bluuebunny/crossref_metadata_2025 huggingface dataset:
Tracking the ancestral functions of erythropoietin: neuroprotection & mitochondria
Authors: Arias‐Reyes Christian , Rojas‐Ruiz Andrés , Soliz Jorge
Published: May 2022

It has long been thought that erythropoietin (Epo) is exclusively involved in erythropoiesis; now, it is known that EPO in mammal's brain plays key roles in the development, maintenance, protection, and repair of the nervous system. Also, EPO in mammals contributes to the efficient use of oxygen through the regulation of mitochondrial bioenergetic. Remarkably, a similar neuroprotective impact of recombinant human EPO (rhEPO) has been found in the brain of grasshoppers, raising questions about the evolutive origin of the EPO and its generic molecular function. The objective of this study is to show that the neuroprotective effect of rhEPO in insects involves the regulation of mitochondrial functions. The experiments were performed in crickets (Acheta domesticus). These insects were exposed under normoxia and hypoxia (5 days; 6% O2) conditions. Before experimentation, the animals were treated with EPO (30 IU/ml ‐ intra‐lymphatic injection) or PBS, as a control. The brains of the crickets were removed, and then we determined the mitochondrial respiration and production of mitochondrial ROS using our system oxygraphy ‐ 2K (ORORBOROS). Our results show that compares to normoxia; hypoxia significantly reduces mitochondrial respiration of complexes 1 and 1&2. On the other hand, the treatment of EPO in hypoxia, despite significantly increasing these parameters, does not recover the levels of mitochondrial respiration under normoxic conditions. In addition, the activity of complex IV (an indicator of the number of mitochondria) does not vary significantly between any of the treatments. Furthermore, we observed that while hypoxia did not significantly affect H2O2 production, the treatment with EPO increased ROS production under normoxic but not hypoxic conditions. Our data suggest that rhEPO regulates in some way the mitochondrial respiration and ROS production in the brain of crickets. Considering that insects appeared during a geological period (Cambrian explosion) in which the atmospheric O2 was increasing, which could cause great oxidative stress due to the change in the metabolism of these animals, this molecule would have appeared as a regulator of mitochondrial functions.
Assembly‐line catalysis in bifunctional terpene synthases: (+)‐Copalyl diphosphate synthase from fungal Penicillium species
Authors: Ronnebaum Trey A. , Eaton Samuel A. , Brackhahn Emily A. E. , Faylo Jacque L. , Gupta Kushol , Christianson David W.
Published: May 2022

The magnificent chemodiversity of terpenoid natural products is largely rooted in two types of terpene synthases: prenyltransferases and cyclases. The unusual diterpene (C20) synthase, (+)‐copalyl diphosphate synthase from fungal Penicilliumspecies (PvCPS, PfCPS),is the first bifunctional terpene synthase identified with both prenyltransferase and class II cyclase activities in a single polypeptide chain. The C‐terminal prenyltransferase α‐domain generates the C20 linear isoprenoid, geranylgeranyl diphosphate, which is then cyclized to (+)‐copalyl diphosphate at the interface of the N‐terminal βγ‐domains. We, first, establish that PvCPS exists as a hexamer – a unique quaternary structure for known αβγ terpene synthases.1 Hexamer assembly is corroborated by 1) crystal structures of the prenyltransferase α‐domain obtained from limited proteolysis of full‐length PvCPS, 2) the ab initio modeling of full‐length PvCPS derived from small‐angle X‐ray scattering data, and 3) preliminary 3D reconstructions of PfCPS by cryo‐EM. Interestingly, biophysical experiments with PvCPS suggest that oligomer formation is dynamic since the hexamer dissociates into lower‐order species at lower concentrations. However, enzyme concentration does not affect prenyltransferase activity in vitro.

Assembly‐line catalysis provides an efficient carbon management system for generating high‐value terpenoid natural products. With the goal of generating different C5n isoprenoid products, we next explored the structure‐function relationship of the prenyltransferase domain within the assembly‐line platform of PvCPS. Steady‐state kinetics, product analysis, and crystal structures of various structure guided site‐directed variants afforded a functional C15 prenyltransferase chimera.2 This variant will be used in future engineering experiments to create bifunctional sesquiterpene synthases.

References

1. Ronnebaum, T.A.; Gupta, K.; Christianson, D.W., J. Struct. Biol.2020, 210 (1), 107463.

2. Ronnebaum, T.A.; Eaton, S.A.; Brackhahn, E.A.E.; Christianson, D.W., Biochem. 2021, 60 (42), 3162‐3272.
Contribution of the Golgi and Endosomes to Proteostasis During Aging in the Nematode Caenorhabditis elegans
Authors: Palumbo Morgan , Saladino Christopher , Youngman Matthew
Published: May 2022

