bluuebunny/crossref_metadata_2025 huggingface dataset:
Signaling Pathways Associated with miR‐106b mediated Radioresistance in the Treatment of Colorectal Cancer
Authors: Rubio‐Guevara Susana , Olascuaga‐Castillo Karyn , Cáceres‐Andonaire Elena , Altamirano‐Sarmiento Dan , Caballero‐Aquiño Olga , Mantilla‐Rodríguez Elena , Hilario‐Vargas Julio , Berezovski Maxim , Morgado‐Díaz José Andrés
Published: May 2022

Tumor radioresistance, or the lack of control of certain tumors with this treatment, can result in locoregional recurrence; therefore, there is great interest in understanding the underlying biology and developing strategies to overcome this problem. MicroRNAs (miRNAs, miRs) are small non‐coding RNAs that regulate gene expression at the post‐transcriptional level and participate in cancer invasion, progression, metastasis, and therapeutic resistance. Emerging evidence indicates that miRNAs play a critical role in modulating key cellular pathways that mediate response to radiation, influencing radiosensitivity of cancer cells through interaction with other biological processes such as cell cycle checkpoints, apoptosis, autophagy, epithelialmesenchymal transition, and cancer stem cells. Today, most studies on patient data report different, results on the miRNAs evaluated for each tumor type, highlighting miR‐106b whose overexpression can determine radioresistance both in vitro and in vivo by inhibiting apoptosis and promotion of cell proliferation. The objective of this study was to find the signaling pathways involved with miR‐106b‐mediated radioresistance.

Methods

CRC gene expression data sets were collected from the public database, The Cancer Genome Atlas (TCGA). In addition, web‐based tools were used to explore TCGA data, specifically that developed by the Memorial Sloan Kettering Cancer Center (MSK) cBioPortal for Cancer Genomics. The public site cBioPortal is hosted at the MSK Molecular Oncology Center, in which the term “colorectal cancer” was searched and 12 studies were selected, creating a single combined study which has 4341 patients and 4488 samples, within which the search for miR‐106b was carried out and the signaling pathways involved with the expression mediated by this miRNA were obtained.

Results

The following signaling pathways involved with miR‐106b were obtained: WNT, TP53, TGF‐Beta, RTK‐RAS, PI3K, NRF2, NOTCH, MYC, HIPPO, and its influence on the cell cycle was also noted.

Conclusion

miRNAs have been shown to play an important role in the regulation of CRC radio resistance, by controlling various signaling pathways, including cell cycle, proliferation, apoptosis, and DNA damage repair. Furthermore, these results are consistent with recent data that have shown that selective modulation of miRNA activity can improve the response to radiotherapy, providing an innovative antitumor approach based on miRNA‐related gene therapy. Therefore, miRNAs could also serve as targets for the development of new therapeutic strategies to overcome radiation resistance in CRC.
Anticancer and chemosensitization effects of Wee‐1 kinase inhibitor Adavosertib (MK‐1775; AZD‐1775) in breast cancer cell lines
Authors: Nieves Aviles Nicole M. , Díaz‐Quiñones Adriana O. , Salgado Iris K. , Silva Walter , Maldonado Hector
Published: May 2022

