bluuebunny/crossref_metadata_2025 huggingface dataset:
Multi‐omics profiling shows BAP1 loss is associated with upregulated cell adhesion molecules in uveal melanoma
Authors: Baqai Usman , Purwin Timothy J. , Chua Vivian , Han Anna , Bechtel Nelisa , Hartsough Edward J. , Kuznetsoff Jeffim , Harbour J. W. , Aplin Andrew E.
Published: May 2022

BRCA1‐associated protein 1 (BAP1) is a tumor suppressor gene that is mutated in cancer, including uveal melanoma (UM). Loss‐of‐function BAP1 mutations are associated with UM metastasis and poor prognosis, but the mechanisms underlying these effects remain unclear. Upregulation of cell‐cell adhesion proteins is involved with collective migration and metastatic seeding of cancer cells. Here, we show that BAP1 loss in UM patient samples is associated with an upregulated gene expression profile of multiple cell adhesion molecules (CAMs), including E‐cadherin (CDH1), cell adhesion molecule 1 (CADM1), and syndecan‐2 (SDC2). Similar findings were observed in UM cell lines and single cell RNA sequencing data from patient samples. BAP1 re‐expression in UM cells reduced E‐cadherin and CADM1 levels. Functionally, knockdown of E‐cadherin decreased spheroid cluster formation and knockdown of CADM1 decreased growth of BAP1 mutant UM cells. Together, our findings demonstrate that BAP1 regulates the expression of CAMs which may regulate metastatic traits.
Monomeric CCTδ Interacts with Dynactin Associated Protein (dynAP)
Authors: Iwuagwu Benedicta U. , Grantham Julie
Published: May 2022

Introduction‐

The chaperonin containing tailless complex polypeptide 1 (CCT) is a molecular chaperone consisting of eight distinct protein subunits. In the monomeric form, several CCT subunits possess additional functions distinct from their oligomeric function [1]. Over‐expression of CCTδ, but not the other seven CCT subunits, results in the appearance of numerous protrusions at the cell surface termed a “protrusion phenotype” which is abrogated by mutations in CCTδ apical domain (G357D) and the ATP binding pocket (D104E) [2]. In addition, it is dependent on CCTδ binding the p150Glued of the dynactin complex. Depletion of either p150Glued or the dynactin complex‐associated transmembrane protein, dynAP prevents GFP‐CCTδ localization to the plasma membrane and blocks protrusion phenotype formation [3]. The work of others [4] shows dynAP binds p150Glued. However, biochemical evidence of CCTδ interacting with dynAP has not been shown. We hypothesized either a direct transient interaction between CCTδ and dynAP or a trimer with p150Glued occurring.

Materials & Method‐

Mouse melanoma B16F1 cells were cultured as described in [2], transiently transfected with CCTδ constructs, and mutants thereof fused to GFP as described in [2], and full‐length human; p150Glued with C‐terminal Myc and dynAP with an N‐terminal FLAG epitope tags as described in [3]. To stabilize transient interactions in‐vivo, confluent cells were crosslinked with 0.5 mM 3, 3′‐Dithiodipropionic acid di (N‐hydroxysuccinimide ester). Post‐nuclear supernatants from lysed cells were applied to GFP‐trap beads (Chromotek), followed by Western Blotting to detect precipitating proteins.

Results‐

Following the double transfection of B16 cells with Flag‐dynAP and wild type (WT) GFP‐CCTδ, there was a significant interaction between dynAP and WT CCTδ versus control, D104E. In addition, following the triple transfection of GFP‐CCTδ/p150Glued‐Myc/Flag‐dynAP, there was a significant interaction between dynAP and WT CCTδ versus control, D104E. Furthermore, following the double transfection of WT CCTδ apical domain of (AD) and Flag‐dynAP, there was a significant interaction between dynAP and WT AD versus control, G357D.

Conclusion‐

Here, we show for the first time an interaction between CCTδ and dynAP via immunoprecipitation representing either a direct interaction or as part of a trimeric complex with p150Glued. This provides biochemical evidence for the mechanism underlying the formation of the protrusion phenotype. This work lends more information on monomeric CCTδ function in cells and would contribute to more understanding of CCTδ’ s role in basic cell and cancer biology. More research is needed to understand the molecular effects of these interactions in cells and in‐vivo.

References

Svanstrom & Grantham (2015) Cell Stress and Chaperones; 21:55–62.

Spiess, M. et al (2015) Journal of Molecular Biology; 427:2757‐2764.

Echbarthi M. et al (2018) Experimental Cell Research; 370:137‐149.

Kunoh T. et al (2010) Molecular Cancer Therapeutics; 9:2934‐2942.
Gap Junction Protein, Connexin 43 is a Key Regulator of Metastasis in Syngeneic Ovarian Cancer Mouse Model
Authors: Mulchandani Vaishali
Published: May 2022

