bluuebunny/crossref_metadata_2025 huggingface dataset:
Small Molecule Anticancer Compound Modulates Cell Cycle DNA Damage Response Pathway and Inhibit Tumorigenesis in Triple Negative Breast Cancer
Authors: Okpechi Samuel C. , Dong Shengli , Yousefi Hassan , Guidry Jessie J. , Otohinoyi David A. , Worthylake David K. , Hicks Chindo , Alahari Suresh K.
Published: May 2022

Objective

To investigate the mechanism of action and therapeutic benefits of NSC001, a small molecule anticancer compound, in the treatment of triple negative breast cancer (TNBC), a clinical subtype of breast cancer that is more aggressive, has poor prognosis. In TNBC, patients harbor tumors that lack expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (Her2).

Hypothesis

We hypothesize that NSC001 will have a beneficial therapeutic effect on TNBC cells through the regulation of specific tumor associated signaling pathways in vitro.

Material and methods

We screened 1360 compounds from the National Cancer Institute (NCI) Diversity and Mechanistic Set library using the cell counting kit‐8 (CCK‐8) reagent from Dojindo Molecular Technologies, Inc. The screening was performed by applying 10µM of each candidate drug upon plates harboring the aggressive TNBC cell line, MDA‐MB‐231. Treatment time for all screening was fixed at 24 hours. The efficacy and potential mechanism of action of one of the potent drug candidates identified (NSC001) was further investigated on three TNBC cell lines (MDA‐MB‐231, MDA231‐LM2‐4175, and MDA‐MB‐468) using mass spectrometry and proteomic analysis. Afterwards, the results from the proteomic analysis were validated using molecular methods including cell viability assays, apoptosis assays, cell cycle assays, and western blotting.

Results

We identified eight compounds that reduced the viability of MDA‐MB‐231 cells by up to 80% following a 24 hour treatment time point. Next, our in vitrodata show that NSC001 reduces the cell viability of TNBC cells in a dose‐and‐time dependent manner. Cell death data show that NSC001 induces potent apoptosis in a dose dependent manner in TNBC cells. Also, the results from colony formation assay show that 100nM [NSC001] is sufficient to inhibit the proliferative capacity of TNBC cells. Mass spectrometry and proteomic data reveals that NSC001 activates the cell cycle G2/M DNA damage checkpoint regulation in all the three TNBC cell lines used. Furthermore, we confirmed this finding in our cell cycle assay where we observed a clear G2/M cell cycle phase arrest. The arrest was potently induced at a dose significantly lower than the IC50 of NSC001. Lastly, we validated our proteomic results using western blotting, which confirmed that this low dose of NSC001 induces expression of p21, a cell cycle checkpoint regulator and tumor suppressor protein which is known to trigger cell cycle arrest in most cancer cell lines.

Conclusion

Overall, our data suggest that NSC001 may be a potent small molecule anticancer compound which should be further investigated for its potential to be used clinically in the treatment of TNBC patients.
Assembly Principles of the Human R2TP Chaperone Complex Reveal the Presence of R2T and R2P Complexes
Authors: Houry Walid A. , Seraphim Thiago V.
Published: May 2022

R2TP is a highly conserved chaperone complex formed by two AAA+ ATPases, RUVBL1 and RUVBL2, that associate with PIH1D1 and RPAP3 proteins. R2TP acts in promoting macromolecular complex formation. Here, we establish the principles of R2TP assembly. Three distinct RUVBL1/2‐based complexes are identified: R2TP, RUVBL1/2‐RPAP3 (R2T), and RUVBL1/2‐PIH1D1 (R2P). Interestingly, we find that PIH1D1 does not bind to RUVBL1/RUVBL2 in R2TP and does not function as a nucleotide exchange factor; instead, RPAP3 is found to be the central subunit coordinating R2TP architecture and linking PIH1D1 and RUVBL1/2. We also report that RPAP3 contains an intrinsically disordered N‐terminal domain mediating interactions with substrates whose sequences are primarily enriched for armadillo repeat domains and other helical‐type domains. Our work provides a clear and consistent model of R2TP complex structure and gives important insights into how a chaperone machine concerned with assembly of folded proteins into multisubunit complexes might work.
Filling in the GAPs‐ Mapping the Spatiotemporal Kinetics of Arf1 and its Regulators
Authors: Manzer Kaitlyn , Fromme J. C.
Published: May 2022

The Golgi serves as a hub for intracellular membrane trafficking. The small GTPase Arf1 coordinates several Golgi trafficking pathways by directly recruiting many effectors, such as cargo adaptors and lipid regulators, to the Golgi at precise and distinct points of cisternal maturation. To accomplish this, Arf1 distribution at the Golgi is tightly controlled by GEFs and GAPs, which activate and inactivate Arf1, respectively. Once active, Arf1 becomes stabilized on the membrane where it can then carryout its functions. Despite the significance of Arf1 Golgi localization in properly orchestrating Golgi trafficking, its maturation kinetics remain largely unknown due to its sensitivity to tagging. Furthermore, while the maturation kinetics of the Arf‐GEFs are well‐characterized, they are yet to be determined for the ArfGAPs. Here, I have utilized time‐lapse fluorescence microscopy and novel approaches for Arf1 visualization to begin to map the spatiotemporal dynamics of Arf1 and its GAPs. I have made a breakthrough in developing a method for directly visualizing the location of Arf1 in living cells that does not interfere with Arf1 function. Additionally, I have established the timing of each of the yeast Golgi‐localized ArfGAPs relative to the major Golgi trafficking pathways. Further, I am investigating the role of a highly conserved sequence in the TGN‐localized ArfGAP, Age2. This work will ultimately provide important insights into fundamental questions about Golgi trafficking and function.
A Biochemistry Course Undergraduate Research Experience (CURE) on Crithidia fasciculata parasites
Authors: Andanje Ronald , Greene Amy S.
Published: May 2022