Current breast cancer classification included cells that express estrogen (ER) and progesterone (PR) receptors, those with ERBB2 (HER‐2/Neu) amplification, and those without expression of ER, PR, or amplification of ERBB2 (referred to as triple‐negative or basal‐like breast cancer). Triple negative breast cancers (TNBC) are generally more difficult to treat, are known to be more aggressive, and with poor prognosis. Several molecular mechanisms have been identified as potential targets for the development of drugs to treat breast cancer including cell cycle checkpoints kinases. The G2/M cell‐cycle protein tyrosine kinase, Wee‐1, plays a crucial role in the checkpoint arrest for DNA repair before mitotic entry. Under normal conditions, cells repair damaged DNA during G1 arrest; however, cancer cells often have a deficient G1/S (p53) checkpoint and depend on a functional G2/M checkpoint for DNA repair. Adavosertib is a well‐characterized Wee‐1 kinase inhibitor with significant evidence supporting its potential use as an anti‐cancer and/or chemosensitizer drug. Working hypothesis proposed for this project was that use of Wee‐1 kinase inhibitors in breast cancer cells can result in profound and significant anticancer and chemosensitization effects. In order to test this hypothesis, we performed a series of dose‐response experiments using a well‐known DNA‐Damaging agent (DNA‐dt: Cisplatin) alone and/or in combination with Adavosertib. Cytotoxic effects of these treatments were evaluated with the MDA‐MB‐231 (TNBC cells) and the non‐tumorigenic MCF10A, as our in‐vitro cell model systems, and the 384‐well plate/96 hours/resazurin (AlamarBlue) cell proliferation assay. Analysis of these results included determination of half‐maximal inhibitory or effective concentration (IC50 or EC50) for each drug treatment and/or combination; and the nature of these interactions (synergy, additivity, antagonism) was determined with the open‐access software CompuSyn, Combenefit, and SynergyFinder. Analysis of the potential for interactions of cisplatin and Adavosertib in MDA‐MB‐231 indicated significant synergism as illustrated by surface and matrix synergy plots, combination index determinations, and isobolograms. However, no significant synergistic interactions were observed when these two drugs are combined in the non‐tumorigenic MCF10A cells. Finally, studies designed to evaluate the possibility that these synergistic effects can also be extended to the levels of expression of several biochemical markers (including pCDC2 and γH2AX) and cell cycle duration are currently underway. These preliminary results are consistent with a potential role of Wee‐1 kinase inhibitors like Adavosertib as anticancer and/or chemosensitizer agents against triple‐negative or basal‐like breast cancer cells.
Pedagogical Lessons Learned During the Time of COVID‐19
Authors: Ortiz Phillip , Dehner Carolyn
Published: May 2022

As COVID‐19 swept the globe colleges and universities opted to suspend face‐to‐face lectures and laboratories as a step to slow the spread of this disease. Rather than cancel their courses instructors worked to deliver content and assess learning via distance learning and online pedagogies. Since this shift occurred mid‐semester and unexpectedly, instructors and curriculum designers needed to make the transition quickly and smoothly. The urgency of the situation was not a call for lowering standards or changing learning outcomes. Rather, it was an opportunity to revisit our courses with a focus on the critical learning outcomes.

During their course transitions many instructors discovered that creating coursework for an online setting is not a transcriptional process, in which only the medium by which the content delivery differed, but a translational one in which they needed to use an entirely different set of tools. For example, while creating video lecture captures or transcribing the text of the lectures may be the quickest way to make the transition, in the long‐term instructors may have used readings from a variety of sources; relatively brief written, audio or video “mini‐lectures”; different textbooks, open educational resources, and other content sources; and importantly, incorporated frequent assessments with rapid feedback.

Delivering laboratory content via online modalities can be especially challenging. Moving it online may have been an opportunity to reconfigure an experience that often requires that students focus much of their efforts on data collection, to one that emphasizes hypothesis formulation, experimental design, and data analysis. It is possible that when this crisis passes some of the insights gained in moving courses online will be useful in enhancing learning in traditional settings.

To help educators transition to online teaching, the leadership of Biochemistry and Molecular Biology Education (BAMBEd) compiled a freely accessible virtual issue that may serve as useful resources for BMB educators who are converting their face‐to‐face courses to the online modality. That content may be accessed at: http://IUBMB.onlinelibrary.Wiley.com/doi/toc/10.1002/(ISSN)1539‐3429.Teaching‐COVID‐19

That virtual collection also marked BAMBEd's commitment to continue to expand this knowledge base and to make new resources available to the community of educators quickly. To that end, we welcomed brief rapid communications from educators on creative and innovative approaches. In addition, we also welcomed submissions by the leadership of professional organizations as they make statements of educational priorities and offer guidance to the field.
S426/S430 phosphorylation accounts for Beta‐arrestin 2‐mediated desensitization for cannabinoid sensitivity and tolerance in mutant mice
Authors: Piscura Mary , Henderson‐Redmond Angela , Sepulveda Diana , Morgan Daniel
Published: May 2022