A gradual breakdown in the cellular networks that assure protein stability, termed proteostasis, contributes significantly to the accumulation of misfolded and aggregated proteins that severely compromises cellular function later in life. A progressive decline in the efficiency of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) causes cells to become particularly susceptible to disturbances affecting the biogenesis, folding, stability, and turnover of membrane proteins. While the dynamics of ER stress response mechanisms during aging have been well‐characterized, less is understood about how the function of other organelles important for membrane protein biogenesis and turnover might change over time. We hypothesized that age‐dependent changes in the function of the Golgi and endosomes could contribute to the collapse in proteostasis during adulthood. Using a reverse genetic approach in Caenorhabditis elegans, we probed the function of these two compartments by knocking down the expression of cogc‐2, a member of the Conserved Oligomeric Golgi (COG) complex and of rme‐8, encoding an endosomal protein important for the retrieval of Golgi residents and the recycling of plasma membrane proteins. A key component of the C. elegans innate immune response is the secretion of immune effector proteins into the intestinal lumen, which can itself trigger the ER stress response due to the increased flux through the secretory pathway. Therefore, as a proxy for the integrity of vesicular transport pathways we measured the survival of juvenile and adult animals infected with the bacterial pathogen Pseudomonas aeruginosa following RNAi treatments targeting cogc‐2 or rme‐8. While there was a negligible effect on late larval stage animals, reduced expression of cogc‐2 and rme‐8 enhanced the sensitivity of post‐reproductive adult animals to infection. This suggests that components of vesicular transport pathways downstream of the ER could also contribute to proteostasis in an age‐dependent manner through their roles in ensuring the proper localization of membrane proteins. Follow‐up studies will use in vivo fluorescent reporters of protein stability to determine whether interfering with protein transport through the Golgi and endosomes accelerates the decline in proteostasis in older animals. Our studies imply that organelles involved in late steps of membrane protein biogenesis and stability play increasingly important roles in maintaining the proteome during aging.
Influence of cotinine on breathing related motor output
Authors: Hernandez‐De La Pena Alessandro G. , Fregosi Ralph F. , Wollman Lila B.
Published: May 2022

Cotinine, the primary metabolite of nicotine, is used as a marker of nicotine exposure due to its long plasma half‐life. Previous research shows that perinatal nicotine exposure alters the regions of the brain that control breathing and results in impaired protective breathing reflexes in exposed human and animal neonates. While it has long been known that nicotine has direct effects on brain function and development, it has only recently been established that cotinine may act on the brain as well. Preliminary data suggests that cotinine, the primary metabolite of nicotine, enhances the effects of nicotine on breathing, consistent with its presumed action as a modulator of nAChRs. Here, to build on this preliminary data, we test the hypothesis that cotinine enhances the effects of nicotine on the frequency and amplitude of respiratory motor bursting in the brainstem spinal cord preparation from 1‐5 day old control rat pups and rat pups that were exposed to nicotine in utero and after birth (developmental nicotine exposure, DNE). Respiratory motor nerve bursting was recorded from the 4th cervical ventral root in brainstem spinal‐cord preparations studied in two experimental groups. In the first group, preparations were perfused with traditional aCSF that did not contain cotinine (Control). In a second group, 400 nM cotinine was added to the aCSF during dissection and throughout the experiment (Cotinine). The frequency and amplitude of bursts were recorded using the following protocol: a 30‐minute baseline recording where amplitude and frequency of bursts were stable, 20‐minutes of 500 nM nicotine application, and a 30‐minute washout. To assess the effects of cotinine alone, additional control experiments were performed where preparations were perfused with traditional aCSF during the baseline period, followed by application of 400 nM cotinine aCSF for 60 minutes. Results from these experiments indicate that: (1)400 nM cotinine alone does not alter burst amplitude or frequency in either control (n=6, 2‐way ANOVA: Amplitude; P=0.4487, Frequency; P=0.1020) or DNE preparations (n=6, Amplitude; P=0.1338, Frequency; P=0.1034) (2) 500 nM nicotine decreased burst amplitude and increased burst frequency in both control (n=8, for Amplitude and Frequency; P<0.0001) and DNE preparations (n=8, for Amplitude and Frequency; P<0.0001); and (3)cotinine had no effect on the nicotine‐ mediated increase in frequency in either treatment group (n=8 for Control; P=0.5105, n=8 for DNE; P=0.0762), but enhanced the nicotine‐ mediated decrease in amplitude in DNE preparations (n=8, P=0.0227) but not in control preparations (n=8, P=0.2716). We conclude that cotinine may modulate the function of specific nAChR subtypes that mediate the inhibitory effects of nicotine on respiratory motor nerve burst amplitude, and that these receptors may be functionally upregulated by DNE.
The Lasting Effects of COVID‐19 on The Progression of Non‐Alcoholic Fatty Liver Disease
Authors: Backer‐Meurke Sean L. , Khanna Deepesh
Published: May 2022

It is estimated that around 30% of the Western population has non‐alcoholic liver disease (NAFLD); a spectrum of pathology (not attributed to alcohol/substance intake) initiated by steatosis and progression towards inflammation and irreversible fibrosis (non‐alcoholic steatohepatitis, or NASH). With inflammation being a key component of the transition to NASH, it raises the question of whether the ongoing COVID‐19 pandemic, which has notoriously induced hyperinflammatory states, may influence the progression of NAFLD. Specifically, it remains unclear if potential chronic sequelae of COVID‐19 in recovered patients may increase predisposition for NASH. Since NASH maintains a high risk for hepatocellular carcinoma, liver failure, and need for liver transplant, potential additive effects of COVID‐19 could prove critical to study. Thus, the objective of this study was to conduct a literature review to examine if COVID‐19 could have chronic sequelae that affect the progression of NAFLD pathogenesis. It was hypothesized that severe cases of COVID‐19 could induce systemic inflammation, metabolic changes, and lasting gut microbiome alterations that lead to inflammatory and fibrotic changes of the liver, similar to those seen in NASH.

A scoping review of literature was conducted utilizing the PubMed database. Studies that examined hepatobiliary pathology, gut microbiome, systemic inflammation, metabolic changes, drug‐induced liver injury, and hypoxia seen in COVID‐19 were included. Human studies of adult cohorts, animal models, and in vitro experiments were included. Genetic components of NAFLD were not examined. Exclusion criteria also encompassed any studies not referencing the hepatobiliary, GIT, portal system, or systemic circulation.