Crithidia fasciculata is a non‐pathogenic parasite related to human‐infective parasites including Trypanosoma bruceiwhich causes African Sleeping Sickness, T. cruziwhich causes Chagas’ disease, and Leishmania spp. which causes leishmaniasis. We developed a Course Undergraduate Research Experience (CURE) on Crithidiaparasitology for a 300‐level biochemistry laboratory. Optical density at 600nm was used to study parasite cell density compared to hemocytometer counts of cells. At 27⁰C the population doubled every four hours. Carbon‐13 NMR was used to study Crithidia fasciculatametabolism of 1‐13C‐glucose. NMR peaks for the fermentation products ethanol and succinate increased over time. Competition experiments demonstrated that sucrose competed with glucose as a carbon source, fructose metabolism was less efficient than glucose, and xylose did not compete. Students also designed their own projects on Crithidia, building a toolbox of interesting stressors and successful assays to study biochemical parasitology with Crithidiaparasites.
Targeting the Contractile Vacuole of Parasites
Authors: Harley Jaquan , Reilly Mikhayla S. , Greene Amy S.
Published: May 2022

The contractile vacuole (CV) is a poorly studied organelle involved in osmotic regulation. In every contraction cycle, the CV expels excess liquid to maintain osmoregulation. CVs are found in single‐celled eukaryotes including parasitic and free‐living ciliates. Parasitic examples include Trypanosoma cruzi, amoebas, and Leishmaniaparasites. We use the free‐living ciliate Vorticella convallaria as a model for CV cycling, which can be observed directly under a microscope. The CV contraction rate in Vorticella convallaria,increases with temperature within physiologically relevant non‐lethal temperature ranges. We performed a historical literature review of CV cycling rates and temperature across ciliates and amoeba. The Arrhenius equation was used to calculate the activation energy barrier for CV contraction. The slope of the Arrhenius graph was similar across all organisms ranging from 6000 to 17000 calories (per mol CV cycle), suggesting that conserved chemical or physical processes control CV cycling across biology. Future research will investigate whether using drugs to inhibit the CV will change the slope of the Arrhenius graph in Vorticella convallaria,and whether those same drugs will exacerbate osmotic cell death in the parasite Crithidia fasciculata. Ultimately, our results may contribute to the development of anti‐parasitic drugs targeting parasites’ unique CV organelle.
Flexible design can help overcome barriers identified when implementing a Course‐based Undergraduate Research Experience (CURE)
Authors: Roberts Rebecca , Hall Bonnie , O’Handley Suzanne
Published: May 2022

Course‐based Undergraduate Research Experiences (CUREs) are recognized for their potential to increase access to research in undergraduate STEM education. CUREs have also been identified as one mechanism for increasing participation and inclusion of underrepresented students in STEM. However, the process through which faculty decide to adopt new instructional approaches is often highly individualized and is affected by personal, institutional, and other contextual factors. Many faculty experience or perceive barriers to CURE implementation at their own institution, including available facilities and equipment, fit with departmental curriculum, and hours per week students spend in the lab. Flexibility in CURE design can help overcome some of these barriers. The Biochemistry Authentic Scientific Inquiry Lab (BASIL) CURE was intentionally designed to be flexible to facilitate adoption in a wide range of settings. We present ways in which this flexibility has been and can be leveraged to overcome real and perceived implementation barriers. We also demonstrate how the curriculum can be adapted, even for extreme situations such as the pandemic necessitating the switch to emergency remote learning.
Glucose‐1‐phosphate regulates orphan nuclear receptor NR4A1 and apoptosis
Authors: Lee Young‐Sam , Shon Hyungjoo , Rockenhaus Abigail E.
Published: May 2022

Mitochondrial metabolic reprogramming is important for many different processes like apoptosis and inflammation. NR4A1 is necessary for many different pro‐apoptotic and inflammatory stimuli to induce mitochondrial metabolic reprogramming. However, despite the importance for small molecule ligands in the regulation of nuclear receptors, biological ligands regulating NR4A1 have not been identified. Here we report that glucose‐1‐phosphate (G1P), an intracellular metabolite from glycogen metabolism and glycolysis, is an NR4A1 ligand. In vitro, G1P specifically bound to NR4A1 ligand‐binding domain (NR4A1LBD) and alters its oligomerization state. In cells, a synthetic cell‐permeable G1P analog regulates transcriptional activity of NR4A1. This G1P analog suppressed mitochondrial respiration and induced apoptosis of H1299 lung adenocarcinoma cells in an NR4A1‐dependent manner. This result suggests that G1P might be an endogenous ligand regulating NR4A1 and NR4A1‐dependent mitochondrial metabolic reprogramming. It represents a rare case where a polar water‐soluble intracellular metabolite modulates a nuclear receptor function.
Changes in Oral Microbial Diversity in a Piglet Model of Traumatic Brain Injury
Authors: Jeon Julie H. , Lourenco Jeferson , Fagan Madison M. , Welch Christina B. , Sneed Sydney E. , Duberstein Kylee J. , Callaway Todd R. , West Franklin D. , Park Hea Jin
Published: May 2022

The dynamic changes in the oral microbiome in response to disease has led to significant interest in utilizing microbiome analysis as a potential diagnostic and prognostic biomarker. Oral dysbiosis has been linked to the progression of neurological diseases such as Alzheimer’s and Parkinson’s diseases, however, no studies have been conducted to evaluate microbiome changes in traumatic brain injury (TBI). This study aimed to examine the changes in the oral microbiome structure during the acute stage of TBI in a clinically translational pig model.