Desensitization of G protein‐coupled receptors, such as the cannabinoid receptor 1 (CB1R), is a key mechanism involved in the in the development of tolerance. CB1R desensitization is mediated via phosphorylation of residues S426 and S430 by a G protein‐coupled receptor kinase (GRK). Following GRK‐mediated phosphorylation at these residues, beta‐arrestin 2 (bArr2) is recruited to facilitate desensitization of the receptor. Previous work demonstrated that tolerance for cannabinoid drugs was reduced in S426A/S430A mutant mice and in bArr2‐KO mice. Since there are other phosphorylatable serine and threonine residues in the C‐terminal tail of CB1R, it was not clear whether bArr2 recruitment to S426 and S430 accounts for all bArr2‐mediated desensitization. To address this question, we assessed cannabinoid acute response and daily tolerance in S426A/S430A x bArr2‐KO double mutant mice. Both S426A/S430A and bArr2‐KO single mutant mice display increased sensitivity and delayed tolerance to the antinociceptive effects of delta‐9‐tetrahydrocannabinol (Δ9‐THC) and the synthetic cannabinoid, CP55,940. Double mutant mice did not differ from the S426A/S430A single mutant model in respect to antinociception, suggesting that the effect of complete bArr2 deletion did not enhance the effect of the S426A/S430A point mutants. However, the hypothermic effects of acute Δ9‐THC dosing were increased in male double mutant mice, relative to S426A/S430A and bArr2 single mutant mice. These results indicate that phosphorylation of S426 and S430 are likely responsible for bArr2‐mediated desensitization, with the exception of hypothermia induced by acute Δ9‐THC dosing. Therefore, these results also suggest sensitivity to cannabinoid‐induced hypothermia is agonist‐ and duration‐dependent.
Unraveling the Roles of Mitochondria and Peroxisomes in Lipid Droplet Utilization in Tetrahymena thermophila
Authors: Krueger Vivienne , Ransom Daaé , Williams Abbie , Carson Emily , Ahn Byunghyun , Kandl Kim , Listenberger Laura
Published: May 2022

There is growing recognition of the lipid droplet as a dynamic hub for intracellular metabolic pathways. However, the cellular mechanisms that facilitate lipid droplet utilization remain unclear. In an effort to unravel these mechanisms, our experiments examine lipid droplets in the single‐celled ciliate Tetrahymena thermophila, an evolutionarily divergent organism with a rich history of experimentation. Like other organisms, Tetrahymenaaccumulate lipid droplets in response to starvation. Within 3 hours of starving Tetrahymena in 10mM Tris buffer (pH 7.4), lipid droplet size and number increase, remain elevated for an additional 72 hours, and then decline. We hypothesize that starved Tetrahymena mobilize lipids in lipid droplets for oxidation in the mitochondria or peroxisomes. Pulse chase experiments using a fluorescent fatty acid analogue (red C12) demonstrate lipid trafficking to lipid droplets in starved Tetrahymena. Further experiments show modest co‐localization of the red C12 fatty acid analogue with fluorescent markers of mitochondria, and at later time points, the peroxisomes. Chlorpromazine, an inhibitor of peroxisomal fatty acid metabolism, significantly decreased the viability and metabolic activity of starved Tetrahymena,as measured by cell density, trypan blue staining, and MTT reduction. On the other hand, inhibition of fatty acid import into mitochondria with the drug etomoxir elicited smaller decreases in viability and metabolic activity. Together, our experiments suggest that lipid mobilization in lipid droplets and fatty acid oxidation in peroxisomes and mitochondria contribute to cell survival in nutrient‐scarce environments.
Alcoholic fatty liver is blunted by rFGF21 administration in mice lacking adipose FGFR1: the role of FGF21 in PPARα‐mediated regulation of adipose tissue mass
Authors: Xu Yunhui , Lu Yongke
Published: May 2022

