bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin Signaling Induces AKR1C3 to Promote Lipid Overload through FASN in SGBS Cells, as a Model of PCOS Adipocytes
Authors: Paulukinas Ryan D. , Penning Trevor M.
Published: May 2022

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age, affecting 8‐13% of women. The characteristic androgen excess (AE) of PCOS is believed to promote comorbidities, such as obesity and type 2 diabetes. AE correlates with insulin resistance (IR) in these women and may be the driving factor for lipid overload. Aldo‐keto reductase family 1 member C3 (AKR1C3) is induced by insulin in PCOS adipocytes and may be central in driving the AE of PCOS. AKR1C3 predominantly catalyzes formation of androgens such as testosterone and 5α‐dihydrotestosterone in peripheral tissues, which are potent agonists for the androgen receptor (AR) and upregulate de novo lipogenesis. Fatty acid synthase (FASN) is induced by androgens and may be responsible for an altered lipid profile in mature adipocytes. Nuclear factor erythroid 2‐related factor 2 (NRF2) is a known transcription factor for AKR1C3 and is regulated by insulin. AKR1C3 overexpression in adipocytes by insulin activation of NRF2 could increase androgen receptor signaling and promote de novo lipogenesis. The resulting lipid overflow could exacerbate insulin resistance through a feedforward mechanism and potentiate the cardio‐metabolic risk seen in PCOS. We hypothesize that insulin signaling induces AKR1C3 to promote lipid overload through FASN in PCOS adipocytes. Using differentiated human Simpson‐Golabi‐Behmel Syndrome (SGBS) adipocytes as a model for PCOS adipocytes, we have confirmed that AKR1C3 is induced >600 fold by insulin. Using pharmacological inhibition, we found that the insulin induction of AKR1C3 and FASN is dependent on PI3K/AKT/mTOR measured by RT‐qPCR and immunoblot. Using NRF2 siRNA and ML‐385, a NRF2 inhibitor, we have determined that AKR1C3 and FASN induction are NRF2 dependent, indicating NRF2 may be one transcription factor that binds to the AKR1C3 promotor region in adipocytes. We also determined that FASN induction is both AKR1C3 and AR dependent using pharmacological inhibition/antagonism and siRNA, where its effect was measured by RT‐qPCR and immunoblot. Pharmacological inhibition with NRF2 or AKR1C3 inhibitors additionally downregulated de novo lipogenesis measured by Oil Red O staining, which is a phenotypic readout for FASN activity. These studies elucidate the signaling mechanism by which AKR1C3 and FASN are induced by insulin in adipocytes. We conclude that upregulation of AKR1C3 may promote lipid overload in PCOS adipocytes by induction of FASN, and that AKR1C3 is a possible therapeutic target to prevent the risk of cardio‐metabolic disease in PCOS women.
Creation of MALDI‐TOF Main Spectral Libraries (MSPs) of Four Distinct Parasitic Flatworm Cercariae
Authors: Goetsch Quinn , Fritz Corinn , Meyer Roiya , VanDomelen Elizabeth , Thao Kao Lee , Jadin Robert , Orlofske Sarah , Lawrencee Jim
Published: May 2022

Parasitic flatworms (Phylum Platyhelminthes: Subclass Digenea) are scientifically important organisms in aquatic habitats because they represent potential human (i.e. Swimmer’s Itch) and wildlife pathogens. Furthermore, larval stages (cercariae) released from snail intermediate hosts represent substantial biomass, contribute to energy flow in food webs, and potentially serve as biological indicators of ecosystem health. Identification of different cercariae is has traditionally been done by morphological traits or classical genotyping. These approaches are difficult, time consuming and expensive. This presents a barrier to researchers in the field working on non‐model organisms. Matrix Assisted Laser Desorption Ionization ‐ Time of flight (MALDI‐TOF) mass spectrometric based identification of microorganisms has been successfully used to identify single‐ and multi‐cellular organisms more quickly and accurately than traditional methods. Here we describe the protein extraction and MALDI‐TOF identification of cercariae from four morphologically distinct parasite taxa. We have also assembled multiple cercariae MALDI‐TOF spectra into individual Main Spectral Libraries (MSP) to differentiate each unique cercariae. Finally, we describe how these MSPs can be used to quickly and accurately identify unknown cercariae and thus remove a barrier to parasitic flatworm research progress.
PP1cβ dephosphorylates cardiac myosin by MYPT‐dependent and independent mechanisms
Authors: Lee Eunyoung , Liu Zhenan , Nguyen Nhu , Nairn Angus , Chang Audrey N.
Published: May 2022

In beating hearts, cardiac muscle myosin is phosphorylated at its regulatory light chain (RLC) at 0.4 mol phosphate/mol RLC. This level of constitutive phosphorylation is necessary for normal cardiac function, and is maintained by balanced activities of the dedicated cardiac muscle‐specific myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). The MLCP is best characterized in the smooth muscle, and is comprised of the regulatory myosin phosphatase target subunit MYPT1, a catalytic subunit PP1cβ, and an accessory subunit of unknown function M20. In smooth muscles in vivo, MYPT1 and PP1cβ co‐stabilize each other and are both necessary for normal smooth muscle contractions. In the cardiac muscle, the ubiquitously‐expressed MYPT1 and the striated muscle‐specific MYPT2 are both expressed in the cardiomyocyte, but the contributions to physiological regulation of cardiac myosin dephosphorylation by PP1cβ are unclear.