Ovarian cancer is the 7th most common cancer of women and the most lethal gynecological cancer that demands rigorous treatments involving increased radiation cycles and higher drug concentration that triggers toxicity and eventually mortality of patients. Understanding of the disease's progression warrants effective therapeutic inventions. Epithelial Ovarian Cancer cell lines ID8 derived from C57Bl/6 mice and ID8 transfected with Vascular Endothelial Growth Factor‐A (ID8‐VEGF) were used in vitro to generate a Syngeneic Ovarian Cancer Mouse Model. Vascular Endothelial Growth Factor‐A (VEGF‐A/VEGF) is the critical regulator of tumor angiogenesis that supports the transport of nutrients, hormones and O2 to the tumor cells and removal of waste products and CO2. It regulates vascular permeability, actin reorganization, cell migration, enhances tumor growth and progression, which may be associated with aberrant cellular metabolism due to altered gap junction intercellular communication (GJIC) contributing to the tumorigenesis. The transmembrane protein Connexin forms hexameric hemichannels known as connexons and docks with another connexon of adjacent cells to form a functional GJIC. Connexin 43 (Cx43) is one of the most ubiquitously expressed connexins and its C‐terminus interacts with various molecules to regulate many biological functions, including modulation of hemichannel activity, apoptotic signaling, cell cycle regulation and, cytoskeletal rearrangements. Our study aims to understand the effect of VEGF expression on the spatial expression of Cx43 and its nexus with other cellular factors contributing to consequent GJIC formation in the ovarian cancer cells as well as in the mouse model. Our findings suggest that upregulation of VEGF in ID8‐VEGF cell lines is associated with accelerated tumor formation in syngeneic mouse models. A series of biochemical and cellular studies revealed that Sodium Phenylbutyrate(4PBA) treatment induced differential upregulation of Cx43 trafficking to the cell surface, rearrangement of cytoskeletal proteins, cell migration, and the formation of GJIC in ID8 and ID8‐VEGF cells. 4PBA treatment on ID8‐VEGF cells significantly reduced the rate of tumor formation. These differential cellular activities denote that VEGF may directly or indirectly interact with cytoskeletal proteins that regulate Cx43 trafficking and its GJIC formation. Overall, The increased capacity of GJIC formation post‐4PBA treatment is associated with reduction in cell migration in vitro (ID8 and ID8‐VEGF) and tumor formation in vivo (ID8‐VEGF). Interactome studies combining RNASeq and IPA are designed to understand the nexus between several cellular pathways, including oxidative stress in the regulation of Cx43 in the context of VEGF, to dissect the cellular and molecular mechanism of metastasis.
Creation of Three‐Dimensional Pedagogical Models of Genetic Mutations
Authors: Paliwal Vipin , Cohan Megan
Published: May 2022

A genetic mutation is a change of one or more bases in the DNA sequence that may alter the structure and ability of the resulting protein product to function properly. In this study, we created three‐dimensional (3D) pedagogical models for teaching genetic mutations. SolidWorks©, 3D modeling software was used to create the models showing the three main types of DNA mutations: (i) point mutation, (ii) frameshift mutation, and (iii) insertion mutation. The models were 3D printed using a selective laser sintering (SLS) machine. Further, the printed models are strong enough to absorb physical mishandling by the users such as accidental drops. User instructions accompanying the models were developed.

The models were created to allow the individual nucleotide bases adenine (A), thymine (T), guanine (G), cytosine (C) and uracil (U) to be attached or removed from the DNA and mRNA backbone and moved around easily by using magnets embedded into the individual pieces. Other ease of use features include color coding and labeling emphasize the difference between the four nucleotides in DNA. Further, as an example of real‐life mutations, we also created a unique model explaining the formation of cyclobutane thymine‐thymine (T‐T) dimers following exposure of DNA to ultraviolet B (UV‐B) radiations. UV‐B radiation in the region of 290‐320 nm is absorbed by skin cells resulting in DNA damage and mutated DNA which can lead to skin cancer. The 3D model showing cyclobutane dimers should help the students gain a deeper understanding of DNA damage, and skin cancer at the biochemical level. Ultraviolet light can break bonds in adjacent thymine bases. New covalent bonds are formed, linking the two thymine bases, forming cyclobutane T‐T dimers. We believe that models will enhance in‐depth hands‐on learning of the genetic mutations.
Synthesis and Characterization of a nano‐bead sunscreen & testing its efficacy
Authors: Paliwal Vipin , Kantarovich Katsiaryna
Published: May 2022

Prolonged exposure to sunlight is one of the major causes of skin cancer due to mutations in the DNA. Repetitive or severe ultraviolet induced DNA damage increases the risk of skin cancer and rate of aging for human skin. Sunscreens are used to protect skin from harmful UV radiations and decrease the risk of skin cancers. There are several advantages of using nanobead sunscreens which include retention of effective UV light‐absorbing capacity and scattering visible light, rendering them transparent on the skin. In addition, their increased stability reduces reapplication. They are also considered less irritating to skin. A cinnamate sunscreen was synthesized through a functional group transformation of cinnamic acid to cinnamate, an ester. After this, the sunscreen was synthesized into a nano‐bead form using flash nanoprecipitation (FNP) technique. Cinnamate sunscreen and sunscreen incorporated nano‐bead product were characterized using UV‐visible spectroscopy scanning in the range of 250‐400 nm. A single peak at 310 nm was observed confirming the high quality of the cinnamate sunscreen. The efficacy of the sunscreen and sunscreen nano‐beads for their ability to chemically block ultraviolet radiation for skin protection was quantified using a customized chamber equipped with a UVB light source that emitted 310 nm light. To simulate human skin conditions, the UVB chamber was equipped with a temperature‐controlled heat retention box that was constantly monitored and maintained between 33 – 37.5 °C throughout the investigation. A thin, even layer of a sunscreen was applied between two thin clear plastic wraps and exposed to the UVB rays. The ability to block UVB radiation was quantified through comparison between the intensity (in mW/m2) of the UVB light going through the plastic wrap before and after the sunscreen applied. It was observed that the sunscreen synthesized had the ability to absorb about 60% of the UVB radiation. Time related decay of the sunscreen after exposure to UVB was investigated. Preliminary results of time decay experiments suggest a possible UV induced hydrolysis of the sunscreen ester product. The results also indicate that sunscreen nano‐beads possibly aggregate to super micellar complexes after exposure to UVB radiation after 3 hours leading to decreased efficacy. Future work includes further investigation of observations in‐depth to improve the nano‐bead sunscreen.
Expression of IDO1 is regulated via Ras signaling pathways
Authors: Lee Eunji , He Felicia , Choi Byeong Hyeok , Dai Wei
Published: May 2022