Fibroblast growth factor 21 (FGF21) regulates lipid metabolism via fibroblast growth factor receptor 1 (FGFR1). FGF21 protects against alcoholic fatty liver, which is still observed in mice lacking liver FGFR1, suggesting the involvement of extra‐hepatic FGFR1 in mediating alcoholic fatty liver. We have previously shown that, in a chronic Lieber‐DeCarli model, the peroxisome proliferator‐activated receptor‐α (PPARα) agonist WY‐14,643 induced FGF21 and adipose atrophy but failed to protect against alcoholic fatty liver in mice lacking adipose FGFR1. In this study we tested the role of the FGF21 in regulation of adipose tissue mass and ethanol induced‐hepatic fat (triglyceride, TG) accumulation in mice lacking adipose FGFR1. In binge alcohol model, male adipose FGFR1 knockout mice (fgfr1adipoQ‐cre) and corresponding normal control (fgfr1fl/fl) mice were fed liquid control diet with or without 20 mg/L WY‐14,643 for 2 weeks followed by a binge ethanol gavage at 5 g/kg, adipose tissue mass and serum free fatty acid were decreased by WY‐14,643 in the fgfr1adipoQ‐cremice but not in the fgfr1fl/flmice. However, in contrast to the chronic model, the binge ethanol‐induced liver TG accumulation was blunted by WY‐14,643 to a greater extent in the fgfr1adipoQ‐cremice than in the fgfr1fl/flmice. Consistently, on top of WY‐14,643 feeding, circulating FGF21 was elevated by binge ethanol to a greater extent in the fgfr1adipoQ‐cre mice than in the fgfr1fl/fl mice. To test the involvement of the FGF21 in adipose atrophy and the less extent of hepatic fat accumulation in the fgfr1adipoQ‐cremice, recombinant mouse FGF21 (rFGF21) was injected intraperitoneally at 0.4 mg/kg to the 3‐week ethanol fed fgfr1adipoQ‐cre mice and fgfr1fl/fl mice during the last 10 days of the ethanol feeding. Consistent with FGFR1‐dependent effects of WY‐14,643 on adipose atrophy and liver fat accumulation, on the top of chronic ethanol diet, rFGF21 administration induced adipose atrophy, blunted liver TG accumulation and serum TG elevation to a greater extent in the fgfr1adipoQ‐cre mice than in the fgfr1fl/fl mice. These results indicated the consistency of adipose FGFR1 dependent effect of WY‐14,643 and FGF21 in PPARα‐mediated regulation of adipose tissue mass and fat mobilization from adipose tissues to the liver.
Assessing the Effectiveness of flowcharts as a teaching and learning tool
Authors: King Ethan , Zimmermann Anthony E. , Bose Diptiman
Published: May 2022

The Integrated Cardiovascular Acute Care disease module in the second professional year at Western New England University, College of Pharmacy and Health Sciences, introduces disease states' pharmacotherapeutics such as coronary artery disease, shock, and arrhythmias. To improve learner understanding and application of this material, we introduced the use of flowcharts in the module. Flowcharts are skeletal outlines of key concepts that help summarize acute cardiovascular diseases by combining pharmacological concepts with evidence‐based pharmacotherapeutic decision‐making. This study aims to evaluate the effectiveness of flowcharts as an instructional tool in the therapeutics module.

A skeletal outline of the flowcharts was provided to the students on the College’s learning management site. The instructors completed the charts using a doc‐cam system during class at the end of each disease state. A pre‐ and post‐survey was administered to evaluate the effectiveness of these flowcharts. The study was initiated in Spring 2019 and data was collected until Spring 2021.

A pre‐survey showed that 83% (n=45) agreed that flowchart use would improve their knowledge; the post‐survey showed that 91% (n=60) agreed that flowcharts improved their overall knowledge in the course. While 90% (n=60) agreed that flowcharts improved their understanding of pharmacology, 95% of the surveyed participants agreed that flowcharts helped them understand the role of drugs in pharmacotherapy. Furthermore, 96% (n=60) of the participants agreed that they would like to see more use of flowcharts in the other pharmacotherapeutics modules. A topic‐wise comparison of the average scores demonstrated significant differences in the mean±SD score on summative assessments for the classes that received flowcharts versus those that did not use flowcharts.