Using a conditional knockout of PP1cβ, we tested the hypothesis that the main catalytic subunit for cardiac MLCP is PP1cβ, and that its activity toward phosphorylated cardiac RLC in vivo is dependent on regulation by striated muscle‐specific MYPT2. We measured the effects of the knockout on the expression levels of other regulators of cardiac RLC phosphorylation, and a cardiac myofibrillar phosphatase assay was developed to measure MYPT‐regulated and MYPT‐independent phosphatase activities toward phosphorylated cardiac myosin. We report that 1) PP1cβ is the main isoform expressed in the cardiac myocyte, 2) cardiac muscle pathogenesis in PP1cβ knockout animals involve upregulation of PP1cα and MYPT1 in activated myofibroblasts, 3) the protein stability of endogenous mouse cardiac MYPT1 and MYPT2 are not dependent on the expression of PP1cβ, and 4) phosphorylated cardiac myosin in intact myofibrillar preparations are dephosphorylated by both myosin‐targeted and soluble MYPT‐independent PP1cβ activities. These results contribute to defining properties of the cardiac MLCP in vivo.
Cellular localization, immunological function and biophysical characterization of Mycobacterium tuberculosis Methyltransferases
Authors: Nazim Tasmin
Published: May 2022

Background

Tuberculosis is the 13th leading cause of death and the second leading infectious killer after COVID‐19 (above HIV/AIDS) around the world. In 2020, an estimated 10 million people fell ill with tuberculosis (TB) worldwide. Extensively drug‐resistant TB (XDR TB) is a rare type of MDR TB that is resistant to isoniazid and rifampin, plus any fluoroquinolone and at least one of three injectable second‐line drugs (CDC). To avoid another pandemic from happening after covid‐19 we must work on drug discovery and vaccines to prevent TB from spreading.

Objective

This work is done on mycobacterium tuberculosis, the causative agent of tuberculosis, using laboratory experiments and computational biological tools to characterize methyltransferase proteins of Mtb. This study is targeted to find out any potential drug or vaccine target in Mtb.

Method

With the help of bioinformatic tools a hypothetical methyltransferse protein was selected from previously reported variety of 121 methyltransferses according to the domain configuration. The protein was verified by the thermal stability, Protein conformational changes, localization, signal peptide chain, structure prediction and function, mutational analysis and molecular docking.

Results

Rv3366 gene was selected based on its secretory characteristic and localisation in mitochondria among 121 methyltransferases. SignalP4.1 and Mitoprot ll were used for the cleavage site prediction. Protein modelled by online server has 95.6% allowed region and validated by Ramachandran Plot Statistics from Richardson's lab.

The biophysical characteristics of the gene were also identified using methyltransferase assay, thermal denaturation assay, spectrophotometry, fluorescence assay. Alarm assay was done for quantitation of viable cell number in proliferation and cytotoxicity in the presence of the target protein. MitoTracker Red CMXRos kit was used to verify the localisation of the protein in mitochondria.

Conclusion

This study covers essential identification of hypothetical methyltransferase that will help in targeting dataset for future studies leading to drug target and TB control of MTB or probably even MDRTB.
The Pattern of Ampakine and Hypoxia Exposure Determines Phrenic Motor Facilitation
Authors: Thakre Prajwal P. , Sunshine Michael D. , Fuller David D.
Published: May 2022

Phrenic motor facilitation (pMF) is a persistent increase in phrenic nerve activity upon the removal of a triggering stimuli. A variety of stimulation paradigms can induce pMF including electrical activation of the carotid sinus nerve, serotonin or adenosine receptor agonists, acute intermittent hypoxia (AIH) and inactivity. Recently, Wollman et al. (2020) demonstrated a new kind of pMF using a combination of a pharmacological agent (ampakine) and a single brief hypoxic episode. A subsequent study confirmed this finding and also showed that pairing ampakine with multiple hypoxic episodes did not increase magnitude of pMF (Thakre et al., 2021). The present study investigated if ampakine‐hypoxia induced pMF is sensitive to the pattern in which ampakine and hypoxia are administered. Phrenic nerve output was recorded from urethane‐anesthetized, mechanically ventilated and vagotomized adult male Sprague‐Dawley rats. Hypoxic exposures were 5 min in duration with moderate severity (arterial O2 partial pressure approximately 45 mmHg). The ampakine used was CX717, given intravenously (i.v.) at 15 mg/kg. We first verified the impact of a single administration of i.v. ampakine followed after 2 min by hypoxia. In agreement with prior work, this paradigm evoked a sustained increase in phrenic burst amplitude (60 min: 73±19% baseline, n=8). In the next experiments, the order of ampakine and hypoxia was reversed such that hypoxia was followed after 2 min by ampakine. This paradigm did not evoke a sustained increase in phrenic burst amplitude (60 min: 11±15% baseline, n=6). In the final series of experiments, ampakine was delivered during an ongoing episode of hypoxia (2 min after onset). This paradigm also did not evoke a sustained increase in phrenic burst amplitude (60 min: 12±14% baseline, n=7). Our results indicate that the mechanisms of pMF evoked by the ampakine‐hypoxia interaction are sensitive to the order in which these stimuli are presented. The presentation of ampakine followed by a single brief hypoxic episode appears to be ideal for producing pMF. These findings could have significant implications in planning hypoxia‐based neurorehabilitation strategies with ampakines as an adjunct therapy.
The Role of Palmitoyltransferase ZDHHC5 in Regulating RAS Function
Authors: Ritho Joan , Dixon Scott
Published: May 2022