Findings indicated a frequent trend of elevated liver enzymes, mild steatosis, Kupffer cell hyperplasia, and hepatobiliary congestion. It was found that direct cytopathic effects on hepatocytes was unlikely, but direct viral insult of cholangiocytes was a potential complication. High serum levels of IL‐1, TNF‐a, and MCP‐1, in COVID‐19 were found as potential risk factors for NASH development. Hypoxia, altered lipid metabolism, and iatrogenic drug‐induced liver injury were also proposed as potential precipitators of NASH development. Notably, lasting changes in gut microbiome were also frequently observed, and correlate closely with those seen in NASH.

It was concluded that future research should target the gut microbiome, hyperinflammation, and hepatobiliary congestion as integral components of potentially accelerated NASH development NAFLD‐comorbid patients following COVID‐19 recovery.
Flavin binding affinity and initial kinetic characterization of DnmZ, a flavin‐dependent N‐oxygenase
Authors: Vaca Valerie , Huynh Katherine , Hinojosa Alejandra , Villanueva Molina Delmy , Palfey Bruce , Vey Jessica
Published: May 2022

Doxorubicin is an anthracycline chemotherapeutic produced by Streptomyces peucetius. It is an effective chemotherapeutic but has severe side effects and high toxicity, and the details of its biosynthetic pathway are largely unknown. Baumycin is a derivatized form of doxorubicin with an additional seven‐carbon acetal moiety attached to the anthracycline backbone of the molecule. In this work, we report our progress on the characterization of DnmZ, a Class D flavin monooxygenase involved in formation of the baumycin acetal. DnmZ is a N‐oxygenase that oxidizes a sugar‐linked exocyclic amine, triggering a nonenzymatic retro oxime‐aldol cleavage of the hexose. We aim to characterize the kinetics of the DnmZ‐catalyzed reaction between flavin and oxygen using stopped flow spectrophotometry. Results from this research can aid in the development of derivatized anthracyclines and the understanding of the class of enzymes known as the class D flavin monooxygenases.

Our transient‐state experiments confirm that DnmZ catalyzes the formation of the C4a‐(hydro)peroxyflavin intermediate (C4a‐OOH). We have identified the appropriate wavelengths to monitor to observe C4a‐OOH formation (378nm) and elimination (450 and 480 nm). With this information we have quantitated flavin binding and determined that DnmZ’s binding affinity for flavin is approximately 13 mM, which compares well with other Class D monooxygenases. Our preliminary results suggest that increasing pH speeds up C4a‐OOH elimination but has no effect on the intermediate’s formation. We are also investigating the effect of mutating active site residues on C4a‐OOH formation and elimination.
Chronic Unpredictable Stress Impairs the Reactivity of the Pudendal Artery and Corpus Cavernosum in Male Mice
Authors: Priviero Fernanda , Arishe Olufunke , Wilczynski Stephanie , Crockett Alexia , Hollis Fiona , Webb Clinton
Published: May 2022

Erectile dysfunction (ED) can be associated with psychological disorders as well as vascular disorders. It is known that stress is associated with erectile dysfunction, however, it is not clear if psychological stressors could lead to a vascular and cavernosal dysfunction that could contribute to a vasculogenic erectile dysfunction. A recent study reported a positive correlation between hyperprolactinemia, stress and erectile dysfunction, and interestingly, prolactin was shown to decrease endothelium nitric oxide and endothelium‐ dependent vasorelaxation of aorta and coronary arteries. In this study, we aimed to evaluate the relaxation of the corpus cavernosum and pudendal artery of mice subject to chronic unpredictable stress (CUS). We hypothesize that CUS will lead to an impairment of the relaxation of the pudendal artery and corpus cavernosum leading to erectile dysfunction.

Methods

Male C57Bl/6 mice were submitted to 28 days of chronic unpredictable stress (CUS). The pudendal artery and corpus cavernosum were removed and mounted in a myograph to evaluate reactivity. Tissues were kept in physiological salt solution at 37°C, constantly aerated with 95%O2/5%CO2 and concentration‐response curves to acetylcholine (ACh; 1 nM – 30 uM)) were obtained. Using non‐linear regression, we obtained the maximal response (Emax) and potency (pEC50) of ACh. Data are expressed as mean ± S.E.M. P<0.05 was considered statistically different.

Results

Neither the potency nor the maximal response to acetylcholine was changed in the pudendal artery of stressed animals (Fig. 1A) whereas in the corpus cavernosum, there was a significant decrease in the maximal relaxation induced by acetylcholine (control: 88 ± 4% vs stress: 75 ± 4%, n=7 each group; Fig. 1B).