In order to gain insight into how the students use flowcharts for learning, we also conducted focus‐group interviews of the students using predesigned questions. A total of 16 students were interviewed in groups of 4‐5. Thematic coding of the interview transcripts is currently being performed. However, preliminary 1st and 2nd round coding identified emerging themes such as “visually useful,” “summarizes material,” “helps retain and memorize”, “helps in exam” etc.

The results from our study indicate that the use of flowcharts improved content delivery and application in an acute care cardiovascular disease pharmacotherapeutics module. The flowcharts provide the students with a decision‐making framework to make informed therapeutic decisions. We believe that the use of these flowcharts benefit student learning and understanding and can be used in other disease state modules.
An Undergraduate Introduction to Physiology Course as an Opportunity to Address Diversity, Equity and Inclusion in Biomedical Science
Authors: Rhinehart Erin
Published: May 2022

If you asked someone to conjure an image in their mind of a scientist, most people envision someone who roughly resembles Albert Einstein. This is largely because we are constantly bombarded with consistent, stereotyped images about what type of person “does science”. In most science courses and textbooks, the people given credit for major scientific advancements are typically white males. In fact, if you search the internet for lists of the most influential or famous scientists, the top ones listed are almost exclusively white males. Whether or not this lack of diverse representations of scientists registers consciously, the impact of this messaging about the types of people who work in science is clear and consistent. Therefore, it is important that STEM faculty recognize the impact of this messaging and develop mechanisms to combat it, especially considering the increasingly diverse array of postsecondary students interested in STEM majors. The first opportunity to combat this dominant narrative should occur on the first day of class by focusing on diversity and inclusion statements included in the syllabus in conjunction with the collaborative development of agreed upon social norms and expectations. In addition, assignments that allow students to connect with and recognize the contributions of non‐stereotypical scientists help to provide concrete examples that are likely to be effective at countering the dominant narrative that the quintessential scientist is a white male. Therefore, I added a Physiologist biography assignment to my undergraduate Introduction to Physiology course with the objective of providing multiple examples throughout the course of the semester that counter scientist stereotypes. For the assignment, students selected a scientist to research from a curated list that included only women and minority physiologists. The assignment required that they write a paper and give an oral presentation to the class about the scientist’s personal life, career trajectory and scientific contributions along with a discussion about how the student related to the scientist personally. To assess the impact of the assignment on student notions of scientist stereotypes and overall scientist relatability, we conducted a pre‐ and post‐course survey, which indicated a significant shift in student biases of scientists being predominantly white and male. Focus group assessment revealed themes indicating that the assignment also improved student identification with people in science‐related careers. Therefore, this pedagogical intervention appears to be beneficial as a step toward creating a more inclusive STEM educational environment.
Upper airways dysmorphology and obstructive sleeping apnea in Down syndrome
Authors: Martínez Abadías Neus , Echeverry Luis Miguel , Maréchal Laura , Giménez Sandra M. , Sevillano Xavier M. , Fortea Juan M. , Heuzé Yann
Published: May 2022

Down syndrome (DS) is a complex genetic disorder caused by trisomy of chromosome 21 that alters the development of multiple systems, including craniofacial dysmorphologies. Mandibular hypoplasia, macroglossia and midfacial flatness are associated with higher prevalence of malocclusion and mouth breathing in DS. In this study, we assessed the morphology of the upper airways and tested whether alterations in the size and shape of the upper airways were associated with Obstructive Sleep Apnea (OSA), which compromises the quality of life in individuals with DS.

To quantify differences in volume and shape of the upper airways, we obtained 3D reconstructions from head magnetic resonances scans in a cross‐sectional sample of 246 adult individuals, including 159 control euploid individuals and 87 individuals diagnosed with DS. Polysomnography tests were available in a subsample of individuals with DS, and AHI Index scores representing the number of apnea and hypopnea events per hour of sleep were used to indicate the severity of sleep apnea.