More than 90% of pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, harbor an activating KRAS mutation which consequently alters its downstream signaling pathways. S‐palmitoylation is a dynamic post‐translational modification that has been implicated in maintaining the altered function of key oncogenes important in cancer progression. S‐palmitoylation is catalyzed by a large family of 23 zinc finger DHHC‐type (ZDHHC) palmitoyl S‐acyltransferases. Our understanding of how specific ZDHHC enzymes regulate tumor progression remains limited. Analysis of data available in TCGA indicates that high levels of ZDHHC5 correlates with a poor survival in pancreatic cancer patients. Notably, we observed in published proximity‐dependent biotin labeling studies that the palmitoyltransferase ZDHHC5 is consistently identified as a novel Ras interactor. Accordingly, we hypothesize that ZDHHC5 may modulate the function of Ras in cancer cells. We find that genetic disruption of ZDHHC5 causes loss of Ras activity and membrane localization, consequently decreasing the MAPK pathway in pancreatic cancer cell lines. Biochemical characterization of ZDHHC5‐dependent regulation of Ras function in pancreatic cancer will provide more information on how to target it and create a therapeutic window that will help to prevent pancreatic cancer progression.
Fatty Gastrocnemius Muscle: A Case Report
Authors: Liu Siyu , Kostek Matthew , Omstead Kailey
Published: May 2022

Background

The gastrocnemius muscle is a superficial two‐headed muscle located in the posterior compartment of the leg, consisting of two heads: medial head and lateral head. The gastrocnemius muscle plays a significant role in our daily activities because its function is plantar flexing the foot at the ankle joint and flexing the leg at the knee joint. We report a very rare case of fatty gastrocnemius.

Methods

The abnormal gastrocnemius muscles were encountered during the dissection of the lower limbs of a 63‐year‐old female cadaver. For histological examination, muscular samples were sectioned from the top, middle, and bottom of two heads of both legs. A total of six muscular slides, nerve slides, artery slides were all stained with Hematoxylin and Eosin (H&E) stains. Slides were analyzed using light microscopy.

Result

For anatomy analysis, the medial heads of both gastrocnemius muscles of the lower limbs are completely infiltrated by adipose tissue. But the lateral heads of gastrocnemius muscles on both sides are almost healthy. Only the bottom of the lateral head of gastrocnemius muscles shows adipose tissue, but the middle and topdo not. Additionally, the medial heads connect to the tibial nerve and arteries normally. No abnormal adipose tissue was observed in other muscles throughout the body. For histological analysis, the entire medial heads and the bottom lateral heads are replaced by adipose cells, but the middle and top of the lateral heads are healthy muscle cells. No abnormalities or pathologies were observed in the nerves or arteries.

Conclusion

Both medial heads of the gastrocnemius muscle appear to have become adipose tissue. While no underlying cause was identified it appears the pathology was spreading to the medial head.
The Effect of Endomucin on Cell Surface Receptor Signaling Is Specific for Vascular Endothelial Growth Factor Receptor 2
Authors: Cano Issahy , Hu Zhengping , Saint‐Geniez Magali , Ng Yin Shan E. , D'Amore Patricia
Published: May 2022

Endomucin (EMCN) is a mucin‐like glycoprotein selectively expressed by venous and capillary endothelium and, as a component of the endothelial glycocalyx, inhibits leukocyte adhesion. Previously our lab has determined that EMCN interacts with VEGFR2 and that its depletion inhibits VEGF165‐induced VEGFR2 clathrin‐mediated endocytosis and angiogenesis‐related functions. We examined whether other tyrosine kinase receptors required the presence of EMCN or if EMCN regulates specifically VEGF165‐induced VEGFR2 signaling. Thus, we investigated the role of EMCN in receptor internalization and signaling for VEGF121/VEGFR2, VEGF165/VEGFR1, and basic fibroblast growth factor (FGF‐2)/FGF receptor 1 (FGFR1). Human retinal microvascular endothelial cell (HREC) migration was determined using a scratch wound‐healing assay in which the extent of cell migration over a 16 hr period was quantified. EMCN mRNA depletion (95%, P<0.05) was achieved using siRNA. Equal molar concentration of VEGF165 (10 ng/ml) and VEGF121 (7.28 ng/ml), and FGF‐2 at 10 ng/ml induced significant migration of HRECs compared to untreated controls (1.15 ± 0.02 vs. 1 ± 0.02, P=0.0027, 1.19 ± 0.03 vs. 1 ± 0.02, P<0.0001, 1.25 ± 0.04 vs. 1 ± 0.03, P<0.0001, respectively and n=12 for all groups). EMCN knockdown resulted in a significant decrease in cell migration stimulated by VEGF165 and VEGF121 compared to control (1.01 ± 0.02 vs. 1.15 ± 0.04, P=0.0061, 1.04 ± 0.04 vs. 1.19 ± 0.05, P=0.0021, n=12). EMCN knockdown did not decrease FGF‐2‐induced migration compared to control (1.218 ± 0.08 vs. 1.248 ± 0.06, P=1.0, n=12). To determine internalization of cell surface receptors HREC surface proteins were labeled using sulfo‐NHS‐SS biotin, isolated using neutral‐avidin beads and analyzed by western blot. Obtained values represent remaining receptor at the cell surface. EMCN knockdown did not influence VEGF165‐induced VEGFR1 internalization (0.625 ± 0.11 vs. 0.386 ± 0.08, P=0.11, n=6), or FGF‐2‐induced FGFR1 internalization (0.734 ± 0.075 vs. 0.769 ± 0.074, p=0.74 n=7). These data suggest that EMCN modulates VEGFR2 internalization and function induced by both VEGF165 and VEGF121 isoforms, but does not impact VEGF165‐induced VEGFR1 or FGF‐2‐induced FGFR1 internalization and function. These data support that the effects of EMCN on angiogenesis occur specifically through modulation of VEGFR2 function.
Disruption of Proteostasis Causes IRE1 Mediated Reprogramming of Alveolar Epithelial Cells in Lung Fibrosis
Authors: Katzen Jeremy , Rodriguez Luis , Tomer Yaniv , Babu Apoorva , Zhao Ming , Murthy Aditi , Morley Michael , Mulugeta Surafel , Pelura Timothy , Rosen Glenn , Beers Michaell F.
Published: May 2022

Pulmonary Fibrosis (PF), a devastating lung disease with rising prevalence, is defined by a physiologic defect in gas exchange owing to two cardinal pathological features: intrusion of myofibroblasts into the distal lung parenchyma and an alteration of normal alveolar epithelial composition most clearly defined by a loss of the squamous alveolar epithelial type 1 cell (AEC1) and the presence of a recently identified aberrant alveolar epithelial “transitional state” enriched in profibrotic mediators. While this transitional cell state has been identified transiently in the epithelium of murine lung injury models, the mechanism by which it arises in human fibrotic lung disease is unknown.