Ras proteins are among the most widely studied proto‐oncogene products. Ras proteins are membrane‐anchored GTPases that participate in multiple signaling cascades regulating crucial cellular processes including cell survival, proliferation, and differentiation. Ras mutations and/or deregulated Ras activities frequently lead to inflammation and malignant transformation. Recent studies have also shown that Ras mutations are associated with immuno‐resistance by positive regulation of PD‐L1 expression. Indoleamine 2,3‐dioxygenases 1 (IDO1) is a heme‐containing enzyme that catalyzes the rate limiting step of conversion of tryptophan into kynurenine (Kyn). Extensive research in the past has shown that IDO1 and its catalytic product Kyn mediate immune tolerance, thus promoting tumorigenesis in vivo. IDO1 expression is activated by immune cytokines including interferon‐γ (IFN‐γ)and interleukin‐6 (IL‐6). However, whether there is a mechanistic link between oncogenic KRas and IDO1 expression and how IDO1 expression contributes to immune invasion of transformed cells with KRas mutations remain unclear. Here we report that oncogenic KRas significantly enhanced IFN‐γ‐induced IDO1 expression. In H358 lung carcinoma cells, IDO1 expression induced by IFN‐γ was at least in part dependent on ERK activation, and treatment with ARS‐1620, a covalent KRasG12C inhibitor, suppressed IDO1 expression induced by IFN‐γαμμα in a concentration‐dependent manner. IDO1 expression was also induced by IFN‐γ in HCT116 colon carcinoma cells that harbored a KRasG12D mutant allele, but not in HCT116 cells with wild‐type KRas, suggesting that IFN‐γ induced IDO1 expression requires Ras activation. In addition, KRas downregulation by specific siRNAs decreased IFN‐γ‐induced IDO1 expression in A549 lung cancer cells. Taken together, our study strongly suggests that Ras/ERK signaling pathway plays an important role in promoting IDO1 expression induced by IFN‐γ.
In Vitro Selection of RNAs That Can Detect Hypoxanthine: A Surprising Technical Issue
Authors: Greene Henry , Morse Daniel P.
Published: May 2022

The goal of this project is to find RNA molecules that detect hypoxanthine with great sensitivity and specificity. Hypoxanthine is the product of a family of RNA editing enzymes called ADARs. A sensor that can detect hypoxanthine will facilitate the study of the regulation of ADAR activity. There are naturally occurring guanine sensors called guanine riboswitches. We are using in vitroselection to find variants of a guanine riboswitch that can detect hypoxanthine rather than guanine. While performing the selection experiment, we encountered a technical problem. Instead of isolating hypoxanthine sensors, a group of related aberrant RNAs dominated the selected RNA pool. These RNAs had many sequence changes and deletions compared to the sequence of the guanine riboswitch, and they could not detect hypoxanthine. The aberrant RNAs were produced not by the selection process, but as a consequence of multiple rounds of amplification. Here we report our attempts to understand why this happened and to prevent the formation of the aberrant RNAs.
Is there a relationship between in‐season injury risk and Y balance or vertical jump in elite youth soccer players?
Authors: Haraldsdottir Kristin , Baer Michael , Brickson Stacey , Watson Andrew
Published: May 2022

Purpose

Injuries are relatively common among elite youth soccer players, and time lost participating in sport due to injury can have detrimental psychological effects on players. The purpose of this study was to determine whether the Y Balance Test (YBT), or vertical jump can identify those players at risk of suffering injury.

Methods

41 female elite soccer players (age 12‐17) performed YBT and vertical jump testing prior to the competitive season. Time‐loss injuries during the following season were recorded. Injured and uninjured athletes were compared with respect to YBT composite scores (absolute and relative to leg length), asymmetry, and vertical jump. Logistic regressions were performed to examine the relationships between YBT, vertical jump, and injury risk.

Results

Twelve athletes (29%) suffered a time‐loss injury during the season. Athletes who suffered an in‐season injury did not differ from uninjured athletes with respect to age (14.9 +/‐ 1.6 v 15.3 +/‐ 1.4 years, respectively), years of experience (9.7 +/‐ 1.7 v 10.2 +/‐ 2.4 years, p=0.26), YBT composite scores (111 +/‐ 16 v 109 +/‐ 17 cm, p=0.99), YBT composite scores relative to leg length (1.29 +/‐ 0.2 v 1.27 +/‐ 0.26, p=0.85), the difference in centimeters between composite scores of each leg (7.9+/‐8.4 v 4.6+/‐3.8, p=0.09) or vertical jump (15.3±1.6 v 15.3±2.6 in, p=0.96). In‐season injuries were not significantly associated with YBT composite scores (OR= 0.99 [95% CI = .95‐1.04] ,p= 0.80), relative YBT composite scores (1.0 [.97‐1.03], p=0.98) or vertical jump scores (0.98 [.72‐1.33], p=.91). Although not reaching statistical significance, higher asymmetry in composite scores showed some evidence of an increased risk of subsequent injury (1.2 [0.97‐1.42], p=0.10).

Conclusions

The composite YBT score and vertical jump did not predict in‐season injury incidence in this sample of elite female adolescent soccer players. Higher YBT asymmetry may be associated with an increased risk of in‐season injury, although this study may have been limited by sample size to identify a statistically significant difference.

Significance

Although the YBT is widely used as an injury risk screening tool, our findings suggest that it may not be a strong predictor of subsequent time‐loss injuries in elite female adolescent soccer players.