Geometric Morphometric analyses based on the 3D coordinates of 15 anatomical landmarks located on the surface of the upper airways showed significant shape differences between DS and control individuals (P‐value=0.001), involving a retraction of the whole upper airways structure and a change in the angular inclination of the oropharynx region in individuals with DS. Volumetric analyses confirmed a significant 40% reduction in total upper airway volume in DS (P‐value<0.0001). Multivariate regression analysis showed that AHI scores explained 10.1% of upper airways morphological variation in DS, with a significant positive correlation between the shape and volume of the upper airways and the severity of OSA. Our study suggests that morphological biomarkers assessed from non‐invasive magnetic resonance images can be useful in diagnosing OSA, a sleep disturbance with high prevalence in DS that if treated correctly, can improve the quality of life of people with DS and prevent the appearance of early dementia.
A Foundation for the Neural Control of Viral Fever
Authors: Madden Christopher J. , Mota Clarissa M.
Published: May 2022

Fever, an acute‐phase neurally‐regulated increase in body temperature, is thought to be an adaptive response to optimize host defenses against invading pathogens. However, fever can be an unpleasant symptom of infection and excessive fever (>41oC) can cause irreversible protein denaturation and result in tissue damage, seizure, coma and death. Common therapeutic approaches to fever include the use of non‐steroidal anti‐inflammatory drugs and cyclooxygenase inhibitors, however these approaches are ineffective for reducing prostaglandin‐independent fevers, can interfere with adaptive and innate immune defenses and are contraindicated for use in some viral infections, including SARS‐CoV2. Although research over the past couple of decades has defined the fundamental central nervous system (CNS) pathways necessary for the lipopolysaccharide model of bacterial fever, the CNS circuits mediating viral fevers are almost completely unknown. Double‐stranded RNA is an intermediary in viral replication and serves as a pathogen‐associated molecular pattern that elicits the acute phase responses to viral infection. Systemic administration of polyinosinic:polycytidylic acid (poly I:C), a synthetic double stranded RNA, mimics the acute phase responses to viral infection. Neurons in the rostral raphe pallidus area (rRPa) and the dorsomedial hypothalamus play important roles in the control of the sympathetic outflow to thermoeffectors, therefore we hypothesized that neurons in these areas would be essential for fever elicited by a model of viral fever, intravenous (iv) poly I:C. Female and male Sprague Dawley rats anesthetized with urethane and α‐chloralose (for BAT sympathetic nerve recording, n=3) or isoflurane (for cutaneous vasoconstriction experiments, n=3) received iv poly I:C (1mg/kg for experiments with sympathetic nerve recording, or 500μg/kg for experiments for cutaneous vasoconstriction). Systemically administered poly I:C increased BAT SNA (+601 ± 161% baseline), BAT temperature (TBAT; +3.1 ± 0.8°C), and expired CO2 (+0.9 ± 0.2%). Poly I:C also elicited CVC, as observed by reductions in paw or tail skin temperature (TPAW/TTail) (‐2.6 ± 1.2°C). A nanoinjection of the ionotropic glutamate receptor antagonists, AP5 and CNQX in the rRPa reversed poly I:C‐evoked increases in BAT SNA (‐596 ± 128% baseline), TBAT (‐1.3 ± 0.2°C), and expired CO2 (‐0.5 ± 0.1%). Nanoinjection of AP5 and CNQX into the DMH reversed the polyI:C‐evoked reduction in TPAW/TTail (+2.8 ± 1.1°C). These data suggest that the activity of neurons in the rRPa and the DMH are necessary for poly I:C‐induced activation of BAT thermogenesis and cutaneous vasoconstriction. These data define critical components of the neural circuits for poly I:C‐induced fever, highlight differences between the neural circuitry mediating bacterial fever and viral fever, and provide a foundation for elucidating additional brain mechanisms responsible for viral fever.
Effects of Microglia APOE Expression on Tau Pathology
Authors: Baker Samantha K. , Wang Na , Bu Guojun , Liu Chia‐Chen
Published: May 2022