We previously reported the in vivo modeling of PF utilizing inducible, knock‐in expression of a clinical Surfactant Protein C (SP‐C) mutation in alveolar type 2 cells (AEC2). Expression of the SP‐C mutation in the adult mouse AEC2s led to activation of Unfolded Protein Response (UPR) signaling pathways, endoplasmic reticulum (ER) stress and spontaneous fibrosis providing proof of concept for disruption to proteostasis as a proximal driver of PF. We hypothesized that disruption of AEC2 protein quality control and specifically UPR signaling causes cell autonomous AEC2 reprogramming to the transitional state in the absence of an exogenous lung injury.

Using two clinical SP‐C mutation models we discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop the recently identified transitional cell state. Using single cell RNA sequencing of the epithelium in our murine models we identify that UPR activated AEC2s develop the transitional cell phenotype in the absence of an exogenous lung injury. Through organoid based modeling we validated that this state arises de novo from intrinsic AEC2 dysfunction. The cell autonomous AEC2 reprogramming is mediated through IRE1 signaling as use of a novel IRE1 inhibitor attenuated the development of the transitional cell state and diminished AEC2 driven recruitment of granulocytes, alveolitis, and lung injury arising from the loss of proteostasis.

We further show that this transitional state persists into the fibrotic phase of our murine models and is enriched in pro‐fibrotic mediators. These findings identify AEC2 proteostasis, and specifically IRE1 signaling, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.
CRISPR‐ethics in the Undergraduate Classroom using CCR5 Gene
Authors: Santisteban Maria S. , Applewhite Leanne , Natvig Lydia , Challa Anil K.
Published: May 2022

Biology education has undergone revolutionary changes in the last two decades. Research on how students learn, personal experiences, and the ultimate recommendation of the Vision and Change report that “Students should have opportunities to participate in authentic research experiences”has moved instructors to adopt active learning strategies that emphasize concepts and skills over details, such as CUREs, where students learn science by doing science.

Active learning should engage students to allow them to develop core competencies. However, even a well‐designed CURE may not fully engage students if they don’t see the relevance. CRISPR‐Cas9 makes an excellent tool to bring authentic research experience in the molecular biology laboratory because is contemporary, the concept is attractive to students, and provides a common set of technologies to teach molecular biology. Multiple iterations are possible by changing the research questions being addressed.

In 2018, after the inaugural CRISPR‐Cas workshop offered in advance at the ABLE meeting, this framework was adopted to teach a summer Molecular Biology course at UNC Pembroke and the research question has been modified every year. This past summer students at UNC Pembroke and the University of Alabama at Birmingham worked on a project to produce various knockouts the human CCR5 gene in vitro, in an attempt to mirror the infamous experiment of the “Chinese twins” whose genomes were edited to be HIV resistant. The CRISPR‐Cas9 context was effective at engaging students and provided opportunities for discussions around ethics. Students in the two institutions connected twice towards the end of the course, once to reflect on their gains and shared experiences, and another time to discuss ethical implications. As part of the assessment, regarding the ethics, students created two products: 1‐ a reflection on the potential broader social and ethical implications of CRISPR‐editing, and their personal views on the subject before and after taking the course; 2‐ a letter to their congressperson where they explain the CRISPR‐Cas9 technology and the bioethical dilemma associate with its use for genome editing and describe and support an action plan that they want the congressperson to take.

In addition to becoming familiar with the CRISPR‐Cas9 technology and potential applications, the students learned about the central dogma and mastered many techniques of molecular biology (primer design, PCR, in vitro transcription, nuclease digestions, and polyacrylamide and agarose electrophoresis). They were introduced to several web platforms, databases, and computer programs, such as Ensembl, Benchling, CRISPRscan, SnapGene and used electronic notebooks.

Beyond the practical and conceptual learning, the approach proved valuable in engaging students through the ethics discussions and by feeling part of a larger effort, which should nurture their science identity.
The Role of Angiotensin 1‐7 in Isolated Human Arterioles with SARS‐CoV‐2
Authors: Nishijima Yoshinori , Hader Shelby , Zhang David , Gutterman David , Beyer Andreas
Published: May 2022

Introduction

The vascular endothelium plays a crucial role to regulate vascular tone. Recently, our laboratory reported severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection significantly reduced endothelial‐dependent vasodilation months after exposed to SARS‐CoV‐2 in human microvessels. SARS‐CoV‐2 enters host cells through angiotensin‐converting enzyme (ACE)2 as a cell surface receptor. Downregulation of ACE2 protein expression by the virus leads to imbalance between angiotensin (ANG) II and ANG 1‐7, causing vasoconstriction, inflammation and increase oxidative stress. With this background, we intend to test our new hypothesis that administration of ANG 1‐7 will improve vasodilation in human arterioles from previously SARS‐CoV‐2 positive subjects.

