice (R120G), a classic model of cardiac proteotoxicity. We detected that myocardial total (0.76±0.07 vs. 1.0±0.19, p<0.005) and insoluble (0.80±0.12 vs. 1.0±0.14, p<0.01) CryAB‐R120G protein levels were lower in S14D::R120G compared to WT::R120G mice (n=4 males+4 females/group), suggesting that PSM activation by PKA decreases the accumulation and aggregation of misfolded proteins in vivo. Echocardiography further revealed that S14D significantly attenuated the decrease of left ventricular (LV) ejection fraction (51.80±11.65 vs. 40.20±13.88 %, p<0.01), stroke volume (38.44±7.30 vs. 28.65±5.69 μl, p<0.005) and cardiac output (17.45±3.09 vs. 12.77±2.46 ml/min, p<0.005) of R120G mice at 6 months of age, which indicates that genetic mimicry of pS14‐Rpn6 ameliorates cardiac proteotoxicity.

Conclusions

We have established for the first time in vivo that pS14‐Rpn6 is required for activation of 26S PSMs by PKA and provides the first genetic evidence that PKA‐induced PSM activation protects against proteotoxicity in animals.
Dissecting the Role of the Six1‐Associated Genes in Craniofacial Development and Disease
Authors: Jourdeuil Karyn , Baxi Aparna B. , Tavares Andre L.
Published: May 2022

Genetic variants of SIX1, a transcription factor implicated in many cellular processes during craniofacial development, and of its co‐factor EYA1 underlie 50% of Branchio‐oto‐renal syndrome (BOR) cases. BOR is an autosomal dominant disorder characterized by various craniofacial defects, including malformed middle ear ossicles leading to conductive hearing loss. Interestingly, some BOR variants in SIX1 also underlie craniosynostosis (CRS) which indicates that SIX1 has a crucial role in craniofacial bone development. One important aspect of SIX1 function is that it interacts with co‐factors that can induce or repress its transcriptional activity. In this work, we expand our knowledge of the Six1 gene regulatory network with respect to craniofacial development. We first used a Six1 knockout mouse line to assess gene expression profiles of dissected E10.5 mandibular arches which give rise to the middle ear ossicles and lower jaw, via RNA‐sequencing. Our transcriptomic analysis led to the identification of 122 genes, including components of the FGF and WNT signaling pathways, known contributors to craniofacial bone development. In parallel, we used in vitro studies to characterize three novel co‐factors of Six1: Sobp, Mcrs1 and Pa2g4 that were recently identified in Xenopus. Our findings indicate that Sobp and Mcrs1 are bona fide Six1 co‐factors in mouse. We are now studying the genes identified in the RNA‐seq and the novel Six1 co‐factors using in vitro culture of mouse pre‐osteoblastic cells (MC3T3‐E1) to identify their role in bone formation. Altogether, our results identified novel Six1‐associated genes that are potential candidate genes for the unresolved cases of BOR and CRS.
The (pro)renin receptor antagonist, PRO20, attenuates high fat diet induced hepatic steatosis and fibrosis in non‐alcoholic fatty liver disease (NAFLD)
Authors: Gayban Ariana Julia , Cooper Silvana , Souza Lucas , Regalado Erick , Feng Earley Yumei
Published: May 2022