Landrace piglets (4‐5 weeks old, male) underwent either a controlled cortical impact surgery (TBI, n=6) or sham surgery (Sham, n=6). TBI was confirmed by magnetic resonance imaging 1 day post‐TBI. Oral swabs of the inside of upper and lower lips, both cheeks, and under the tongue were collected to sample the oral mucosa pre‐surgery and 1, 3, and 7 days post‐surgery (PS). Bacterial DNA was extracted and the V3‐V4 region of 16s rRNA gene was amplified and sequenced using an Illumina NovaSeq platform. Alpha‐, beta‐diversity, and oral microbial composition at the phylum (P), family (F), genus (G), and species (S) level were examined using Qiime2 plugins. Mixed effects ANOVA was used to compare differences between the groups and time points.

Significant time effects were identified in several alpha‐diversity indexes during the acute stage of TBI: The number of observed features, Faith’s Phylogenetic Diversity, Shannon Index, and Pielou’s Evenness. However, no group effects were found between TBI and Sham groups, except for Faith’s Phylogenetic Diversity. Faith’s Phylogenetic Diversity showed group and time‐by‐group effects including a significant difference between groups at 7 days PS. In both TBI and Sham groups, beta‐diversity was significantly different between time points and distinct microbial patterns were observed between TBI and Sham groups at 1, 3, and 7 days PS, with the most apparent difference at 7 days PS. Changes in oral microbial composition did not differ between TBI and Sham groups at any taxonomic level (P, F, G, and S), while significant time effects were found in several microbial taxa. Interestingly, significant time‐by‐group effects were found in P_Proteobacteria, F_Leptotrichiaceae, F_Micrococcaceae, and G_Rothia.

In conclusion, species diversity as measured by Faith’s Phylogenetic Diversity was lowest at 7 days PS in TBI group compared to Sham group, and TBI and Sham groups showed significant differences in beta‐diversity. With respect to individual microbial taxa, the oral microbial composition was not significantly different between the two groups during the acute stage of TBI. Future studies are needed to investigate further changes in oral microbiome during the chronic stages of TBI.
Nicotine Alters cAMP Signaling in Arterial Myocytes
Authors: Singhrao Navid , Nieves‐Cintron Madeline
Published: May 2022

Arterial myocytes receive many inputs from sympathetic nerves that regulate vascular reactivity by activating GS protein‐coupled receptors (GSPCRs). The ubiquitous second messenger 3′,5′‐ cyclic adenosine monophosphate (cAMP) is synthesized by adenylyl cyclases (ACs) in response to the activation of various GSPCRs and plays a critical role in integrating the myriad of signals elicited. Importantly, arterial myocyte cAMP signaling has been traditionally linked with relaxation. We found that arterial myocytes from mice exposed to secondhand smoke (SHS) had an attenuated cAMP synthesis in response to the β‐adrenergic receptor (βAR) agonist isoproterenol (ISO) (unpublished data), which could alter vascular reactivity. Tobacco smoke contains over 7,000 chemicals, including nicotine. Nicotine, the primary addictive component in tobacco cigarettes is a strong sympathomimetic with significant vasoactive effects and have been shown to alter cellular cAMP production. However, how nicotine impacts cAMP signaling in the vasculature, particularly in arterial myocytes, and the implications of these changes in vascular reactivity are unclear. We are tackling this knowledge gap by expressing a FRET‐based cAMP biosensor in cultured arterial myocytes to determine cAMP signaling in response to nicotine/cotinine. We also developed a mouse expressing a new cAMP sensor specifically in SM (CamperSM) to investigate cAMP dynamics in native VSM/arteries. We hypothesize that nicotine impairs receptor‐mediated cAMP signaling in arterial myocytes leading to altered vascular reactivity. Our data using unpassaged arterial myocytes expressing a cytosolic FRET‐based cAMP sensor exposed to cotinine (the primary metabolite of nicotine) show a reduction in ISOinduced cAMP production. Similar results were observed in arterial myocytes from CAMPERSM mice infused with nicotine. These results suggest similar effects of cotinine in vitro or nicotine in vivo on βAR‐mediated cAMP synthesis in arterial myocytes. Intriguingly, we found that inhibition of phosphodiesterases rescue cAMP synthesis in CAMPERSM arterial myocytes exposed to nicotine. We correlated the nicotine‐induced reduction in arterial myocytes cAMP synthesis with impaired vasodilation in response to ISO. Our data suggest that nicotine impairs receptormediated cAMP signaling in arterial myocytes leading to altered vascular reactivity. Nicotineinduced cAMP dysregulation leading to altered arterial myocytes excitability could be an underlying factor contributing to cardiovascular disease in individuals exposed to SHS and users of nicotine containing products.
Regulation of GPCR‐induced Hippo pathway signaling by ARRDC3 in breast carcinoma
Authors: Caplan Mika , Trejo JoAnn
Published: May 2022