Methods

Fresh human tissues were obtained as de‐identified surgical discarded specimens. Control subjects (n=9) were SARS‐CoV‐2 negative and without known virus exposure. We had 11 previously SARS‐CoV‐2 positive subjects or post‐COVID. For all post‐COVID patients, a negative COVID test was obtained prior to collection of surgical specimens. We isolated arterioles (100‐200 µm) and cannulated onto glass micropipettes under 60 mmHg and examined for diameter changes to the endothelial‐dependent vasodilator acetylcholine (ACh; 10−9 to 10−5 M) using videomicroscopy. Flow‐mediated dilation (FMD) was recorded at steady‐state during varying intraluminal pressure gradients (5‐100 cm H2O). All post‐COVID vessels were incubated in a culture media at 37°C for 12–15 hours with or without 1 nM of ANG 1‐7.

Results

Dilation to ACh and FMD in arterioles from post‐COVID was significantly reduced (ACh max. dilation at 10−5 M: 74±6% vs. 93±4% in control, n=7‐9, P<0.001; FMD max. dilation at 100 cm H2O: 49±3% vs. 86±2% in control, n=6‐11, P<0.001) while overnight incubation of 1 nM ANG 1‐7 significantly improved the dilation (ACh max. dilation at 10−5 M: 74±6% in post‐COVID vs. 88±5% in post‐COVID + AND 1‐7, n=11, P<0.001; FMD max. dilation at 100 cm H2O: 49±3% in post‐COVID vs. 76±3% in post‐COVID + ANG 1‐7, n=11, P<0.001).

Conclusion

Administration of ANG 1‐7 significantly improved vasodilation in isolated arterioles from post‐COVID subjects.
Pressure Modulation of Vascular L‐type Calcium Channels: implications to the Myogenic Response
Authors: Mironova Galina Y. , Baudel Miguel M‐A , Flores‐Tamez Victor A. , Brett Suzanne E. , Navedo Manuel F. , Welsh Donald G.
Published: May 2022

L‐type Ca2+ channels govern smooth muscle [Ca2+] and myogenic tone development through mechanisms traditionally tied to voltage control. While essential, questions remain as to whether voltage is the sole regulatory influence or whether L‐type Ca2+ channels are additionally mechanosensitive. In this context, this study defined whether and by what mechanisms pressure stimuli modulate L‐type Ca2+ channels in rodent (rat & mouse) resistance arteries. Experiments extended from single smooth muscle cells (whole‐cell/single‐channel patch clamp; immunohistochemistry; proximity ligation assay) to intact arteries (pressure myography). Whole‐cell patch clamp electrophysiology revealed that pressure stimuli increased the L‐type Ca2+ current, a phenomenon that single channel analysis attributed to enhanced functional coupling. Subsequent work noted that pressure‐enhanced functional coupling is tied to: 1) PKC‐mediated cooperative gating; and 2) subunit trafficking to caveolae. Functional experiments, using 30 mM KCl to elicit a set depolarization (~‐40 mV), revealed that tone and cytosolic [Ca2+] responses were more pronounced in arteries pressurized to 80 rather than 20 mmHg. These functional results are in alignment with patch clamp observations and suggest that L‐type Ca2+ channels are indeed mechanosensitive. Cumulatively, our results suggest that there is more to L‐type Ca2+ channel regulation than voltage and that pressure sensitivity must be carefully weighed in the healthy and diseased setting. Work continues to precisely define the pressure‐sensitive signaling complexes within vascular smooth muscle.
Domain‐swapped dimeric γ‐crystallin: the missing link in the evolution of oligomeric β‐crystallins
Authors: Thorn David C. , Serebryany Eugene , Birrane Gabriel , Kaya Ali I. , Shakhnovich Eugene I.
Published: May 2022

Maintaining the transparency and proper functioning of the eye lens throughout adulthood is achieved by the high kinetic and thermodynamic stabilities of the long‐lived crystallin proteins, which must retain their soluble, folded conformations to avoid diseases of the lens such as cataract. Vertebrate βγ‐crystallins adopt two homologous domains connected via a linking peptide which is considered a key determinant of their tertiary and quaternary arrangements. In vitro, γ‐crystallins are monomeric whereas β‐crystallins form mainly dimers that are thought to associate further via domain‐swapping to form larger oligomers. By mutating the interdomain linker in γD‐crystallin, we have engineered a β‐crystallin mimic whereby it forms dimers in solution. The crystal structure of the γ‐crystallin mutant shows a domain‐swapped dimer, distinct from the compact ‘face en face’ dimeric structure of β‐crystallin in solution. While preserving its native domain structure, the domain‐swapped dimer exhibits reduced thermodynamic stability, suggesting that this arrangement is unfavorable in an aging lens where the risk of protein misfolding and aggregation is pronounced due to cumulative post‐translational modifications. However, as higher‐order oligomers of β‐crystallin are domain‐swapped, the γ‐crystallin mutant may be representative of a short‐lived, conformational intermediate in the oligomerization of β‐crystallins.
Potassium Hydroxide Activated Titanium Dioxide Nanoparticles Diminishes Streptococcus mutans Biofilm Formation
Authors: Caudle K. , Morris M. , Bhalla S. , Kositangool P. , Falahati K. , Barragan J. , Hong B.Y. , Lee S. , Serizawa A. , Cervantes J.
Published: May 2022

Dental caries represents one of the most common oral diseases. Streptococcus mutans is an oral bacterium involved in the process of caries development, forming a biofilm to promote bacterial adherence to the tooth surface and to other bacteria. Photocatalytically activated titanium dioxide (TiO2) has been shown to have antimicrobial properties on oral pathogens as well as on biofilm formation.

We aimed to evaluate the effect of potassium‐incorporated TiO2 (KTiOx) nanostructures, a high performance photocatalyst, on biofilm formation by S. mutans.