Acknowledgements

We are grateful to the adolescent soccer players who participated in the study.
A Single Glucocorticoid Injection Accelerate Erythropoiesis but Does Not Improve Performance
Authors: Bejder Jacob , Bonne Thomas C. , Breenfeldt Andersen Andreas , Sørensen Henrik , Nordsborg Nikolai B.
Published: May 2022

The World Anti‐Doping Agency prohibit glucocorticoid (GC) administration during competition but allow administration in periods out of competition. However, GC administration may accelerate erythropoiesis and resemble blood volume manipulation methods, which are prohibited at all times. Here, we investigated whether a single GC injection accelerate erythropoiesis, increase total hemoglobin mass (Hbmass) and improve exercise performance.

In a randomized, double‐blinded, placebo‐controlled crossover design (3‐month washout), eight trained males (maximal oxygen uptake 60±5 ml O2·min‐1·kg‐1) were injected with 40 mg triamcinolone acetonide (GC group) or saline (PLA group) in the gluteal muscles. Venous blood samples collected before and 7‐10h, 1, 3, 7, 14 and 21 days after treatment were analyzed for [Hb] and reticulocyte percentage (ret%). Hbmass and mean power output in a 450‐kCal time trial was measured before as well as one and three weeks after treatment.

A 27±28% and 54±41% higher ret% was evident three (P<0.01) and seven (P<0.001) days after the GC administration, respectively, compared with PLA, whereas [Hb] was similar between groups. Additionally, Hbmass was 20±28g higher (P<0.05) one week after GC administration compared with PLA, but similar after three weeks. The mean power output was similar between groups one (GC: 285±70 W, PLA: 279±69 W) and three (GC: 279±72 W, PLA: 276±71 W) weeks after treatment.

In conclusion, a single intramuscular injection of triamcinolone acetonide was sufficient to accelerate erythropoiesis and increase Hbmass, but did not translate into improved aerobic exercise performance in the present study. Future research should confirm whether anti‐doping authorities should consider glucocorticoid injections as a method of blood volume manipulation.
Vaping /E‐liquid Exposure Causes Dysregulation of Neutrophil Extracellular Trap formation
Authors: Abdelwahab Sabri , Ghosh Arunava , Wrennall Joe , Tarran Robert
Published: May 2022

Introduction

Electronic cigarettes (e‐cigarettes) vaporize e‐liquids containing propylene glycol and vegetable glycerine (PG/VG), nicotine, and flavors. The inhaled aerosol from e‐liquids may be potentially toxic or harmful. Although the short and long‐term health consequences of e‐cigarettes use are poorly understood, their use has risen steeply among adolescents and young adults. Neutrophils play an essential role in innate immune and are the first responders during infections. Thus, this study aims to test whether vaping can affect neutrophil function. We hypothesized that vaping e‐cigarettes and exposure to e‐liquids activate neutrophils, cause degranulation, and induce extracellular traps (NETs) through altered calcium signaling.

Method and Materials

Neutrophils were isolated from human blood samples of non‐vapor control (n=9) and e‐cigarette users (n=5) and studied within 2 hours. To measure changes in cytosolic calcium, neutrophils were loaded with Fluo‐4. Intracellular calcium responses were monitored on a Tecan multi‐plate reader following stimulation by 1 µM thapsigargin and 1 µM fMLP/100ng per ml LPS. In addition, since elevations in intracellular calcium trigger neutrophil activation and degranulation, elastase activity in the media samples was measured using a specific fluorogenic substrate (MAA‐3133, © vivitide, LLC.) at excitation/emission wavelengths 380/460 nm. To study NET formation, neutrophils were labeled with SYTOX Green dye and then exposed to different concentrations of Juul e‐liquids (menthol, classic tobacco flavors) and Bidi Stick flavors (Arctic, Gold, and Solar). Data acquisition was performed with Cytation‐5 Cell Imaging Plate Reader, then analysis and representative images were captured by confocal microscopy.

Results

Neutrophils from e‐cigarette users (n=5 vapers) showed significantly (P < 0.001) increased intracellular calcium responses to thapsigargin and fMLP/LPS treatments compared to the non‐user controls (n=9). Additionally, vapers' neutrophils also secreted more elastase (P < 0.001) compared to the control non‐users, indicating that e‐cigarette users may be susceptible to increased systemic protease activity. Our data also indicated that Juul and Bidi Stick e‐liquids stimulated neutrophils to induce NETs in a dose‐dependent manner. Furthermore, the study revealed that Juul e‐liquid menthol flavor with 5% nicotine could significantly (P < 0.001) release NETs than equivalent nicotine ± benzoic acid alone.

Circulating neutrophils are normally quiescent. However, our data indicated that circulating neutrophils from vapers were abnormally active and degranulated by released elastase. Additionally, e‐cigarette liquids dysregulated neutrophils by increasing NETs production. Overall, we speculate that vaping imbalanced innate immune system and may lead to abnormal responses to the infection, inappropriate inflammatory responses, and adverse health effects.
Physical contact with breast cancer weakens endothelial cell chirality
Authors: Hang Benson X. , Mohammed Hanan , Wan Leo , Fan Jie
Published: May 2022