Conclusion

Our findings demonstrate that CUS for 4 weeks impairs acetylcholine‐induced relaxation of the corpus cavernosum. This may contribute to the development of erectile dysfunction in stressed males.
Syncytialization of BeWo trophoblasts induces changes in angiogenic signaling and response to hypoxia
Authors: Murphy Hayley A. , Lartigue Elecia M. , Chapman Heather M. , George Eric M.
Published: May 2022

During pregnancy, the placenta produces significant amounts of pro and anti‐angiogenic factors, notably VEGF, PlGF, and sFlt‐1. The exact cell population(s) most responsible for this production is not definitively known. In the event of placental insufficiency, placental ischemia can cause alterations in the production of these factors, favoring the anti‐angiogenic sFlt‐1 protein. This mechanism is believed to be a major driver of preeclampsia. Within the placenta, the maternal/fetal interface is defined by a layer of specialized syncytiotrophoblasts (ST) which line the maternal side of the underlying fetal chorionic vessels. These large multinucleated cells define the selectivity of nutrient/waste exchange from the maternal to the fetal circulation. In studying trophoblast function, immortalized trophoblast‐derived cell lines are commonly utilized, such as the BeWo choriocarcinoma cell line. These cells however, do not spontaneously syncytialize, and therefore do not likely reflect in vivo ST function. We hypothesized that inducing syncytialization of BeWo cells would alter their production of angiogenic factors in vitro. Syncytialization of BeWo cells was induced with forskolin treatment for 48 hours. At 48 hours, cellular morphology shifted from dense cobblestone patterning into majority large, multinucleated cells. Syncytialization caused ~5 fold increase in both VEGF and PlGF mRNA (p<0.0001). There were significant (p<0.0001) increases in the levels of sFlt‐1 splice variants 2 (~5 fold), 3 (~50 fold), and 4 (~3fold). As a result, there were significant (p,<0.005) ~2 fold increases in secreted VEGF and PlGF protein, while sFlt‐1 went from not detectable to 53 pg/ml as measured by ELISA. Perhaps most interestingly, when cultured in oxygen concentrations mimicking healthy and ischemic placentas (8% and 1% respectively), there was differential regulation of these factors at the transcriptional level. There was no change in VEGF, while sFlt V2 and V3 were significantly upregulated (2‐fold each, p<0.0005), while PlGF was significantly down regulated by 40% (p<0.0001). Taken together, these data suggest that syncytialization significantly alters expression of both pro and anti‐angiogenic factors in vitro. Future studies should take syncytialization state into account when extrapolating from cell culture to in vivo placental function.
PACER lncRNA Regulates COX‐2 Expression in Lung Cancer Cell Lines
Authors: Desind Samuel Z. , Iacona Joseph R. , Yu Christina Y. , Mitrofanova Antonina , Lutz Carol S.
Published: May 2022

Long noncoding RNAs (lncRNAs) are known to regulate gene expression; however, in many cases, the mechanism of this regulation is unknown. One novel lncRNA relevant to inflammation and arachidonic acid (AA) metabolism is the p50‐associated COX‐2 extragenic RNA (PACER). We focused our research on the regulation of PACER in lung cancer. While the function of PACER is not entirely understood, PACER is known to play a role in inflammation‐associated conditions. Our data suggest that PACER is critically involved in COX‐2 transcription and dysregulation in lung cancer cells.

Our analysis of The Cancer Genome Atlas (TCGA) expression data revealed that PACER expression is significantly higher in lung adenocarcinomas than normal lung tissues. Additionally, we discovered that elevated PACER expression strongly correlates with COX‐2 expression in lung adenocarcinoma patients. Specific siRNA‐mediated knockdown of PACER decreases COX‐2 expression indicating a direct relationship. Additionally, we show that PACER expression is induced upon treatment with proinflammatory cytokines to mimic inflammation. Treatment with prostaglandin E2 (PGE2) induces both PACER and COX‐2 expression, suggesting a PGE2‐mediated feedback loop. Inhibition of COX‐2 with celecoxib decreased PACER expression, confirming this self‐regulatory process. Significant overlap between the COX‐2 promotor and the PACER promotor led us to investigate their transcriptional regulatory mechanisms. Treatment with pharmacologic inhibitors of NF‐ϰB or AP‐1 showed a modest effect on both PACER and COX‐2 expression but did not eliminate expression. These data suggest that the regulation of expression of both PACER and COX‐2 is complex and intricately linked.
Alternative Approach to NADPH Oxidase Inhibition: Challenging Selectivity through Fragment Based Drug Design
Authors: Develin Angela M. , Fuglestad Brian
Published: May 2022

The regulation of cellular signaling across plasma membranes is a well‐organized process through cross‐talk of biomolecules such as proteins and lipids. Peripheral membrane proteins (PMPs) are a class of water soluble, membrane associated, proteins that play a critical role in transducing these signals. Due to the dynamic nature of their membrane interactions, relatively smooth surfaces, and lack of deep ligand binding pockets, PMPs are commonly associated with the ~85% of the human proteome that is classified as ‘undruggable’ by current drug discovery methodologies. One such PMP is p47phox that acts as an organizer/scaffold subunit essential for activation of transmembrane enzyme NADPH oxidase 2 (NOX2). The NOX2 complex is activated by translocation of the PX domain of p47phox (p47phox‐PX) through recognition of phospholipids (PIPs) localized in the plasma membrane. NOX2 is implicated in diseases such as cardiovascular, cancers, and neurodegenerative disorders, however, despite its importance, past efforts at inhibiting NOX2 activation have failed. The challenge lies in a lack of specificity among the NOX family of enzymes due to a highly conserved catalytic pocket. Inhibition of the complex prior to the activation event by targeting p47phox‐PX presents a novel alternative strategy for reviving NOX2 inhibition. Fragment‐based drug design (FBDD) was developed over the past twenty years and has already shown promise at targeting ‘undruggable’ proteins. Here, we initiate a FBDD study against p47phox‐PX. We discovered a small molecule binder, myo‐inositol‐1‐phosphate (I1P), that mimics the headgroup of p47phox‐PX’s natural PIP ligand. I1P has an ~800uM affinity towards the PX domain, as displayed by nuclear magnetic resonance spectroscopy (NMR) titrations, which will act as a scaffold for inhibitor design. Through a NMR based fragment screen, we identified 13 additional fragments that bind to p47phox‐PX. Initial characterization of fragment hits displayed a number of high uM to low mM affinities within the lipid headgroup recognition pocket, which is promising for fragment advancement towards inhibitors. Future endeavors to probe the interactions between fragments and the PX domain of p47phox will further optimize the affinity of the inhibitor to p47phox‐PX and increase selectivity against other PX domains. An array of biochemical, biophysical, and cellular assays will establish and optimize our inhibitor as a novel p47phox‐PX small molecule drug and deactivator of the NOX2 complex.
Transcriptional Role of the Cdk8 Kinase Module with Protein Synthesis Machinery Before and After Nitrogen Starvation
Authors: Friedson Brittany , Cooper Katrina
Published: May 2022