G protein‐coupled receptors (GPCRs) are a large and diverse family of cell surface receptors implicated in cancer progression. In particular, overexpression of the GPCR protease‐activated receptor‐1 (PAR1) is associated with poor patient prognosis and metastasis of breast carcinoma. However, there remains a critical gap in knowledge of how PAR1 expression leads to breast cancer progression. Recently, the alpha‐arrestin, arrestin domain‐containing protein 3 (ARRDC3), was identified as a tumor suppressor in invasive breast carcinoma. In non‐invasive cells, ARRDC3 is essential for PAR1 trafficking and lysosomal degradation. However, ARRDC3 expression is lost in invasive breast carcinoma, resulting in dysregulated PAR1 trafficking and persistent signaling that promotes invasive breast cancer progression. Re‐expression of ARRDC3 in invasive breast carcinoma is sufficient to restore PAR1 lysosomal degradation and normal signaling, and thereby suppresses breast cancer invasion and metastasis. Moreover, ARRDC3 regulates PAR1‐stimulated Hippo pathway signaling independent of its function in regulating receptor trafficking. The Hippo pathway is driven by the activity of two transcriptional co‐activators: yes‐associated protein (YAP) and transcriptional co‐activator with PDZ‐binding motif (TAZ), and is linked to cancer progression. ARRDC3 regulates GPCR‐induced Hippo pathway signaling by binding and sequestering TAZ in the cytoplasm, thus preventing nuclear translocation and transcription of genes associated with tumor metastasis and progression. Thus, ARRDC3 exhibits multiple tumor suppressor activities to control GPCR signaling in breast cancer. However, it remains unclear how GPCR activation regulates ARRDC3 function to control Hippo pathway signaling in invasive breast cancer. Here, I will present studies that interrogate various mechanisms by which GPCRs regulate Hippo pathway signaling. The results of these studies will define fundamental mechanisms by which GPCRs promote breast cancer progression via Hippo pathway signaling and vital regulatory mechanisms of the Hippo pathway that may lead to the development of new and effective therapies.
Loss of Pkm2 Alters Myocardial Metabolism and Increases Oxidative Stress After Infarction
Authors: Lee Katie C. , Williams Allison L. , Shohet Ralph V.
Published: May 2022

Introduction

Pyruvate kinase (PKM1) directs pyruvate to the Krebs cycle for oxidative metabolism in the healthy heart. Our lab described a hypoxia‐mediated switch to the alternatively spliced isoform PKM2, enhancing pyruvate to lactate conversion. Recently, we have also found that Pkm2 knockout (KO) mice had profound depletion of basal glucose in the heart compared to control mice. Pkm2 has also been shown to reduce oxidative damage and promote cardiomyocyte cell proliferation after myocardial infarction (MI). We hypothesize that upregulation of PKM2 can alter metabolic pathways by promoting glycolysis, and that after injury, this can preserve ATP production, protecting the heart from the stresses of hypoxia and injury.

Methods

Global Pkm2 KO mice were subjected to permanent ligation of the left anterior descending coronary artery to mimic an MI. RNA‐seq analysis of left ventricles from control (n=8) and Pkm2 KO mice (n=8) before and 3 days after sham or MI surgery was performed. Semi‐quantitative real‐time PCR was used to confirm changes in selected genes of interest.

Results

Loss of Pkm2 moderately altered gene expression at baseline (q<0.05, FDR<0.05). Notably, the mitochondrial gene COX3 was downregulated in Pkm2 KO hearts. 68 genes were differentially expressed in Pkm2 KO hearts after MI, not observed in control MI hearts. MI of Pkm2 KO hearts resulted in considerable reduction of transcripts of enzymes in the insulin signaling pathway, mitochondrial oxidative phosphorylation, mitochondrial uncoupling, fatty acid metabolism, and increase in transcripts encoding enzymes in the pentose phosphate pathway, response to oxidative stress, and apoptotic signaling. Semi‐quantitative PCR of selected genes involved in glucose metabolism confirmed RNA‐seq results.

Conclusions

RNA‐seq analysis of Pkm2 KO hearts demonstrated that loss of Pkm2 altered gene expression of metabolic and mitochondrial enzymes. Pkm2 KO hearts also showed increased abundance of pro‐apoptotic markers which may be a result of increased oxidative stress.
AI/ML‐driven simulation of the skin retention of opioid analgesics following topical administration in normal skin and cutaneous lesions
Authors: Khotimchenko Maksim , Antontsev Victor , Chakravarty Kaushik , Hou Hypatia , Varshney Jyotika
Published: May 2022

Topical drug administration in the treatment of skin disorders is known to have high efficacy with a favorable safety profile in clinical settings. Disposition of the active pharmaceutical ingredient (API) in the skin layers plays a critical role for the dose regimen calculator required for desired therapy efficiency. Experimental measurements of the API skin concentration are complicated and inaccurate and can be replaced with physiologically based pharmacokinetic modeling predicting local and systemic drug exposure. Opioid analgesics are commonly used in treating severe pain and it is frequently associated with putative adverse effects. Topical applications of opioids were shown to have high efficacy with a favorable safety profile in clinical settings. Clinical investigations of transdermal drug formulations, especially in patients with skin lesions, are often hindered due to the patient intolerance, high cost of the studies, and ethical aspects.

BIOiSIM, a hybrid artificial intelligence computational platform integrating machine learning optimization with mechanistic modeling was implemented to accurately simulate in vivo plasma concentration profile and predict partition coefficients and API skin retention for two opioid analgesics in healthy skin and simulated skin lesions. Publicly available drug parameters and physiological data were used for the model building.

The platform successfully predicted API systemic exposure and skin concentration following the epicutaneous application of central opioid agonist buprenorphine and peripheral opioid agonist oxycodone in healthy human skin and simulated subjects impaired skin. Experimentally observed plasma concentration for both drugs demonstrated validity of the in‐silico methods. The results indicated that buprenorphine permeates rapidly through the stratum corneum and partitions into the dermis, where its concentration slowly declines as the reservoir in the compound vehicle is depleted. Thus, the impact of lesions is not as significant. In contrast, the hydrophilic oxycodone, where the rate‐limiting stratum corneum layer prevents a significant portion of the compound from permeating deeper in healthy patients. Hence, concentration of oxycodone sharply declines over the first hours of exposure in deeper indicating poorer accumulation capacity in the skin in the presence of lesions.