Cells have been recently discovered processing a phenotype‐specific chirality which can be seen as directional cell alignment, rotation, or migration. Endothelial cells (ECs) exhibit a strong right bias (or clockwise chirality) under normal conditions. Enervating this chirality impairs EC junction formation and leads to significant leakage of the blood vessels. Malfunction of endothelial barrier induced by malignant tumor cell (TC) facilitates metastasis; however, the role of EC chirality during its crosstalk with TCs is still poorly understood. Using a Transwell model, we evaluated the chirality of human umbilical vein endothelial cells (HUVECs) on a ring‐shaped micropattern with direct or indirect co‐culture of TCs. Our data show a significant loss, or partial reversal of EC chirality when MCF10A‐HRAS or MCF10A‐HER2 cells were directly introduced to EC populations, while indirect co‐culture with either type of TCs only caused a minor decrease in EC chirality. Furthermore, we characterized the bias of each EC on the micropattern using a cell organelle‐based labeling and image analysis. We found the ECs in direct contact with TCs trend to reverse their bias, while the “spaced ones” do not. Together, our results suggest direct physical contact with malignant TCs could cause the EC chirality weakening, which potentially compromises the overall endothelial integrity and contributes to cancer spread.
Extracellular Vesicles from iPSC derived Muscle Progenitor Cells Rejuvenate the Dysfunctional Muscle Stem Cells during Aging
Authors: Xuan Wanling , Tipparaju Srinivas M. , Ashraf Muhammad
Published: May 2022

Background and Objective

Sarcopenia is defined as the loss of skeletal muscle mass and function due to age, which is a major concern for life long health and wellbeing in elderly population. Age‐associated decline in the performance of muscle stem cells (MuSC) contributes to the deterioration of skeletal muscle during aging. Using small molecule Givinostat and CHIR99021 we previously demonstrated the generation of muscle progenitor cells (Givi‐MPC) of human Induced pluripotent stem cells (iPSC) from multiple cell lines. We found that extracellular vesicles (EVs) from these MPC have a specific cargo compared with human myoblasts. Here, we investigated whether these EVs can rejuvenate the aging dysfunctional muscle stem cells.

Methods and Results

Using size exclusion chromatography EVs were isolated and purified from iPSC derived Givi‐MPC and primary human myoblasts. The EVs were visualized by transmission electron microscopy. Next, we isolated primary MuSC from 25 months old aged C57/B6 mice, which were confirmed by the expression of myogenic transcription factor, Pax7. We found the aged MuSC pretreated with EVs from MPC promoted clonal expansion and restored myogenic potential in vitro with more myotubes formed after 72h differentiation in medium with 2% horse serum compared with PBS pretreated MuSC (MuSC (Fusion index: 40.3±5.2% vs.15.0± 2.1%, n=3).). To test the regenerative potential of the EVs on aged MuSC, 8 weeks old mdx/scid mice (dystrophin deficient mice) were irradiated to destroy the host MuSC and then injured their tibialis anterior (TA) muscle with cardiotoxin (CTX). 24 hours after injury, we transplanted the aging MuSC (5000 cells) either with EV or PBS pretreatment into TA muscles, respectively. 5 days after transplantation, the EVs pretreated MuSC increased muscle regeneration with muscle fibers positive for embryonic myosin heavy chain compared to PBS pretreated MuSC. One month later, we found transplantation of EVs pretreated MuSC restored dystrophin expression in these mdx mice. We further tested the effects of EVs pretreatment on MuSC in 25 months old mice following CTX injury. Consistent with the results in mdx mice, pretreatment of mice with EVs from Givi‐MPC showed enhanced regeneration (6‐fold increase in number of new muscle cells with central nuclei) in CTX injured aged muscle accompanied by an increase of Pax7 positive MuSC compared with PBS treated mice or treatment with EVs from human myoblasts (number of Pax7 positive MuSC: EVs‐Givi‐MPC 10±5/mm2; PBS 2±1/mm2; EVs‐human myoblast 6±2/mm2, n=3).

Conclusion

The regenerative potential of aging MuSC is restored with the treatment of EVs from Givi‐MPC allowing stem cell renewal and differentiation upon activation.
An Underlying Mechanism by which Hepatic Steatosis Drives the Development of Hypertension
Authors: Bruggink Stephanie , Xiao Yao , Geisler Caroline , Ghimire Susma , Strom Joshua , Renquist Benjamin
Published: May 2022

Hepatic steatosis, a consequence of obesity, is closely associated with metabolic and cardiovascular diseases, including hypertension. Even in patients that are not hypertensive, the amount of fat in the liver is positively associated with blood pressure. We hypothesized there was an underlying mechanism linking excess fat in the liver with increased blood pressure. We have previously established that liver lipid content is positively associated with hepatic production and release of the inhibitory neurotransmitter GABA, a process dependent on both the GABA shunt and electrogenic GABA transporters. Because GABA is co‐transported with 1‐2 net positive charges, hepatocyte depolarization, common in obesity, encourages hepatocyte GABA release. To investigate the effect of hepatocyte depolarization, we used an adeno‐associated virus to induce hepatocyte specific expression of an artificial chimeric channel that opens in the presence of an exogenous ligand causing depolarization. Inducing hepatocyte depolarization increases release of GABA and decreases hepatic vagal afferent nerve activity. We established that administering the depolarizing ligand acutely (15‐40 minutes after IP administration) increased systolic (30.08 ± 5.6 mmHg), diastolic (16.47 + 4.3 mmHg), and mean blood pressure (17.85 + 4.4 mmHg) measured via telemetry devices. In hepatic vagotomized mice hepatocyte depolarization had no effect on blood pressure, establishing the key role of afferent hepatic vagal signals in regulating blood pressure. Pharmacological inhibition of the GABA shunt by daily administration of ethanolamine‐O‐sulfate (4 days; 8 mg/mouse/day) limited liver slice GABA release and decreased systolic, diastolic, and mean (10 ± 3.07 mmHg; P < 0.05) blood pressure in diet‐induced obese mice during the first hour of the dark cycle. We subsequently knocked down GABA‐transaminase with bi‐weekly IP deliver of an anti‐sense oligonucleotide targeted to GABA‐transaminase (12.5 mg/kg). We have previously established that this decreases liver GABA‐transaminase mRNA expression by 97% without affecting mRNA expression in the pancreas or brain. GABA‐transaminase knockdown decreased 24h mean, systolic, and diastolic blood pressure (17 + 3, 20 +2, and 14 + 4 mmHg respectively) in obese, angiotensin II induced hypertensive (continuous delivery of angiotensin II 800 ng/kg/min by Alzet® osmotic minipump) male mice. Together this data supports a role of hepatocyte GABA production and release in the hypertension that accompanies obesity. Moreover, our results provide a mechanism by which hepatic lipid content can affect blood pressure, identifying potential targets for the treatment and prevention of obesity‐induced hypertension.
Enhancer regulation by H3K4me1 methyltransferases MLL3/MLL4
Authors: Ge Kai
Published: May 2022