Translation is tightly coupled to growth status. Efficient protein synthesis is necessary for cell growth in nutrient rich environments, while global translation inhibition combined with selective translation of stress‐responsive mRNAs helps limit growth in times of stress. How does the cell alter protein synthesis machinery to survive in stress such as nutrient deprivation? A few mechanisms in nitrogen starvation include selective degradation of specific mRNA‐binding translation factors, inhibition of activators of genes whose products are required for general translation, and the primarily cap‐independent selective translation of mRNAs necessary for the stress response. How and when these occur, however, have remained elusive. Here, we demonstrate in Saccharomyces cerevisiae that the highly conserved Cdk8 kinase module (CKM) of the mediator complex (cyclin C, Cdk8, Med13, and Med12) transcriptionally upregulates genes encoding 60S ribosome proteins and cap‐dependent initiation factors such as eIF4G1 in physiological conditions. Yeast CKM is known to predominantly repress stress response genes (SRG), and our previous findings revealed that SRG suppression is relieved through the degradation of Med13 and cyclin C following both cell survival and death cues. Our recent data suggest that degradation of the CKM following nitrogen starvation may play a transcriptional role in altering the levels of proteins no longer needed for general translation. Additionally, while cyclin C has a secondary role of promoting mitochondrial fragmentation in the cytoplasm following oxidative stress, we have previously found that Med13 goes to the cytoplasm to be degraded via autophagy following nitrogen starvation. Our preliminary data also shows Med13 associating with proteins involved in translation regulation in nitrogen starvation, suggesting it may also have a more direct role in controlling translation. The CKM is thus a multi‐faceted hub that can provide insight to how the cell adapts to stress on both the level of transcription and translation.
Preliminary Assessment of the Combination Studies of Artesunate with Paclitaxel on Human Prostate Cancer Cell Proliferation
Authors: Fabian Juan , Pierce Taylor , Brown Shenell , Payne Gloria , Adedeji Dolapo
Published: May 2022

Prostate cancer (PC) is the most common malignant cancer and the second leading cause of cancer‐related deaths in men. Approximately 18.9 out of 100,000 men per year die of prostate cancer. Common treatments such as androgen deprivation therapy (ADT,) surgical castration, and radiation therapy are used to counter PC; however, a proportion of patients relapse within a median of 2‐3 years with castration‐resistant prostate cancer (CRPC). In this study, combined effect of artesunate (ART) with paclitaxel (PTX) on human prostate cancer cell lines – LnCaP (androgen‐ dependent) and PC‐3 (androgen‐independent) were examined. In vitro anti‐proliferative (MTT) assay was used to assess the cytotoxic effects of ART, PTX, and in combination ratios 1:1, 1:2 and 2:1 on LnCaP and PC‐3 after 72‐ hour exposure. The IC50 values of 25.1 μM and 3.98 μM for ART and PXT on PC‐3 while the IC50 values of 2.13 μM and 0.05 μM on LnCaP were observed respectively. The average IC50 values when ART was combines with PXT were 0.499 μM and 0.084 μM on PC‐3 and LnCaP respectively. Overall, this study demonstrates that combining ART with PTX at different combination ratios has more effect on LnCaP than PC‐3 ‐ low IC50. In conclusion, combining ART with PTX displayed cytotoxicity regardless of the type of prostate cancer cell line. This may offer a promising new therapeutic option for the treatment of metastatic hormone‐refractory prostate cancer, aid in reducing cytotoxicity exerted by PTX alone and low prostate cancer mortality rate in men.
Role of PTP1B in the Regulation of Cholesterol Homeostasis
Authors: Sagabala Reddy S. , Londhe Avinash D. , Marquart Krisann , Pappin Darryl , Boivin Benoit
Published: May 2022