An AI/ML‐driven PBPK model was applied for accurate simulation skin concentrations of two opioid analgesics – buprenorphine and oxycodone. The results have confirmed that both these drugs have good skin retention and can stimulate opioid receptors located in skin. The results suggest that buprenorphine showing substantial skin retention capacity can be applied topically for the treatment of skin disorders in in patients.
Comprehensive analysis of NFAT5 associated gene expression in the renal collecting duct
Authors: Edemir Bayram , Petrillo Federica , Chernyakov Dmitry , Fenton Robert
Published: May 2022

Background

The renal cortico‐medullary osmotic gradient is a crucial factor for the urine concentration mechanism and is mainly generated by sodium chloride and urea. Due to this, the cells in the renal inner medulla are challenged with a hypertonic environment. The adaptation of these cells is mediated by the action of the tonicity‐responsive enhancer‐binding factor (TonEBP, also known as nuclear factor of activated T cells 5, NFAT5). NFAT5 induces the expression of genes that lead to accumulation of organic osmolytes within the cells. The cortico‐medullary osmotic gradient also induces a specific spatial gene expression pattern within the kidneys. So far, a systematic analysis of the contribution of NFAT5 on gene expression in the kidneys was missing, which was accomplished here.

Methods

We performed next generation RNA sequencing (NGS) using NFAT5 deficient and wild type control primary cultivated inner medullary collecting duct (IMCD) cells. As a second approach, we used renal cortex and renal inner medulla from principal cell specific NFAT5 deficient mice and control mice for NGS analysis. Differentially expressed genes were analyzed by pathway, functional and gene enrichment analysis.

Results

In primary IMCD cells hyperosmolality induced massive changes in gene expression. Aquaproin‐2 (Aqp2) was one of the transcripts with the highest level of induction. Loss of NFAT5 abolished hyperosmolality‐induced expression of Aqp2. Most of the hyperosmolality induced transcripts had the highest expression in the inner medulla. Loss of NFAT5 affected the expression of more than 3,000 genes in renal cortex and more than 5,000 genes in the inner medulla, compared to control kidneys. Again, loss of NFAT5 was associated with decreased expression of Aqp2. We observed increased expression of kidney injury marker genes, like lipocalin‐2 or kidney injury molecule 1. Gene enrichment analysis also indicated that loss of NFAT5 is associated with a renal inflammation‐like phenotype.

Conclusion

This is the first study addressing the role of NFAT5 on hypertonicty‐induced changes in gene expression in the kidney. NFAT5 is the master regulator of osmolality induced Aqp2 gene expression and in vivoloss of function induces a kidney injury‐like phenotype through currently unknown mechanisms.
Molecular Basis for Antiviral Action of EDP‐235: A Potent and Selective SARS‐CoV‐2 3CLpro Inhibitor for the Treatment of Covid 19
Authors: Balakrishnan Anand , Reyes Archie , Shen Ruichao , Bisht Nalini , Sweeney Joyce , Levene Rachel , McAllister Nicole , Cressey Tessa , Manalo Nathan , Rhodin Michael H. , Vaine Michael , Wang Guoqiang , Or Yat Sun , Goodwin Bryan
Published: May 2022

To date, there are no approved oral antiviral therapies that can be administered early in the course of COVID‐19 to suppress progression of the disease or for prophylaxis. EDP‐235 is a potent and selective inhibitor of SARS‐CoV‐2 3C‐like protease (3CLpro). EDP‐235 inhibits SARS‐CoV‐2 3CLpro protease activity with an IC50 of 5.8 ± 3.7 nM and retains its activity against variant 3CLpro proteins from multiple SARS‐CoV‐2 lineages (IC50range of 2.8­–5.8 nM). 3CLpro protease activity progress curves showed significant curvature in a time‐ and EDP‐235‐concentration‐dependent manner indicative of slow‐onset inhibition. Slow reversal of inhibition of SARS‐CoV‐2 3CLpro enzyme activity was observed in a jump dilution experiment. Michaelis‐Menten kinetic studies with a FRET peptide substrate in the presence of EDP‐235 indicated that EDP‐235 is a substrate‐competitive inhibitor of SARS‐CoV‐2 3CLpro with an overall dissociation constant Kiof 3.0 ± 1.6 nM. SARS‐CoV‐2 3CLpro was crystallized bound to a close analog of EDP‐235 and structure elucidation revealed that the ligand bound at the active site and interacted with side chains of conserved residues Cys‐145, His‐163, and Glu‐166. EDP‐235 also potently inhibits 3CLpro enzymes from other α‐coronaviruses (IC50range of 2–4 nM) and β‐coronaviruses (SARS‐CoV IC50 of 5.4 nM, MERS‐CoV IC50 of 70 nM) which cause disease in humans to date. EDP‐235 resistance mutations in HCoV‐229E map to the active site of 3CLpro close to the predicted binding site and offer additional support to the mechanism of inhibition. EDP‐235 also showed a favorable selectivity profile (>300 selectivity index) when tested against a panel of 30 mammalian proteases. In summary, EDP‐235 acts as a slow‐onset, slow‐reversible, substrate‐competitive inhibitor of SARS‐CoV‐2 3CLpro. The outstanding preclinical profile of EDP‐235 supports its further evaluation as an oral therapeutic for the management of COVID‐19.
Guanine Nucleotide‐Dependent Conformational Selection Regulates Distinct Alternate Ribosome Bound States of the Translation Factor BipA
Authors: Montovano Giancarlo , Buckley Theresa S. , Sharawy Mahmoud , May Eric R. , Anand Ganesh S. , Robinson Victoria L.
Published: May 2022