Enhancers control cell type‐specific gene expression and are marked by H3K4me1. Active enhancers are further marked by H3K27ac. We identified MLL3/MLL4 as major H3K4me1 methyltransferases and CBP/p300 as the H3K27 acetyltransferases in mammalian cells (EMBO J 2011; eLife 2013). During differentiation of adipocytes, myocytes and ES cells, MLL3/MLL4 co‐localize with lineage‐determining transcription factors (LDTFs), CBP/p300, Brd4 and MED1 on active enhancers. MLL3/MLL4 are required for enhancer activation, enhancer‐promoter interaction, cell type‐specific gene expression, and cell differentiation (eLife 2013; PNAS 2016; Nat Comm 2017). MLL3/MLL4 control cell differentiation by orchestrating CBP/p300‐mediated enhancer activation (NAR 2017). Ectopic expression of H3.3K4M, an inhibitor of H3K4 methylation, or deletion of the enzymatic SET domain, destabilizes MLL3/MLL4 proteins, prevents enhancer activation, and impairs cell differentiation and tissue development (NAR 2019).

Using proteomic approaches, we found that endogenous MLL4 complex associates with SWI/SNF chromatin remodeling complex components in cells. Using adipogenesis as a model system, we establish an interdependent relationship between SWI/SNF complex BAF and MLL4 in promoting cell type‐specific enhancer activation by LDTFs, which rectifies seemingly conflicting results from previous studies (Nat Comm 2021).

We showed that MLL4 protein, rather than MLL4‐mediated H3K4me1, controls p300 recruitment to enhancers during ES cell differentiation, suggesting that MLL4 regulates enhancer activation independent of H3K4me1 (PNAS 2016). We have generated enzyme‐dead MLL3/MLL4 double knockin ES cells and mice by CRISPR. Initial analyses indicate that MLL3/MLL4 regulate ES cell differentiation and mouse embryonic development through both enzymatic activity‐dependent and ‐independent mechanisms.
Synergistic Effects of Resveratrol Derivatives with Bortezomib on Reducing Viability and Inducing Apoptosis in Multiple Myeloma Cells
Authors: Staskiewicz Anna , Wong Erica , Farhin Begum , Tucker Michael , Wang Xinyu
Published: May 2022

Multiple myeloma (MM) is a cancer of plasma cells in the bone marrow characterized by bone lesions, hypercalcemia, anemia, and renal failure. Bortezomib (BTZ), a common treatment for MM, is a proteasome inhibitor that induces apoptosis in MM cells. However, high doses of BTZ can be toxic, signifying a need for a synergistic drug combination. Resveratrol (RES) is a phenolic compound found in grapes shown to inhibit MM cell growth and induce apoptosis in these cells. We hypothesize that combining BTZ with a RES derivative at certain concentrations is synergistic in reducing viability and inducing apoptosis in MM cells. To address this hypothesis, we performed viability assays to assess the effects of increasing concentrations of RES derivatives and BTZ on human MM cell viability over time (24, 48, 72 hours) and western blots to assess the compounds’ effects on inducing apoptosis in MM cells following 24 hour treatment. We found that BTZ as well as RES and its derivatives pinostilbene (PIN) and piceatannol (PIC) decreased MM cell viability in a dose‐ and time‐dependent manner and increased expression of cleaved proapoptotic proteins PARP1 and caspase‐3 in a dose‐dependent manner. We screened different combinations of RES derivatives with BTZ for synergistic effects on MM viability using CompuSyn and selected the combination of 5 nM BTZ and 5 μM PIN for further assessment as a synergistic treatment. In conclusion, a combination of low doses of BTZ and PIN synergistically decreases MM cell viability, possibly through inducing apoptosis.
The Impact of Removal of Exercise for 3 Days on Glycemic Control in Older Active Adults
Authors: Reynolds Leryn , Williams Troy , Harden Joel , Twiddy Hannah , Kearney Monica
Published: May 2022