S.mutans was cultured in the presence of KTiOx 1M and 10M nanoparticles, as well as a elemental Ti control. Biofilm formation was assessed using a crystal violet biofilm formation after 24, 48, and 72 hours.

A decrease in biofilm formation was observed in a dose response manner after 48 hours incubation with 32, 64, and 128 ug/mL of KTiOx.

Our findings show that KTiOx nanoparticles have anti‐biofilm properties which may be applied to combat biofilm containing S.mutans, This novel approach may be a promising therapeutic alternative in caries prevention, though reduction of bacterial adhesion to dental surfaces.
TLR4 Promotes ER stress and Unfolded Protein Response in Hypertension‐Induced Kidney Injury
Authors: Pushpakumar Sathnur , Juin Subir Kumar , Whitehead Jaden , Sen Utpal
Published: May 2022

Hypertension is the second leading cause of chronic kidney injury in the world. Endoplasmic reticulum (ER) is an important cell organelle that is involved in the synthesis, folding, and modification of various proteins to maintain homeostasis. ER stress is reported in hypertension‐induced damage of kidney, heart and brain. Further, TLR4 activation and signaling is linked to the development and progression of hypertension induced damage in various organs; however, the interplay between TLR4 and ER stress in renal pathology remains unknown. In the present study, we investigated whether TLR4 activation contributes to renal ER stress and inflammation in Angiotensin‐II (Ang‐II) induced hypertension and whether TLR4 deficiency protects the kidney by suppressing ER stress. C3H/HeouJ (Normal TLR4, TLR4N) and C3H/HeJ (Dysfunctional TLR4, TLR4M) aged 12‐14 weeks were treated with Ang‐II (1000 ng/Kg/min. x 28 days) using osmotic pumps. We found renal function was impaired in TLR4N mice compared to TLR4M. The expression of ER stress markers (ATF6, p‐IRE1α and p‐PERK), renal inflammation (TNFα, IL‐1β, CCL2) and transcription factor p‐NFkB was upregulated in TLR4N kidneys compared to TLR4M. Further, ER stress in TLR4N mice was associated with mRNA changes in GRP78, sXBP1, ATF4, CHOP genes that are involved in unfolded protein response. In TLR4N mice, renal inflammation was predominant in the tubular area and ER stress was observed in both glomerular and tubular area. TLR4M mice showed reduced ER stress and inflammation in the kidney. Taken together, our results suggest significant interaction between TLR4 and ER stress that may act as an obligatory step in mediating renal inflammation and damage, and TLR4 deficiency attenuates injury by regulating ER stress and unfolded protein response in Ang‐II‐induced hypertension.
Insight into Parkinson's Disease From a Yeast Model: How Three Alpha‐Synuclein Mutants (A18T, A29S, & A53V) Generate Toxicity
Authors: Grassel Amanda , Borland Carris , Bertolotti Federica , Osselborn Ryan , Nassuna Tracey , Zabat Bryce , DebBurman Shubhik
Published: May 2022

Parkinson’s Disease (PD) is a fatal and incurable neurodegenerative disorder linked to the loss of dopaminergic neurons in the midbrain. A key pathological marker of PD is the presence of Lewy bodies, which are composed of misfolded α‐synuclein protein. α‐Synuclein is a highly post‐translationally modified protein in both healthy and diseased states. Modifications on it include phosphorylation, nitration, SUMOylation, acetylation, and glycation. Regulation by lipids is also suggested to modify its toxicity. The toxicity of wildtype α‐synuclein and six well‐known mutant versions linked with familial PD (A30P, E46K, H50Q, G51D, A53T, A53E) is well‐modeled in budding yeast systems. In contrast, the toxicity mechanisms of three newer PD‐linked α‐synuclein mutants (A18T, A29S, and A53V) is poorly understood; particularly, the impact of post‐translational modifications and lipid homeostasis on their toxicity is unknown. Using our lab’s budding yeast model of PD, we investigated the toxicity potential of these three new mutants by assessing the impact of the mutations on yeast growth and α‐synuclein localization in various yeast strains. We report that: 1) All three mutants are toxic to yeast, but to different degrees and, surprisingly, less than wild‐type α‐synuclein; 2) Yeast strains with altered triglycerides reduce α‐synuclein toxicity; 3) Yeasts strains with enhanced acetylation or reduced glycation environments rescue α‐synuclein toxicity; 4) and enhanced SUMOylation is protective against α‐synuclein toxicity; 5) and, finally, yeasts strains deleted for specific PD‐associated genes differentially affect α‐synuclein toxicity. This study expands the evaluation of genetic α‐synuclein mutants linked with PD in yeast models and supports the relevance of covalent modifications, lipid homeostasis, and PD‐associated genes on α‐synuclein toxicity.
In Silico Identification of a β2 Adrenergic Receptor Allosteric Site that Selectively Augments Canonical β2AR‐Gs Signaling and Function
Authors: Shah Sushrut D. , Lind Christoffer , De Pascali Francesco , Penn Raymond B. , MacKerell Alexander D. , Deshpande Deepak A.
Published: May 2022

Objective & Hypothesis

Activation of β2‐adrenergic receptors (β2ARs) leads to airway smooth muscle (ASM) relaxation and bronchodilation. While this facilitates β2ARs agonists (β‐agonists) as the front‐line treatments for asthma and related obstructive airway diseases, their therapeutic efficacy of β‐agonists is limited by agonist‐induced β2AR desensitization and activation of non‐canonical β2AR signaling involving β‐arrestin. Accordingly, we undertook the identification of an allosteric site on β2AR and potential ligands, and ascertain their effects in‐vitro. We hypothesize that activation of specific allosteric site on β2AR could selectively modulate the activity of β‐agonists to overcome these limitations.