Apolipoprotein E (APOE) is a lipid transporter involved in neural cholesterol metabolism. Highly expressed in astrocytes and microglia, APOE4, one of the three isoforms of the transporter, is a strong genetic risk factor for Alzheimer’s disease, while APOE3 is considered moderate risk and APOE2 protective. Despite recent advances in Alzheimer’s disease research, the exact mechanism by which APOE4 influences the progression of the disease has yet to be determined. One hallmark pathological feature of Alzheimer’s disease is the accumulation of hyperphosphorylated tau protein in the brain. To investigate the role of APOE isoform expression in the accumulation of hyperphosphorylated tau protein, a microglia‐specific cre‐ recombinase‐mediated APOE inducible mouse model was used. Phosphorylated‐tau accumulation, microglia activation, and neurodegeneration of the hippocampus and piriform cortex upon specific expression of APOE3 were quantified. APOE3 expressing mice had exacerbated neurodegeneration and tau accumulation in the hippocampus. Additionally, it appeared that APOE3 expressing mice had increased microglial activation. Thus, APOE expression may be involved in the activation of microglia in Alzheimer’s disease, eliciting an overactive immune response that accelerates disease progression.
Gold Nanoparticles as a Drug Delivery Vehicle System Against ErbB2+ Breast Cancer
Authors: Kouneski Samantha
Published: May 2022

Breast cancer is the most common and second leading cause of cancer related death among women. Currently, chemotherapy treatments involving heat shock protein 90 (Hsp90) inhibitors (17‐AAG) are not FDA approved due to their lack of clinical efficacy. Hsp90 inhibitors decrease binding and stabilization of clients that aid in tumor formation and growth. ErbB2, a Hsp90 client, is overexpressed in ErbB2+ breast cancers and promotes tumor progression. Cul5 is downregulated in ErbB2+ breast cancer cells making them resistant to chemotherapy treatments involving Hsp90 inhibitors. Cul5 is required for degradation of ErbB2 in response to Hsp90 inhibitors. Hsp90 inhibitors are not efficacious alone and require Cul5 for effective antitumor response. I have synthesized gold nanoparticles coated with anti‐ErbB2 antibody, 17‐AAG, and Cul5 DNA as an intracellular drug delivery mechanism. This targeted drug delivery vehicle has been evaluated in a cell culture model of drug resistance. We have constructed a breast cancer cell line in the past that is resistant to the HSP90 inhibitor 17‐AAG and we used this model system to evaluate Cul5 expression. Our hypothesis is that gold nanoparticles coated with Cul5 DNA, 17‐AAG, and anti‐ErbB2 antibodies will increase the sensitivity and specificity of Cul5 deficient ErbB2+ breast cancer cells to 17‐AAG. This has been achieved through comparing payload efficacy of differently sized gold nanoparticles and evaluating targeting of anti‐ErbB2 antibodies to ErbB2+ breast cancer cells. Specifically targeting and making breast cancer cells susceptible to Hsp90 inhibitors will be a milestone in improving therapeutic treatment for those diagnosed.
Nandrolone Use During Adolescence Increases Cocaine Sensitization And Impairs Reproductive Function In Adult Female Rats
Authors: Rivero Carlos J. , Freire Jaime A. , Siaca Paula , Santiago Ivan , Segarra Annabell C.
Published: May 2022

Anabolic androgenic steroids (AAS) are drugs that mimic the chemical structure of testosterone. The most commonly used AAS by young adults is nandrolone. Use of AAS has been associated with increased risk of steroid dependence, mood disorders and polysubstance use. Indeed, cocaine use among AAS users is approximately 33% compared to 4.9% in the rest of the population. The effect of nandrolone (ND) use during adolescence on female motivational behaviors and on the reproductive system has not been investigated. Adolescent female rats were divided into two groups, one that received nandrolone decanoate (20mg/kg/sc) and another that received sesame oil (vehicle) for 10 successive days starting on day 28. On day 38 rats were tested on an Open Field and on day 39 in an Elevated Plus Maze. On day 40, the rats were then divided into 2 additional groups for a total of 4 groups: Oil‐Saline, ND‐Saline, Oil‐Cocaine and ND‐Cocaine and tested for locomotor sensitization to cocaine. From days 40‐44 and at days 52 and 62 rats received an injection of cocaine (15 mg/kg/ip) or saline. Their locomotor response to cocaine was measured at days 40, 44, 52 and 62. Our data show that nandrolone treated rats spent more time in the center of the open field, have no preference between open and closed arms of the plus maze and show augmented locomotor response to cocaine on days 5 and 13 compared to oil treated rats. This suggests that nandrolone increases risk taking behavior and the psychoactive properties of cocaine while having no effect on anxiety. In addition, nandrolone reduced ovarian weight and increased the formation of ovarian cysts. In contrast, in the uterus, it increased uterine weight and hindered endometrial growth. These data show that exposure to supra‐physiological levels of androgens during adolescence induces neurochemical changes in brain circuitry that regulate emotions and increases the psychoactive properties of cocaine. Nandrolone also severely impairs reproductive function, inducing formation of ovarian cysts and altering uterine morphology, an effect that is exacerbated when paired with cocaine.
Effect of Transporter Genotype on the Rate of Caffeine Absorption
Authors: Gjeci Iliana , Hegel Justin , Millard Julie T.
Published: May 2022