BipA is a conserved translational GTPase necessary for securing bacterial survival and successful invasion of the host. Structural and biochemical studies indicate that GTP and ppGpp compete for binding to BipA to promote differential association of BipA to either the 70S or 30S ribosomal species. Exactly how guanine nucleotide binding to BipA prompts a change in the association of this protein with the ribosome is not understood. Crystallographic models show local structural rearrangements occur near the nucleotide‐binding pocket but unexpectedly the overall domain arrangement, and therefore intramolecular contacts are similar in the various guanine nucleotide bound states. One explanation is that the lattice contacts in the crystal restrict the conformational space available to the protein. Another is that the GTP‐ and ppGpp‐bound forms of BipA are dynamic meta‐stable or intermediate states poised to bind the ribosome. This model would account for a reverse flow of information evidenced by the increase in BipA’s rate of GTP hydrolysis upon ribosome binding.

BipA’s hydrodynamic radius as measured by analytical ultracentrifugation and heat capacity changes assessed by isothermal titration calorimetry experimentally support our hypothesis that limited domain movements occur in solution upon guanine nucleotide binding. MD simulations utilizing the conformational end‐states provided by our GTP‐ and ppGpp‐bound crystal structures, coupled to a description of the solution dynamics of BipA using amide hydrogen/deuterium exchange mass spectrometry (HDX‐MS) indicate that this protein exists in an ensemble of states in solution as a metastable entity. The mutually exclusive binding of GTP or ppGpp produce unique allosteric relays within the protein directing the equilibria to alternate distinct conformations resulting in the formation of the two BipA:ribosome complexes. This intrinsic conformational fluidity is the origin of BipA’s ability to respond to ever changing environmental conditions synchronizing them with cellular physiology facilitating adaptation and long‐term survival of the bacteria.
Flavonoids Ameliorate Ferroptotic Cell Death in Cardiomyocytes through ROS Scavenging and Lipid Peroxide Reduction
Authors: Zhang Kai‐Cheng , Liu Chin‐Hung , Yang Kun‐Ta
Published: May 2022

Myocardial ischemia‐reperfusion injury (MIRI) is a lethal disease that occurs after percutaneous intervention which may lead acute myocardial infarction patients to heart failure. Therefore, it is an important issue to find solutions for MIRI. A newly discovered cell death named ferroptosis has been associated with MIRI. Ferroptosis, which is characterized by iron‐dependent accumulation of lipid peroxide is distinct from necrosis and apoptosis. Lipophilic antioxidants like ferrostatin‐1 have been discovered to inhibit ferroptosis effectively. Flavanoids, such as Fisetin and Kaempferol (KMP), are natural compounds that are found in many edible plants. They are lipophilic antioxidants that show cardio‐protective effects through scavenging ROS, inhibiting inflammation in MIRI models. However, the effect of flavonoids in inhibiting ferroptosis associated with MIRI remains unclear. This study investigated the ability of Fisetin and KMP to attenuate ferroptosis in H9c2 cells. H9c2 cells were treated with ferroptosis inducers erastin, RSL3, and Fe‐SP. Fisetin and KMP could protect cells against ferroptosis caused by these ferroptosis inducers by ROS scavenging, lipid peroxide reduction, and decrease the mRNA levels of the ferroptosis marker Ptgs2. Thus, Fisetin and KMP play a protective role in H9c2 cells against ferroptosis which may be an effective candidate to treat MIRI.
Myoglobin knockout mice experience less damage and more immune infiltration in muscle pressure injury
Authors: Nasir Nurul Jannah M. , Heemskerk Johannes A. , So Peter T. , Tucker‐Kellogg Lisa
Published: May 2022

Introduction

Muscle pressure ulcers are slow to heal and classified as “chronic” wounds, but the reasons for slow healing are not well understood. Elevated levels of extracellular hemoglobin have been implicated in the pathogenesis and impaired healing of sickle cell and vascular ulcers. We question whether extracellular release of myoglobin after muscle pressure injury might contribute to a hostile wound environment, analogous to hemoglobin. We hypothesise that knockout of myoglobin will decrease tissue damage and improve the microenvironment of muscle pressure ulcers.

Methods

We developed germline Myoglobin knockout (Mb–/–) mice via CRISPR deletion. Similar to previous studies, our adult Mb–/– mice show no phenotypic differences to Mb‐wildtype (Mb+/+) mice. Pressure injuries were created in mice by applying a pair of 12 mm magnets to the dorsal skinfold and panniculus carnosus muscle, in two intervals of 12 hours. We compared pressure ulcers in elderly 20‐month‐old mice carrying Mb–/– or Mb+/+. Ex vivo wound tissues were analysed at day 3 after injury.

Results

Mb‐wildtype mice had high levels of ferric iron in the wound localized to the extracellular space and infiltrating cells (presumably macrophages). In knockout mice, iron was undetectable. The knockout mice had smaller pressure ulcers post‐injury and 50% less tissue damage at the wound centre (p < 0.01), compared to control Mb+/+ mice. In addition, the wounds of Mb–/– mice show a marked decrease in protein nitration (2.7‐fold decrease in nitrotyrosine levels; p < 0.0001) compared to Mb+/+ mice. Furthermore, Mb‐knockout wounds exhibited an almost 21‐fold decrease in DNA oxidation, measured by oxidised guanine (8‐oxoguanine; p < 0.0001) compared to Myoglobin+/+. We found 3.5‐fold greater infiltration of immune cells (p < 0.01) and higher CCL7 chemokine expression (p < 0.01) in Mb‐knockout tissues than Mb‐wildtype. Furthermore, Mb–/– wounds displayed a four‐fold decrease in citrullinated histone 3 (p < 0.0001), a marker of extracellular traps, compared to Mb+/+.