Postprandial glycemia (PPG) predicts cardiovascular disease, and 3 days of physical inactivity impairs PPG in young, active adults. Whether this occurs in older, active adults who may be more prone to bouts of inactivity is unknown. This study determined if PPG was impaired in active older adults (old) following a 3 day removal of exercise compared to active young adults. In this randomized, crossover study, 10 older (73.0 ± 1.9 yrs) and 9 young (32.8 ± 1.8 yrs) habitually active (≥90 min/week of exercise) adults completed a 3 day period of exercise removal (NOEX) and a 3 day period of normal exercise maintenance (EX), separated by ≥ 1 week. Diet was standardized across phases. Glycemic control (GC) (2 hour PPG, peak glucose values, and standard deviation (STDEV) of blood glucose values) was assessed via continuous glucose monitoring (IPro2, Enlite Sensors, Medtronic Inc., Northridge, CA) during both phases. No differences in body mass index (Old: 26.5 ± 1.3, Young: 24.8 ± 1.0 kg/m2, p>0.05) existed between groups. Both groups had a similar reduction in activity during NOEX (Old: ‐64.6% ± 6.2, Young: ‐55.7% ± 4.9, p>0.05). There were significant main effects (p<0.05) in PPG of age (Old: 116±1.2 mg/dL, Young: 106 ± 1.3 mg/dL), phase (Inactive: 113 ± 1.3 mg/dL, Active: 109 ± 1.2 mg/dL), and time (0 min: 99 ± 2.0, 30 min: 118 ± 2.0, 60 min: 117 ± 2.0, 90 min: 112 ± 2.0, 120 min: 108 ± 2.0 mg/dL), but no interaction (p>0.05). Further, a trend (p=0.06) for significant main effect of age (Old: 149±5.6, Young: 133±5.9 mg/dL) and phase (p<0.05; Inactive: 145±4.4, Active: 136±4.4 mg/dL) were found for peak glucose values. Significant main effects (p<0.05) exist for age (Old: 15.3±1.2, Young: 11.3±1.2) and phase (Inactive: 14.4±0.97, Active: 12.2±0.97) for blood glucose STDEV. These results indicate that older adults have impaired GC compared to young adults and that removal of exercise impairs GC. Yet, the impairment in GC with exercise removal is not different between old and young adults when the decrease in daily physical activity is similar.
Applying Crystallography and 19F NMR to investigate dynamics and partner protein interactions in a long chain Flavodoxin
Authors: Khan Sharique , Ansari Ahmadullah , Miller Anne‐Frances
Published: May 2022

Flavodoxin (Fld) is a small FMN containing protein that is involved in single electron transfer. The long‐chain flavodoxin in Rhodopseudomonas palustris bacteria replaces ferredoxin as a low‐potential electron carrier when iron is scarce. Thus it is proposed to interact with the bifurcating electron transfer flavoprotein (ETF) that yields low‐potential electrons. A surface loop on Fld interacts with another of Fld's partner proteins, so we hypothesize that it also mediates Fld's interaction with ETF. To monitor interactions with ETF directly and investigate dynamics in this loop, we are using 19F NMR in solution. 19F is hyperresponsive to changes in its chemical environment with a chemical shift range of >300 ppm. To provide a static reference point and assess structural heterogeneity, we are also exploiting X‐ray crystallography.

In this study we selectively fluorinated the five tyrosine residues in Fld. We obtained resonance assignments from 19F spectra of Fld variants in which individual tyrosine residues have been replaced. We obtain well resolved signals for each residue, but the resonances' linewidths indicate dynamics that affects some resonances more than others. Y90 residue displays two resonances demonstrating two different conformations that interconvert slowly on an NMR time scale. Meanwhile the crystal structure solved at 2.1Å resolution reveals two molecules per asymmetric unit providing two perspectives on the details of the structure. The crystal symmetry is monoclinic in contrast to most of the other Flds, which are orthorhombic. Interestingly, the long loop bearing Y121 and Y123 is not well resolved in chain B of the crystal structure, and the NMR line of Y123 is exceptionally broad, both indicating that the loop is dynamic and capable of altering its conformation to accomodate binding to a partner protein.

Future directions include monitoring the changes in the 19F NMR of the Fld when titrated with the partner protein, temperature dependence of the NMR spectrum and relaxation studies to evaluate time scales of motions.
Understanding the Role of the Peroxiredoxin Family in the Regulation of Ferroptosis in Colorectal Cancer
Authors: Alshahrani Aziza , Jiang Hong , Thapa Pratik , Ding Na , Hao Yanning , Wei Qiou
Published: May 2022

Ferroptosis, a new form of regulated cell death, is typically characterized by excessively iron‐dependent accumulation of lipid peroxidation, which causes the breakage of plasma membrane. Dysregulation of ferroptotic cell death has been linked to the development of cancer, and inducing ferroptosis has been proposed as a potential strategy for cancer treatment. Although several important regulators involved in ferroptosis have been discovered, the molecular mechanism behind this process has yet to be completely understood. Members of the peroxiredoxin (Prx) family function as leading cellular antioxidants that react with hydrogen peroxide to keep redox homeostasis as well as contribute to oxidative signalling under both physiological and pathological conditions. In this study, we investigated the role of Prx family members in the regulation of ferroptosis in colorectal cancer (CRC) cells using loss‐of‐function and gain‐of‐function experiments. Firstly, immunoblotting was used to examine endogenous levels of Prx family members in different colorectal cancer cells and cells with high or low expression of Prxs were selected for study. Secondly, each of the endogenously expressed 2‐Cys containing Prxs including Prx1, Prx2, Prx3 and Prx4 in representative CRC cell lines was depleted by stably expression of ShRNA targeting their coding regions. The consequences of Prx knockdown in these cells in response to ferroptosis inducers, including erastin and RSL3, were examined by cell viability and colonogenic assays. Cell death due to ferroptosis in these cells was demonstrated by the measurement of lipid peroxidation using the sensor BODIPY 581/591 C11 and differentiated with other modes of cell death such as apoptosis, autophagy and necrosis by the combined application of various inhibitors. Finally, overexpression and rescue experiments were used to further validate the results observed in Prx‐depleted cells. In conclusion, we found that 2‐Cys containing Prxs are widely expressed in CRC cell lines, and depletion of Prx1, Prx2 or Prx4 but not Prx3 sensitizes CRC cells to erastin induced cell death. However, only depletion of Prx4 but not other Prxs sensitizes CRC cells to ferroptotic cell death that is characterized by the accumulation of lipid peroxidation and can be rescued by the treatment with inhibitors of ferroptosis. In consistence, overexpression of Prx4 also leads to the resistance of CRC cell to ferroptosis. Thus, our findings reveal an essential role of Prx4 in protecting colorectal cancer against ferroptosis and provide a potential target to enhance the antitumor activity of ferroptosis‐based treatment.
Characterizing BfpU, an Essential Protein in the Type IV Pilus of Enteropathogenic Escherichia coli
Authors: Little Janay I. , Shabon Samara , Zhao Jinlei , Donnenberg Michael S.
Published: May 2022