Protein tyrosine phosphatase 1B (PTP1B), encoded by the PTPN1 gene, is a well‐known negative regulator of insulin signaling. Since insulin, via the activation of the insulin receptor, is involved in cholesterol synthesis by stimulating genes like HMG‐CoA reductase (HMGCR), we enquired whether PTP1B also played a role in the regulation of cholesterol homeostasis. Interestingly, knocking out PTP1B in hepatocytes, where most of the circulating cholesterol is synthesized, generates mice that have reduced circulating cholesterol levels. Moreover, SNPs in the human PTPN1 gene are associated with higher levels of low‐density lipoprotein cholesterol, total plasma cholesterol, and with the insulin sensitivity index and fasting glucose levels. As a first step, to better understand the function of PTP1B in cholesterol homeostasis, we used Mouse Embryonic Fibroblast (MEFs) cells derived from WT or PTP1B KO mice and measured cholesterol levels utilizing an Amplex red assay. As previously observed in liver PTP1B KO mice, we observed a ~ 50% decrease in cholesterol levels in PTP1B KO cells when compared to WT cells. Supporting this, Western blot analysis also indicated that proteins regulating cholesterol homeostasis are differently expressed in WT and KO MEFs at basal. We observed decreased expression of LDLR, PCSK9, HMGCR, SREBP1 and Cav‐1 in PTP1B KO cells. In addition, cholesterol treatment also showed a decreased adaptive response in PTP1B KO cells. Interestingly, Src, a substrate of PTP1B, was hyperphosphorylated in PTP1B KO MEFs treated with cholesterol and these cells were more sensitive to cholesterol‐induced toxicity as indicated by caspase‐3 cleavage at lower concentrations of cholesterol. Quantitative mass spectrometry analysis using isobaric tag for relative and absolute quantitation (iTRAQ) further revealed that several proteins involved in cholesterol homeostasis (e.g. NPC2, NPC1L1, DHCR7, NCEH1, ApoA, ApoB, ApoC, ApoE) were overexpressed in PTP1B KO MEFs. We also observed that proteins involved in retinoic acid homeostasis, which have also been involved in the regulation of insulin resistance and lipid homeostasis, were overexpressed in PTP1B KO MEFs. We are currently validating these findings and exploring underlying mechanisms by which PTP1B gene inactivation regulates cholesterol homeostasis in mammalian cells.
Exploring additional sleep roles for the PERIOD homolog LIN‐42 using the auxin‐inducible degradation system in Caenorhabditis elegans
Authors: Varano Arianna , Nelson Matthew D.
Published: May 2022

Sleep has been observed in all animals with a nervous system, however, the neural and molecular mechanisms that regulate it are not fully understood. The genetically tractable nematode Caenorhabditis elegans has a simple and fully mapped nervous system and has emerged as a powerful sleep model. C. eleganssleeps concurrently with each larval molt, called developmentally timed sleep (DTS), which is timed by the PERIOD(PER) homolog LIN‐42. C. elegans also sleep during adulthood in response to cellular damage incurred by stressful stimuli, called stress‐induced sleep (SIS). Based on LIN‐42’s central role in the timing of DTS, we sought to examine its role in the timing of SIS. Null mutations of lin‐42are lethal, so to study the requirement of LIN‐42 during SIS, we used CRISPR/Cas9 to insert the auxin‐induced degron and green fluorescent protein sequence in frame with each isoform of the lin‐42 gene. In the absence of auxin these worms are viable and grow successfully to adulthood. We are currently constructing transgenic strains that will allow us to degrade the LIN‐42 in different subsets of cells. To do this we are expressing the coding sequence for the auxin perceptive F‐box protein TIR1 from different tissue‐specific promoters, including those expressed in all cells, and specific sleep‐regulating neurons. Once these strains are constructed we will degrade LIN‐42 protein and measure the effects on SIS.
The Role of Ideonella sakaiensis PETase in the Degradation of PET Plastics: a Structural Comparison of the Wild Type and S238F/W159H Double Mutant
Authors: Alfieri Britney , Alfieri Madeline , Kelly Miles , Kilcoyne Siyara , Poprik Lauren , Sanyal Ananya , Smith Ally , Takkar Mehr , Variankaval Rohan , Pousont Jennifer
Published: May 2022

Polyethylene terephthalate (PET), a synthetic polymer, is one of the most commonly used plastics today and can be found in over 97% of packaging materials. PET’s durability as a plastic also makes it resistant to biodegradation. According to the National Association for PET Container Resources, only 29.1% of PET packaging waste was recycled in 2018, resulting in 27 million tons of plastic in landfills. These alarming statistics indicate a need for new technology to process discarded PET. A recently discovered bacterium, Ideonella sakaiensis 201‐F6, expresses the enzyme PETase, which binds and hydrolyzes PET. The structure of PETase closely resembles enzymes in the cutinase and lipase families. Similar to cutinases, PETase has an a/β hydrolase fold, but with a more open active‐site cleft. The catalytic residues in the active site of PETase hydrolyze PET plastics into intermediates which are then further broken down by the enzyme MHETase. Wild type PETase breaks down crystalline PET inefficiently, due to the rigid and dense structure of this plastic. In an effort to improve PETase performance, scientists made amino acid substitutions to the PETase active site. As a result, they discovered that the S238F/W159H double mutant narrowed the binding cleft, making the enzyme more active and therefore more efficient. The Pingry School MSOE Center for BioMolecular Modeling MAPS Team used 3‐D modeling and printing technology to examine structure‐function relationships of the PETase wild‐type and S238F/W159H double mutant. These models give an in‐depth view of the structure of PETase and illustrate how it can be utilized to degrade the highly prevalent PET plastic that has littered our planet. Furthermore, comparison of the wild‐type and double mutant structures provides molecular level details about structural changes that can enhance the performance of this enzyme. Gaining this perspective on PETase enzymes could lead to further advances in biotechnology solutions to the degradation of PET and related polymers.
Irisin Improves Differentiation through Preserved Mitochondrial Function in Mouse C2C12 Skeletal Muscle Cells
Authors: Slate‐Romano John J. , Yano Naohiro , Kulthinee Supaporn , Wang Lijiang , Zhang Ling Zhang , Zhao Ting
Published: May 2022

PURPOSE

In response to muscle contraction, irisin, a myokine released by skeletal muscle and white adipose tissue (WAT), is cleaved from the transmembrane protein fibronectin type III domain‐containing protein 5 (FNDC5) and has been shown to upregulate expression of uncoupling of protein 1 (UCP 1), inducing the browning of WAT. Prior research has demonstrated the protective role of irisin against insulin resistance, ischemic injury, and inflammation. We aimed to investigate the effects of Irisin on the differentiation of skeletal muscle myocytes.