Conclusion

In the absence of myoglobin, pressure‐injured tissues displayed less oxidative and nitrosative damage. Interestingly, they also showed higher immune infiltration and decreased extracellular traps, meaning that increased immune infiltration co‐occurred with decreased oxidative stress. We conclude that extracellular myoglobin contributes to oxidative stress and immune dysfunction in muscle pressure ulcers, and its removal causes less tissue death. This has important implications for the use of iron chelation therapy in the treatment of muscle pressure ulcers.
Computational Study on the Electrostatic Interactions between Uracil‐DNA Glycosylase (UDG) and DNA
Authors: Xie Yixin , Li Lin
Published: May 2022

In DNA repair mechanism, Uracil‐DNA glycosylase (UDG) is one of the most important enzymes that are involved in the process of base excision repair (BER), the function of UDG is to repair the uracil‐induced DNA lesion by removing uracil from damaged DNA. Uracil in DNA may occur due to the cytosine deamination or deoxy uridine monophosphate (dUMP) residues misincorporation during DNA synthesis. Medical evidences haven shown that an abnormal expression of UDG is related to different types of cancer, including colorectal cancer, lung cancer, liver cancer, etc. Therefore, the research of UDG is crucial in cancer treatment and prevention, as well as other clinical activities. Here we applied multiple computational methods to study the UDG in several perspectives: Understand the stability of UDG enzyme in different pH conditions; study the differences in charge distribution between the pocket side and non‐pocket side of UDG; analyze the field line distribution at the interfacial area between UDG and DNA; perform electrostatic binding forces analyses of special region of UDG (pocket area) and target DNA base (uracil); as well as investigate the charged residues on UDG binding pocket and binding interface. Our results show that it is the whole UDG binding interface rather than the UDG binding pocket area alone who provides the binding attractive force to a damaged DNA at uracil base.
Effect of Alternative Therapeutic Exercise on the Older Adults – A Pilot Study
Authors: Sadacharan Chakravarthy M.
Published: May 2022

Introduction

Regular physical activity is essential for sedentary older adults (aged 65 years and above) who are at the risk for multiple comorbidities. By performing physical activity, older adults can maintain or improve their physical function and mobility that may help them to be independent for a long time and delay the occurrence of disability in the future.

Objective

To assess the effect of the eccentric exercises prior to tai chi and yoga performance in the activities of daily living (ADL), balance and gait, and muscle strength in older adults.

Methods

Nine sedentary independent older adults aged between 60 and 90 were recruited. The study included the medical pre‐protocol assessment, the eccentric (ECC) exercise protocol with and without tai chi and yoga, subjective (Health activities of daily living (ADL) difficulty scale), and objective (Tinetti balance and gait, muscle strength) assessments, and medical post‐protocol assessment.

Results

The functional limitations, Tinetti balance and gait score, manual muscle strength testing, and active range of motion (AROM) were improved in the medical post‐protocol assessment. Muscle strength showed statistically significant differences on the shoulder abductors (P=0.035), the elbow extensors (P=0.046), the hip abductors (P=0.028) and the hip extensors (P=0.03).

Conclusions

Our findings show low perceived exercises (ECC) prior to tai chi and yoga training are well suited to the sedentary independent older adults with chronic conditions.
Internal and External Iliac Arterial Variations in a Single Human Cadaver: A Case Study
Authors: Teran Chavez Morgan E. , Lesciotto Kate
Published: May 2022

Introduction & Objective

The common iliac arteries are the main source of blood supply to the pelvis, perineum, and gluteal region via the internal iliac arteries (IIAs) and lower limbs via the external iliac arteries (EIAs). While variation in the branching patterns of the IIA and EIA is common, previously documented studies only focus on one type of variation. The objective of this study was to document the combination of multiple IIA and EIA variations found in one human cadaver. During herniorrhaphies and other pelvic surgeries, surgeons must enter the retropubic space. This space often has significant variation in collateral circulation, making vasculature in the area susceptible to injury, especially those that cross or reside near the superior pubic ramus. Knowledge of variant IIA and EIA branching patterns will assist surgeons and interventional radiologists in modifying their approach to procedures involving the pelvic area and lower limb (e.g., hernia repairs, fixation of pelvic fractures, etc.), thereby improving patient morbidity and mortality.

Methods

Detailed dissections of the IIA and EIA and their respective branches were performed in a single human cadaver following notation of bilaterally aberrant obturator arteries. All arterial variations were documented through notes, diagrams, and photography. An in‐depth literature review was performed to evaluate the significance and frequency of the observed variations.

Results

The left lower extremity had the following variations: absence of an inferior gluteal artery, an aberrant path of the superior gluteal artery, the inferior epigastric artery sharing a common trunk with the obturator artery from the profunda femoris artery, and anomalous branching of the femoral artery consisting of three larger branches, one noted to be the medial circumflex artery and the other two noted to be unusually large muscular branches. The right lower extremity had a similar aberrant obturator artery, although the common trunk shared with the inferior epigastric artery arose from the EIA.