Methods

We employed the site identification by ligand competitive saturation (SILCS) computational method to map the entire 3D structure of an in silico‐generated β2AR intermediate conformation. Further, in‐silico database was screened to identify potential ligands using SILCS‐PHARM approach. Levels of cyclic‐AMP were used to assess activation of β2AR‐Gs activation using ELISA. Activation of downstream effector of cAMP, protein kinase A (PKA) was assessed by western blotting and luciferase assay. β‐arrestin recruitment to β2AR was assessed using Bioluminescence Resonance Energy Transfer (BRET) assay. ELISA and immunofluorescence were used to assess cell surface expression. Mutagenesis analysis was used to investigate the potential binding site for the allosteric modulators. Lastly, functional effects of the allosteric modulators were determined by assessing relaxation of human ASM cells embedded in 3D‐collagen gels and murine airways.

Results

Database screening using SILCS identified not only putative allosteric site but also potential drug‐like compounds that can bind to the site. Experimental assays in HEK293 cells and human ASM cells identified positive and negative allosteric modulators (PAM and NAM respectively) of β2AR as assessed by generation of cAMP. Co‐stimulation of cells with PAM or NAM modulated β‐agonist‐induced PKA activation. Allosteric modulators had no effect on β‐agonist‐induced recruitment of β‐arrestin to β2AR or β‐agonist‐induced loss of cell surface expression in HEK293 cells expressing β2AR. Mutagenesis analysis of β2AR confirmed the SILCS identified allosteric modulator binding site that comprises of F282, Y219, and R131. Finally, functional studies revealed augmentation of β‐agonist‐induced relaxation of contracted human ASM cells and bronchodilation of contracted murine airways by PAM.

Conclusion

These findings identify a novel allosteric binding site on the β2AR, whose activation selectively augments β‐agonist‐induced Gs signaling and increases relaxation of ASM cells, the principal therapeutic effect of β‐agonists in obstructive lung disease such as asthma and COPD.
Seven structures of atypical chemokine receptor 3 reveal the molecular bases for its promiscuity and signaling bias
Authors: Yen Yu‐Chen , Schafer Christopher T. , Gustavsson Martin , Eberle Stefanie A. , Dominik Pawel K. , Deneka Dawid , Zhang Penglie , Schall Thomas J. , Kossiakoff Anthony A. , Handel Tracy M. , Tesmer John J.
Published: May 2022

Atypical chemokine receptor 3 (ACKR3) is a seven transmembrane (TM) spanning heptahelical receptor that is involved in different stages of cancer progression. Unlike canonical G protein‐coupled receptors (GPCRs), ACKR3 does not couple to heterotrimeric G proteins when activated by its endogenous ligand, CXCL12. It is therefore a naturally occurring fully arrestin biased receptor. ACKR3 however desensitizes normally and contributes to cancer progression through arrestin‐mediated signaling pathways and/or by active sequestration of chemokine ligands, which blocks CXCL12‐mediated downregulation of CXCR4. Here, we report seven cryo‐electron microscopic structures of ACKR3 bound to three different agonistic ligands, including CXCL12, a potent CXCL12 variant (CXCL12LRHQ), and a small molecule agonist (CCX662). Although the chemokines bind to ACKR3 in a truly unique way, all three ligands still extend deep in the orthosteric pocket. In all the structures, the TM spans of ACKR3 assume a configuration like those of active GPCRs, although the cytoplasmic pocket is more compact in ACKR3. In addition, compared to other chemokine receptors, the structure of the intracellular loop 2 (ICL2) of ACKR3 is quite different and lacks a kink at the cytoplasmic end of TM4. This is predicted to prevent ACKR3 from binding heterotrimeric G proteins in a canonical way, thereby blocking nucleotide exchange. In summary, our structures provide insight into the promiscuous recognition of ACKR3 relative to other chemokine receptors, and into its distinct functions relative to other canonical chemokine receptors. The structure models may also guide future drug discovery efforts targeting ACKR3 for cancer therapy.
Effect of Black Fly Larva Diet Supplement on Goldfish (Carassius auratus) Reproduction in an Aquaponics System
Authors: Wheat Elijah L. , Ortiz William , Huertas Mar
Published: May 2022

Aquaponics has become a major focus of aquaculture and related research in recent years. Advancements have been made in increasing production efficiency, however, research has focused on reared fish over ornamental fish, despite an estimated worth of over $1 billion per year in this industry. Our model organism, goldfish (Carassius auratus), are ornamental fish and closely related to koi, another valuable ornamental fish. Additionally, goldfish are often used as a model species in laboratory environments. They are resistant to variations in temperature, water quality, diet, and are tolerant of handling and experimental stress. A common problem in stressful environments, including aquaponics systems, is to supply a diet adequate for reproduction. Black Soldier Fly larvae (BSF) are often used as feed in aquaponics systems because of their nutritional content and the fish preference over pellet food. In previous studies, BSF has been shown to increase the total spawned eggs in Zebrafish (Danio rerio) when 25% of their diet was replaced with BSF. However, there was a significant decrease in the total spawned eggs when 50% of their diet was replaced with BSF supplement. In order to maximize viable spawned eggs and minimize the BSF supplement, we want to hone in on the ideal percentage of BSF meal in the diet of goldfish. We hypothesize that goldfish fed BSF meal will have more total spawned eggs than goldfish fed the control diet of pellet food. Additionally, we hypothesize that goldfish fed 20% BSF meal will have more total spawned eggs than the 40% BSF meal replacement. To test our hypothesis, 40 reproductive fish will be placed into four identical aquaponics systems (300 L/ tank, 10 fish /tank) that we assembled and tested at Texas State University (TXST). We already developed the culture of BSF and spawning goldfish techniques at TXST. The first and second tanks will act as controls being fed a pellet diet. The latter two tanks each will have an experimental diet of 20% BSF meal or 40% BSF. Additionally, each tank will be attached to a separate lettuce grow bed. The mating behavior and sex hormone/pheromone water release in tanks three and four will be compared to the control tanks and will determine what impact their diet has on mating. We will be collecting the eggs and counting how many eggs were laid in each treatment and counting the total spawned eggs. Additionally, any impacts on the growth of the lettuce will be determined. Aquaponics is a growing industry and increasing the efficiency of fish and crop growth while minimizing expense is paramount. Fish physiological studies in aquaponics will allow developing optimal fish rearing techniques necessary for the advance of the industry.
Using Q‐Methodology to Evaluate Student Perceptions of Online Anatomy in the Time of COVID‐19
Authors: Saini Jessica , Brewer‐Deluce Danielle , Akhtar‐Danesh Noori , Saraco Anthony N. , Bayer Ilana , Pitt Courtney , Wainman Bruce
Published: May 2022