Non‐alcoholic fatty liver disease (NAFLD) comprises a spectrum of liver damage directly related to diabetes, metabolic syndrome, and obesity. The (Pro)renin receptor (PRR) in the liver recently has been shown to play an important role in hepatic steatosis. In this study, we test our hypothesis that PRR antagonist, PRO20, reduces hepatic steatosis and fibrosis. Wild‐type mice in C57Bl/6J background (n=9‐10/group) were fed with either HFD (60% calories from fat) or normal fat chow (NFD, 10% calories from fat) with matching calories for 6 weeks. Methionine choline deficient (MCD) diet was used to study fibrosis development in C57BL/6J mice for 8 weeks. Two weeks following respective diet modifications, mice were implanted with a subcutaneous osmotic pump (4 or 6 week release model) containing either PRO20 (700ug/kg/d) or saline. Liver tissues were processed for Oil Red O (ORO), H&E, and Masson’s trichrome histology staining for lipid and collagen accumulation. The presence of lipids, indicative of steatosis, was quantified using ImageJ/FIJI software and presented as % of ORO staining to the total area. We found that 6 weeks of HFD (15.90 ± 1.49 %) induced a significant elevation on liver ORO staining compared with the liver from mice fed with NFD (3.60 ± 1.61 %, p<0.0001). More importantly, PRO20 treatment (9.91 ± 1.46 %; p = 0.001) significantly reduced the ORO staining in mice treated with HFD. Hepatic triglyceride concentrations were quantified with colorimetric assay and shown to be significantly lowered in PRO20 treated mice (2.110 ± 0.6175) compared with scramble peptide treated (6.470 ± 0.3553, p<0.0001) mice under HFD. Collagen area was also found to be reduced in MCD PRO20 (4.970 ± 0.9096 %) treated mice compared with the control mice (8.901 ± 0.2782, p = 0.0033). Moreover, PRO20 treatment (258 ± 53.12 U/L) decreased liver ALT levels in MCD‐fed PRO20 mice compared with the control mice (682 ± 116.4 U/L). De novo lipogenesis genes thought to contribute to the development and progression of NAFLD were evaluated by real‐time PCR and presented as fold‐change expression normalized to β‐actin. The glycerol‐3‐phosphate acetyltransferase 3 (GPAT3) (1.835 ± 0.1798, p = 0.0183) expression was found to be downregulated in the livers of HFD PRO20 treated mice. Our findings indicate that PRO20 reduces HFD‐induced hepatic steatosis and MCD‐induced liver fibrosis and liver dysfunction. We conclude that PRR antagonism using PRO20 might be an effective therapeutic approach for treatment of NAFLD.
MDH protein interactions and phosphorylation: Engaging students with a novel scientific theme using CUREs
Authors: Provost Joseph , Hecht David , Huisinga Kathryn , Peterson Celeste , Parente Amy , Bell Jessica , Bell Ellis
Published: May 2022

Using CUREs to integrate research into the teaching environment has significant impact on motivation and persistence of STEM students as seen from traditional undergraduate research experiences. Specifically, involvement in undergraduate research provides opportunities to think and act as scientists, bolsters feelings of belonging, and improves confidence in STEM. THE MDH CURES Community is an NSF funded community of faculty using malate dehydrogenase (MDH) to engage students in CUREs. One of several themes of MDH CUREs is protein‐protein interactions and the impact of post‐translational modification. Here we present how several primarily undergraduate and comprehensive universities and a community college, several of which are Hispanic Serving Institutions (HSIs), have included MDH‐CS interactions in their classroom. Interactions between cytosolic MDH and four other proteins thought to share substrate/product were explored in silico at a community college in a first‐year chemistry course. At Malone university, students used MS phosphorylation information to predict potential interactions which they explored by mutagenesis. Students at Mercyhust University have used in‐silico and experimental methods to design and then test CS and MDH interactions using pull down assays. Students at Suffolk University created a novel bacterial two‐hybrid model to test interactions between cytosolic and mitochondrial MDH with CS in a genetics course. In a horizontally integrated sequence, students at University of San Diego modeled potential interactions between MDH and CS and the effects of various mutations. In a subsequent semester, students used that information to analyze the mutation’s functional impact and effect on interactions with MDH‐CS using a pull‐down assay. Each project is an ongoing effort working as part of the MCC network. Approaches and lessons learned will be shared.
Mitochondrial oxidative phosphorylation fuels endothelial control of blood vessel tone
Authors: Wilson Calum , Lee Matthew D. , Buckley Charlotte , Zhang Xun , McCarron John G.
Published: May 2022

Arteries and veins are lined by non‐proliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis to fuel each of these energy‐demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells in angiogenesis, and little is known about energy production in endothelial cells within the fully‐formed vascular wall.

Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial oxidative phosphorylation at mitochondrial complex V disrupts calcium‐dependent, nitric oxide‐mediated endothelial cell control of vascular tone. Basal, mechanically‐activated, and agonist‐evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. This effect is mimicked by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. The role for endothelial cell energy production is independent of species, sex, or vascular bed.

These data show that mitochondrial ATP is necessary for the obligatory role of endothelial cells in the control of blood vessel diameter, and validate the idea of targeting endothelial cell metabolism to treat endothelial cell dysfunction in cardiovascular disease.
Neuroprotective Role of Hyperoxia During the Critical Period of Retinal Development in Normal Mice and Mice with Retinitis Pigmentosa
Authors: Villegas Monica Anne Faye , Gdovin Eric , Tilvawala Megha , Brahmbhatt Rushil , Sutera Christopher , Lecker Paul , Berstein Jake , Tsai E‐Jine , Martin Mark , Kim Jessica , Morrison Nathan , Gorski Grzegorz , Antonello Zeus , George‐Weinstein Mindy , Bravo Nuevo Arturo
Published: May 2022

Background

The retina utilizes more oxygen than any other tissue in the body. Photoreceptors located in the retina's outer nuclear layer (ONL) are culled during the “critical period” of eye development when the number of photoreceptors exceeds the supply of oxygen. Retinitis Pigmentosa (RP) is a disease that first affects the rod photoreceptors, causing night blindness and reduced peripheral vision. In mice, RP often starts during the critical period. The objective of this study was to assess the effects of hyperoxia during the critical period on visual function and morphology of the retina in normal mice and mice with RP.