Conclusion/Significance

Additional arterial variations of the EIAs and IIAs were found upon further dissection differing bilaterally. It is essential for these variations to be documented along with the many others discovered previously. Lack of knowledge about arterial variation can lead to complications, such as femoral head osteonecrosis or significant hemorrhage during herniorrhaphy, fixation of pelvic fractures, and other pelvic/lower limb procedures. Therefore, knowledge of possible arterial variations of the human body can improve patient morbidity and mortality during routine surgical procedures, emergent surgical procedures, and various injuries in patients with vascular anomalies such as those documented in this study.
Novel SARS‐CoV‐2 Inactivation Formulation Supports Extended COVID‐19 Testing/Surveillance
Authors: Yu Tian , Holt Stewart , Davis Ryan , Baker Tony
Published: May 2022

Background

With the spread of highly infectious strains such as the Delta variant of SARS‐CoV‐2, rapid antigen testing and variant tracking are gaining more attention from infectious disease specialists and the general public as essentials for disease control and prevention amidst the COVID‐19 pandemic. The lateral flow based antigen tests provide fast turnaround of results within minutes, but it’s open ‐faced assay format means that the test operators are more prone to accidental exposure to pathogen‐containing samples. The viral inactivation transport media (ITMs) provide much needed protection against exposure to highly transmissive live viruses during sample collection, transport, and storage. However, the current ITMs were designed for PCR tests and share a harsh chemical formulation that denatures protein analytes, making them incompatible with antigen tests.

We developed an innovative viral transport media that is designed for a variety of tests, such as antigen testing and PCR. The new formulation can be used to safely inactivate SARS‐CoV‐2 virus while maintaining compatibility with many different formats beyond rt‐PCR.

Design

To evaluate the viral inactivation performance of the novel inactivation transport media formulation, a cytopathic effect assay was conducted using VERO E6 cells in presence of SARS‐CoV‐2 samples incubated in our novel formulation and log reduction were reported.

To assess its compatibility with different SARS‐CoV‐2 related assays, the novel formulation was used as inactivation transport media in antigen, PCR, and NGS based Swab‐Seq tests with known controls.

Results

The results for the inactivation study confirmed > 3.0 log reduction in viral titer after 30 min exposure in novel transport media formulation and demonstrated 99.5% effectiveness in SARS‐CoV‐2 inactivation. The results for the PCR and antigen test compatibility studies showed comparable and/or superior performance in detection limits for the novel formulation when compared to other sample media. The Swab‐Seq result offers preliminary support on the novel formulation’s compatibility with NGS based assays.

Conclusion

Using a novel viral transport medium, we were able to completely inactivate the SARS‐CoV‐2 virus. The new formulation was compatible with both nucleic acid and protein assays while protecting cells from infection. This viral transport media could allow more test types to be done from a single specimen, resulting in safer and reliable outcomes. Additionally, it would reduce the need for multiple samples collected from patients while supporting SARS‐CoV‐2 variant tracking via NGS.
Autophagic activity in response to nanoparticle exposure of A549 cells
Authors: Sipos Arnold , Kim Kwang‐Jin , Crandall Edward D.
Published: May 2022

We have shown previously that lung adenocarcinoma (A549) cells internalize polystyrene nanoparticles (PNP). Once intracellular, PNP stimulate autophagy in A549 cells and over time are delivered to lysosomes, with subsequent egress from the cells by lysosomal exocytosis. We also found evidence that A549 cell exposure to either PNP or ambient air pollution ultrafine particles (UFP) leads to lysosomal dysfunction. In the current study, we assessed if there is a potential limit to autophagic capacity in A549 cells. In the presence of chloroquine (40 μM, 1 hr), A549 cells were exposed to a fluorescent marker, DAPRed (Dojindo; Rockville, MD), that labels autophagosomes and autolysosomes. Autophagic activity was assessed based on the fluorescence intensity of DAPRed, which was measured in serial z sections over the entire cell volume in live, single cells using confocal laser scanning microscopy. Single exposure (50 nM Rapamycin as positive control, 80 μg/mL of 20 nm carboxylated PNP, or 1 μg/mL of UFP (<180 nm, collected in Los Angeles)) and double exposure (Rapamycin + PNP or Rapamycin + UFP) models were utilized to determine the kinetics of autophagic activity over 24 hr. Data on autophagic activity were also obtained by measurement of fluorescence intensities using LC3‐GFP‐RFP in response to PNP/UFP exposures. LC3‐GFP‐RFP was transduced (700:1 virus particle per cell ratio; Thermo Fisher Scientific; Waltham, MA) into A549 cells and expression of LC3‐GFP‐RFP was assessed using confocal microscopy. We found that autophagic activity remained relatively constant in unexposed A549 cells (negative control) as opposed to a rapid increase (>3 times negative control) with a peak at ~3‐5 hr post exposure in Rapamycin‐exposed cells. Both PNP and UFP exposure resulted in gradual elevations in autophagic activity, reaching a steady state (at ~2.5 times negative control) at ~8‐10 hr post exposure with no appreciable change from ~10 to 24 hr. Combining Rapamycin with PNP or UFP caused a rapid rise in autophagic activity (peaking <5 hr post exposure); however, no difference in steady state autophagic capacity (~2.5 times negative control) was observed in comparison to either PNP or UFP exposure over 24 hr. Similar time courses of elevated autophagic activity were observed by increased fluorescence of LC3‐GFP‐RFP in response to 24 hr exposure to Rapamycin, PNP or UFP. In addition, higher expression of LC3‐RFP indicated higher levels of autophagosome fusion with autolysosomes (increased autophagic flux). These data suggest that environmental stressors might exert their harmful effects, at least in part, by limiting available autophagic capacity, thereby making nanoparticle‐exposed cells more susceptible to secondary injury.