METHODS

Mouse C2C12 skeletal muscle cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM) until cell confluency reached 60%. Once the plates reached 60% confluency, 2% Horse Serum was added to the medium to induce differentiation. The cells were incubated at 37 degrees C. and 5% CO2 for three days. Cells were then incubated in medium containing irisin at differing concentrations: 0 ng/mL, 10 ng/mL, and 20 ng/mL for an additional three days. Significance was established at p < 0.05. The number of myotubes were compared days three and six in each group. Immunofluorescent staining of actinin, myosin heavy chain, myocyte enhancing factor‐2C (MEF2C), and mitochondrial capture was performed for each group.

RESULTS

The average number of myotubes per field for the no‐irisin group was 8.95 (+/‐ 7.85), compared to 13.6 myotubes (+/‐ 11.02 myotubes) in the 10 ng/mL irisin group and 30.5 myotubes (+/‐ 9.92 myotubes) in the 20 ng/mL irisin group. There was no statistical significance between the no‐irisin group and the 10 ng/mL group, however the number of myotubes in the 20 ng/mL group was statistically greater than both the no‐irisin group and the 10 ng/mL irisin group at p < 0.05.

CONCLUSION

Treatment of C2C12 myocytes with irisin at 20 ng/mL concentration induced increased myotube differentiation. Irisin appears to play a significant role in muscle regeneration and growth.
The Role of Mitophagy in Quadriceps Atrophy after Non‐Invasive Anterior Cruciate Ligament Injury
Authors: Ahn Ahram , Davi Steven M. , White McKenzie S. , Lepley Lindsey K. , Kwon Oh Sung
Published: May 2022

BACKGROUND

Many patients that suffer from anterior cruciate ligament (ACL) injury have persistent quadriceps atrophy even after considerable rehabilitation. Our previous finding has revealed that mitochondrial dysfunction and redox disturbances are causal events in the initiation of muscle atrophy and central to maintenance of a healthy mitochondria is the removal of damaged mitochondria through mitophagy. However, the extent to which mitophagy play a key role in quadriceps muscle atrophy after ACL injury has yet to be explored. If mitophagy is found to play a central role in directing muscle atrophy after ACL injury than it may be an attractive therapeutic target.

PURPOSE

Using a pre‐clinical non‐invasive ACL injury model, our objective was to use a time course study to investigate the potential role of mitophagy in quadriceps muscle atrophy after ACL injury.

METHODS

48 Long Evans rats (n=8 per group; 4m/4f) underwent non‐invasive rupture of the right ACL and were euthanized at 7, 14, 28, 56 days post‐injury. 8 rats (4m/4f) served as healthy controls (HC). Mitophagy‐related cellular components of the vastus lateralis were analyzed by Western Blot analysis. One‐way ANOVAs with LSD post‐hoc were used to determine differences between groups (P < 0.05).

RESULTS

Dynamin‐related protein 1 (DRP1), a protein regulating mitochondrial fission, was increased significantly at 56 days post‐injury [HC: n=8; 1.294 ± 0.364, 56D: n=7; 2.093 ± 1.519 (A.U), P<0.05]. Lysosomes are the terminal step in mitophagy and one of the lysosomal markers, Lysosomal‐associated membrane protein 1 (LAMP‐1) expression was significantly elevated at 56 days post‐injury [HC: n=8; 1.270 ± 0.840, 56D: n=8; 2.266 ± 1.137 (A.U), P<0.05]. Upstream autophagy marker, Beclin‐1 expression was also significantly increased at 56 days post‐injury (HC: n=8; 1.117 ± 0.339, 56D: n=6; 2.158 ± 1.274, P<0.05).

CONCLUSION

Collectively, these results imply that long‐term ACL injury dysregulates mitochondrial quality control including key mitophagy markers, which contributes to ACL injury‐induced quadriceps atrophy. Therefore, targeting mitophagy may be the one of the potential therapeutic interventions to prevent muscle atrophy in patients with an ACL injury.
Virus‐induced Hepatic Expression of an Oxidized Phospholipid‐binding Antibody Fragment Prevents Initiation of Hepatic Steatosis and Progression to Fibrosis
Authors: Upchurch Clint M. , Yeudall Scott , Pavelec Caitlin M. , Manjegowda Mohan , Bochkis Irina M. , Scott Michael , Perez‐Reyes Edward , Leitinger Norbert
Published: May 2022