Method

Thirty five male and female wild‐type (C57BL/6J) mice and 15 mice with an rd1 mutation (C3H/HeJ), which causes a rapidly progressing form of retinitis pigmentosa, were exposed to hyperoxia (75% O₂), hypoxia (12% O₂), and normoxia (21% O₂) from postnatal (P) days 7 to 20. At P26, electroretinography (ERG) was used to assess the visual function of the photoreceptors (A‐wave) and the inner retinal cells (B‐wave). At P27, optical coherence tomography (OCT) and histology were used to observe retinal structure and the effects of the different O₂ groups. For histology, eyes were extracted at P10, P12, P16, and P28. Cell death was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).

Results

In ERGs from C57 mice, both A‐wave and B‐wave amplitudes were significantly lower in the hypoxic condition indicating that hypoxia was detrimental to visual function compared to the normoxic group. OCT data showed that the hyperoxic groups of C57 mice had thicker ONLs compared to the other treatment groups. TUNEL cell counts in the ONL and INL indicated a significant decrease in apoptotic cells during the critical period in the hyperoxic group compared to the hypoxic group. In C3H mice, hyperoxia improved the A and B waves compared to untreated mice.

Discussion

This study suggests that administration of oxygen during the critical period of retinal development reduces the pruning of cells in the retina and increases visual function in both normal mice and mice with an aggressive form of RP. Oxygen treatment during the critical period of retinal development, which occurs from P8 to P16, may slow retinal degeneration in RP. The effects of hyperoxia on the completion of vascular development must be taken into account when considering the use of hyperoxia therapeutically.
Genetic Predisposition to Type II Diabetes in the Hispanic Population of Orange County in Relation to SNP rs1333051 on the CDKN2 Gene
Authors: Nguyen Jacquelyn H. , Tena Melanie , Gallo Jessie , Calleja‐Macias Itzel
Published: May 2022

Thirty‐four million individuals cope with diabetes within the United States, 95 percent of which have Type II Diabetes (T2D). Hispanics are particularly affected by diabetes and being the largest minority in the U.S., it is a significant public health issue. Since there is a diverse genetic variation between different ethnicities, researchers study genes through single nucleotide polymorphism (SNPs) to determine an individual's risk of developing T2D. The SNP rs1333051 located on the CDKN2 gene correlates with T2D. Evidence shows a nucleotide change of A allele by T allele. The allele A is also reported to be associated with coronary heart disease in T2D patients. The project’s primary goal is to find the prevalence of SNP rs133051 in 24 controls and 23 diabetic patients in the Hispanic population of Orange County. DNA was extracted, taken from a buccal swab sample, and subjected to a polymerase chain reaction (PCR), followed by gel electrophoresis to confirm PCR products. Enzymatic digestion was done using Bsrl to detect the alleles based on restriction fragment length patterns. The alleles A and T were identified in both groups. However, the allele T was predominant in the diabetic group. External factors possibly play a larger role than genetic factors. To know more about the association of the T allele, increasing the number of samples and applying statistical analysis is necessary. The overall knowledge in the identification of SNPs associated with T2D allows for preventative measures to be taken within the Hispanic population at risk for this disease.
The Correlation Between the SNP rs1049594 and Type 2 Diabetes
Authors: Tena Melanie , Nguyen Jacquleyn , Gallo Jessie , Macias‐Calleja Itzel
Published: May 2022

Type 2 Diabetes (T2D) is the fifth leading cause of death within the Hispanic population in the United States. Composed of Puerto Ricans, Mexicans, Cubans as well as Central and South Americans, 50% of US Hispanic adults are expected to develop T2D. Diabetes can induce several serious health complications if left untreated such as cardiovascular disease and kidney disease. Single nucleotide polymorphisms (SNPs) are genetic markers used in research to associate risks of disease. SNPs such as rs1049549 have been linked to Type 2 Diabetes in current studies and the risk allele associated within this specific SNP is the T>C allele. The primary objective for this study was to discern whether or not rs1049549 is linked to Type 2 Diabetes in the Hispanic population of Southern California. We tested 32 control and 24 diabetic samples taken from buccal swabs and the DNA extracted from these specimens were amplified using the polymerase chain reaction (PCR). The PCR amplicons were visualized using gel electrophoresis, digested using the enzyme BstN1, and visualized under gel electrophoresis to distinguish between both versions of the allele. The results from this study will be used to better understand the relationship of this SNP and diabetes within the Hispanic population in Southern California.