Background

Since the seventeenth century, the primary approach to teaching anatomy has involved hands‐on learning using cadaveric specimens. However, the ability to use this long‐standing tradition was curtailed in the 2020‐2021 school year due to the COVID‐19 pandemic. Many institutions closed physical classrooms entirely, launching experiential courses, such as anatomy, into the online space.

Hypothesis

We hypothesized that Q‐methodology could be used to uncover student perceptions of an introductory anatomy and physiology course that was offered online for the very first time.

Methods

Q‐methodology, considered the study of subjectivity, is an approach that statistically uncovers groups of individuals with shared perceptions within a larger cohort. Instructors can use Q‐methodology to identify groups of students with shared needs, allowing for more specific and productive course reform. In the current study, Q‐methodology was used as a means of course evaluation in the fall 2020 and winter 2021 semesters. Students were asked to sort 44 opinion‐based statements in a quasi‐normal table based on their level of agreement. By‐person factor analysis of 166 responses revealed three statistically distinct groups of students.

Results

The three groups were assigned the following monikers: Connected and Contented (CC), Disconnected and Disgruntled (DD), and Interconnected and Collaborative (IC). CC students (n=66) felt generally ambivalent toward course components and were comfortable with the technology skills required to participate in the online course space. DD students (n=50) were deeply unhappy with several elements of the course, including lectures, assignments, and evaluations. These students also felt as if they were teaching themselves. Finally, IC students (n=29) looked favourably upon the tutorial space and the role of teaching assistants. Analysis also revealed that some sentiments were shared across all three groups, including the preference for physical rather than virtual specimens, and the desire for more practice questions from faculty in order to prepare for bellringer exams. Interestingly, cohort opinions did not remain static across both semesters. There was a positive attitude shift as more students felt “Disconnected and Disgruntled” in the fall, and “Connected and Contented” in the winter.

Conclusions

These findings are useful for anatomy instructors interested in transitioning courses to an online or blended space, particularly in the face of ever evolving public health restrictions. The current study also models the wealth of information that can be uncovered using Q‐methodology ‐ useful for anyone interested in the previously amorphous study of subjectivity.
Rapid Hypoxia‐Induced Upregulation of Glutathione‐Related Genes May Protect Elephant Seal Endothelial Cells Against Oxidative Stress
Authors: Allen Kaitlin , Li Alexander , Vázquez‐Medina José Pablo
Published: May 2022

Northern elephant seals (Mirounga angustirostris) experience extreme fluctuations in blood oxygen levels during repeated bouts of deep diving. Such fluctuations characterize human cardiovascular pathologies including myocardial infarction and ischemic stroke, though seals do not incur vascular injury during the repetitive hypoxia/reoxygenation cycles associated with diving. Marine mammals diving at sea remain inaccessible for most real‐time in vivo biochemical investigations, thus we developed a proliferative arterial endothelial cell culture system from expelled placentae from elephant seals and humans to assess the molecular and biochemical response to oxidative stress. Seal endothelial cells generate oxidants when pharmacologically stimulated but remain protected against lipid peroxidation when treated with tert‐Butyl hydroperoxide (t‐BOOH, 300 uM), while human cells exposed to the same conditions display a doubling in lipid peroxidation levels (p=0.0002). Seal endothelial cells maintain viability at moderate (100 uM) doses of t‐BOOH while human cells display significantly reduced viability at the same dose. Interestingly, protein expression of the only two enzymes capable of repairing lipid peroxidation – glutathione peroxidase 4 (Gpx4) and peroxiredoxin 6 (Prdx6) – decreases in both seal and human endothelial cells after t‐BOOH treatment (Human: Gpx4 17% decrease, p=0.08; Prdx6 37% decrease, p=0.007; Seal: Gpx4 53% decrease, p=0.036; Prdx6 35% decrease, p=0.007). Additionally, seal endothelial cells display lower baseline glutathione peroxidase activity than human cells (p<0.0001) and activity in human (p=0.0002) but not seal cells declines after t‐BOOH treatment. Seal endothelial cells exposed to 1% O2 for up to 6 h upregulate expression of genes in the KEGG glutathione metabolism pathway within 30 minutes of hypoxia onset, while expression of these genes returns to baseline levels beyond 1 h in hypoxia. Among those genes contributing to the overrepresentation of the glutathione pathway are several glutathione S‐transferases (GSTM3, GSTK1, GSTO1, MGST2), suggesting that glutathione S‐transferases may limit oxidative damage in seal endothelial cells during hypoxia/reoxygenation bouts. That this upregulation occurs absent the reintroduction of oxygen into the system may suggest that the seal glutathione system is “primed” for rapid response to protect against oxidative injury during the repeated hypoxia/reoxygenation cycles associated with diving.