Oxidized phospholipids have been implicated in chronic pathologies. It had been shown that hyperlipidemic LDL‐R‐deficient mice transgenic for a single chain fragment of an oxidized phosphatidylcholine (OxPC)‐recognizing IgM E06 (scFv‐E06) were protected from developing non‐alcoholic fatty liver disease (NAFLD). To investigate the role of OxPCs in initiation of diet‐induced hepatic steatosis as well as independently in progression to fibrosis, we used an adeno‐associated virus serotype 8 to induce hepatic expression scFv‐E06 in a cre‐dependent manner (AAV8‐scFv‐E06) in B6. Speer6‐ps1Tg(Alb‐cre)21Mgn/J(Alb‐cre) mice. Virus‐induced expression of scFv‐E06 protected mice from diet‐induced hepatic steatosis by reducing liver triglyceride content and tissue damage as measured by plasma liver enzymes. Separately, expression of scFv‐E06 in mice with established hepatic steatosis prevented the progression to hepatic fibrosis. We developed a targeted, mass spectrometry‐based oxophospholipidomic platform to identify and measure OxPC species that were potentially altered by expression of scFv‐E06. We discovered that several OxPCs in the plasma were significantly reduced by scFv‐E06 expression in both steatosis and fibrosis and identified unique OxPC species that were present in both disease settings. Additionally, we show that the identified OxPC species reduced by scFv‐E06 expression dysregulate gene expression and metabolic function of hepatocytes and hepatic stellate cells in vitro. Together, we show that virus‐induced hepatic expression of scFv‐E06 is sufficient to prevent hepatic steatosis as well as intervene in progression from hepatic steatosis to hepatic fibrosis. Furthermore, we identified unique plasma OxPC species that are reduced by expression of scFv‐E06 and may drive initiation of hepatic steatosis and independently progression to hepatic fibrosis. These data demonstrate an important role for individual OxPCs in NAFLD and may provide clinically relevant biomarkers for diagnosis of hepatic steatosis and fibrosis.
Neuronal Nitric Oxide Synthase expression in brain from male spontaneously hypertensive rats. Implication in the control of cardiovascular function
Authors: Peñaloza Grecia N. , Cortes Gallardo Ernesto , Bocanegra Alfaro Ana , Gómez Ma de Jesus
Published: May 2022

Neuronal nitric oxide synthase (nNOS) in brain has been implicated in the regulation of sympathetic tone and control of cardiovascular functions, then in this study it was determined the nNOS expression and nitrites levels in brain from spontaneously hypertensive rats (SHR). Male Wistar Kyoto (WKY) and (SHR) strains at 4 months old were used, half of the experimental animals were administered with reserpine (R), (1 mg/kg of weight, for two weeks) and the others received vehicle (V) administration (dimethyl sulfoxide). Four experimental groups (WK+V, WKY+R, SH+V, SH+R) were followed and after cardiovascular parameters determination, brain was obtained. The expression of nNOS through western blot in frontal cortex‐FC, occipital cortex‐OC, cerebellum‐CB, medulla oblongata‐MO, spinal cord‐EC was determined and nitrites levels in tissue homogenate from the same brain regions were measured through Griess reaction. Heart rate (HR), systolic arterial blood pressure (SABP) and body weight, were analyzed. The results show that reserpine decrease significantly the body weight in g (WK+V 335.2±17.6 versus WK+R 280.4±9.87; SH+V 334.8±9.01 versus SH+R 269.3±8.65), the HR in beat/min (WK+V 302.2±11.6 versus WK+R 264±9.13; SH+V 354±17 versus SH+R 224±16), SABP in mmHg (WK+V: 165.8±4,964, versus WK+R 118.2±7.52; SH+V 215.4±5,074 versus SH+R 139.6±3.75), in both WKY and SHR (P<0.05 for all parameters). nNOS protein expression was high in WK+V in comparison with the SH+V, in FC and CB, whereas in the groups with reserpine administration the nNOS expression was higher in SH+R versus SH+V in FC, CB and MO. Tissue nitrites levels from different regions of the brain were reduced significantly in the SHR in comparison with WKY, particularly in FC (WK+V‐5.11 ±0.60 versus SH+V 3.53±0.53) and MO (WK+V 7.84±0.84 versus SH+V 3.24±0.48), however in the groups SHR+R a significantly increased of nitrites levels was observed in comparison with their own control, particularly in CB (SH+V 3.91±1.04 versus SH+R 7.53±0.83) and MO (SH+V 3.72±0.44 versus SH+R 5.8±0.39) (P<0.05 for all parameters). Increased nNOS expression in frontal cortex and cerebellum produces a significant decrease in the heart rate and systemic arterial blood pressure suggesting a key role of nNOS in the regulation of cardiovascular function in the SHR.
Loss of Renal Mass Exacerbates Renal Damage in a Mouse Model of High‐Renin Hypertension
Authors: Ehmke Heimo , Neuss Torben , Maurer Alva , Meyer‐Schwesinger Catherine , Wenzel Ulrich O. , Seniuk Anika
Published: May 2022

Chronic renal failure is a growing public health problem. Experimental and clinical data suggest that the loss of functional renal mass itself is a major driver for the progression of renal failure. Surprisingly, mice appear to be largely resistant to this pathogenic mechanism. In patients, chronic renal failure usually develops slowly over years, and increased arterial blood pressure and an activated renin‐angiotensin system are common comorbidities of the clinical phenotype. Hence, we formulated the hypothesis that coexistence of both changes over a prolonged period is required to induce sensitivity to a reduction in renal mass in mouse kidneys. To test this, we examined the effects of a moderate reduction in renal mass (unilateral nephrectomy) in SV‐129 renin transgenic mice, which have chronically elevated blood pressure levels subsequent to a genetically clamped high renin. The reduction in renal mass induced albuminuria and histopathological changes (glomerulosclerosis and protein casts) in renin transgenic animals, which were further enhanced when the mice were fed a high‐salt diet. In contrast, fibrosis of the myocardium and aortic wall was not increased in renin‐transgenic compared with control animals. We conclude that loss of renal mass can be an effective pathogenic factor in the development of chronic renal damage in mice. Renin transgenic mice may be a relevant model for chronic renal failure associated with high‐renin hypertension.