Type IV pili (T4Ps) are a family of surface appendages that are important for adhesion, colonization, biofilm formation, virulence, twitching motility and many other functions. BfpU is an essential periplasmic protein of the T4P of Enteropathogenic Escherichia coli. Previous crosslinking and localization studies have shown that BfpU might interact with the outer membrane secretin BfpB, which is also essential for T4P biogenesis. To date, BfpU has no known homologs and its function is unknown. To purify BfpU, we performed affinity and size exclusion chromatography. Preliminary characterization of BfpU via thermal stability assay and circular dichroism suggests a melting temperature of 80 °C, and its secondary structure is comprised of ~40% anti‐parallel β‐sheet and ~45% random coil. The secondary structure profile is reminiscent of PilP, an essential periplasmic protein anchored in the membrane that interacts with secretins in other T4P systems. Additionally, random mutagenesis is in progress to assess essential residues of BfpU that may be involved in protein interactions within this T4P system. Single residue mutations from this mutagenesis screening will be used to confirm interaction sites within BfpU. The results of these studies will help clarify the role of BfpU in T4P biogenesis and its interactions with BfpB.
Heterologous expression of methyl‐coenzyme M reductase reveals the importance of organism‐specific accessory proteins for proper assembly
Authors: Gendron Aleksei , Allen Kylie
Published: May 2022

Methyl‐coenzyme M reductase (MCR) is the key rate‐determining enzyme of methanogenesis as well as the anaerobic oxidation of methane, the essential energy metabolisms of methanogenic archaea and anaerobic methanotrophs (ANME), respectively. MCR is a dimer of heterotrimers with a 2α, 2β, 2γ configuration, and requires the nickel tetrapyrrole prosthetic group, coenzyme F430. The requirement of a unique cofactor, various unusual post translational modifications, and many remaining questions surrounding assembly and activation of MCR has so far largely limited in vitro experiments to native enzymes. To allow further investigation into the catalytic properties and mechanistic aspects of different MCRs, as well as facilitate the development of optimized biocatalytic systems to convert methane to more usable liquid fuels and other valuable compounds, we are developing methods for the heterologous expression of recombinant MCRs in Methanococcus maripaludis, a model methanogen with robust genetic tools.

In methanogens, MCR is encoded in the highly conserved MCR gene cluster mcrBDCGA, which encodes two accessory proteins (McrD and McrC) in addition to the MCR‐encoding genes (McrA, McrB, and McrG). The accessory proteins are proposed to be involved in the assembly and activation of MCR. Interestingly, most ANME lack one or more accessory proteins in their MCR gene clusters. We have created a series of MCR expression constructs containing the MCR operons from several ANME organisms as well as several methanogens, with and without accessory protein(s). All constructs contain a his‐tag on the C‐terminus of McrA which allows the purification and determination of proper assembly. We have successfully purified a recombinant ANME‐2d MCR that is assembled and binds F430, whereas in the case of the ANME‐1 MCR, we can only recover the his‐tagged McrA. Since the ANME‐2d MCR operon contains McrD and the ANME‐1 operon does not, this result supports the importance of McrD for proper assembly in vivo. Combined with other results from the expression of recombinant methanogenic MCRs, our results indicate that MCR accessory proteins are organism specific. Current work is focused on demonstrating the role of McrD through in vitro binding studies as well as further elucidating the importance of mcrDand mcrC for recombinant expression of diverse MCRs.
Janus Kinase 3 phosphorylation and the JAK/STAT pathway are positively modulated by follicle‐stimulating hormone (FSH) in bovine granulosa cells
Authors: Zareifard Amir , Beaudry Francis , Ndiaye Kalidou
Published: May 2022

Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor‐mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was shown as differentially expressed in granulosa cells (GC) of bovine preovulatory follicles and downregulated by the luteinizing hormone. These observations suggested JAK3 regulation could modulate GC proliferation, steroidogenic activity and activation/inhibition of downstream targets. To investigate the mechanisms of JAK3 actions in GC, we used JANEX‐1, a pharmacological JAK3 inhibitor, and FSH treatments and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3 and previously identified JAK3 partners CDKN1B/p27Kip1 and MAPK8IP3/JIP3. Cultured GCs were treated with or without FSH in the presence or not of JANEX‐1. Total RNA and proteins were extracted and analyzed by RT‐qPCR, western blotting and UHPLC/MS‐MS. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was significantly upregulated in GC treated with FSH and both were significantly decreased when JAK3 was inhibited as compared to control. Proliferation markers CCND2 and PCNA were significantly reduced in JANEX‐1‐treated GC and upregulated by FSH. Western blots analyses showed that JANEX‐1 treatment significantly reduced pSTAT3 amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX‐1‐treated GC. UHPLC/MS‐MS analyses showed phosphorylation and additional modifications of specific amino acid residues within JAK3 as well as its binding partners CDKN1B and MAPK8IP3 revealing possible activation or inhibition of JAK3 following FSH or JANEX‐1 treatments, respectively. Abundance of JAK3 total protein was increased post‐FSH treatment and significantly decreased, along with MAPK8IP3, in JANEX‐1‐treated GC while CDKN1B total abundance was altered post‐FSH and increased post‐JANEX‐1. We show that JAK3 influences GC activity through phosphorylation of target proteins in response to stimulations such as FSH, which leads to the activation of JAK/STAT and likely modulating other signaling pathways involving CDKN1B and MAPK8IP3.