Caffeine is the most widely used psychoactive drug in the world, with the majority of the world’s population consuming it on a daily basis. Individuals respond very differently to caffeine, with both environmental factors and genetic variation of proteins involved in metabolism, mode of action, and absorption believed to modulate caffeine’s effects. For example, a single nucleotide polymorphism (rs762551) of the gene for the liver enzyme CYP1A2 has been reported to influence caffeine outcomes, with “slow” metabolizers having reduced benefits of caffeine during exercise and increased risk of heart attack. Our working hypothesis is that single nucleotide polymorphisms in the nucleoside transporter protein ENT1, encoded by SLC29A1, may play a role in caffeine absorption, a topic that has widespread medical significance. In this study, human subjects were genotyped at the SLC29A1 locus for SNP rs760370, previously suggested to modulate the efficacy of nucleoside analogue therapy. Subjects were also phenotyped via ELISA to determine how fast they actually absorb caffeine. We have observed significant variability in tmax (time to maximum concentration) among our subjects and are assessing a potential relationship between tmaxand a subject’s genotype to determine whether some genotypes correlate with faster rates of caffeine absorption. Overall, this work may contribute to a better understanding of the variable effects of caffeine consumption, as well as inform the timing of caffeine dosing for those seeking to enhance alertness and/or performance.
Transcriptome profile of white, brown and beige adipose tissue
Authors: Paz Henry , Zhong Ying , Pilkington Anna‐Claire , Shankar Kartik , Wankhade Umesh
Published: May 2022

Adipose tissue dysregulation lies at the core metabolic syndrome. White adipose tissue (WAT) has the capacity to store energy, whereas brown adipose tissue (BAT) can dissipate energy through thermogenesis. Newly discovered beige adipocytes are brown like adipocytes recruited in WAT in response to b3‐adrenergic stimulation (CL316,243) or cold temperatures. Despite the important metabolic role, information regarding differential expression of molecular markers between different adipose tissue, especially beige and other depots is scarce. The objective of this study was to identify the shared and unique transcript signatures that characterize WAT, BAT and beige adipose tissue. We collected inguinal WAT (iWAT), gonadal WAT (gWAT) and BAT from male C57BL6/J mice fed a control diet (17% fat) for 13 weeks. Beige WAT (iWATCL) was isolated from mice treated with b3‐adrenergic agonist CL316, 243 for 7 days (1 mg/kg BW, ip). RNA sequencing was used to generate transcriptional profiles. Using gWAT as control, differentially expressed genes (DEGs) were determined with the DEseq2 and considered when the false discovery rate corrected P‐value was ≤ 0.05 and the fold change ≥ 4. Principal components analysis revealed that the transcriptional profiles differed across BAT, gWAT and iWAT and iWATCL. Across adipose tissues, 208 DEGs were shared. Between BAT and iWATCL, 223 DEGs were shared and were associated with muscle related pathways. Unique DEGs were 1130, 549, and 40 for BAT, iWAT, and iWATCL, respectively. Top upregulated unique genes included Lncbate10, Zic1, and Gm32468 in BAT; Dsc3, Cd22 and H2‐M2 in iWAT; and Cacng5, Mup11 and Mup9 in iWATCL. Among other processes, genes upregulated in BAT were associated with cellular respiration and fatty acid biosynthesis, in iWAT with immune responses and in iWATCL with sodium ion homeostasis and cellular response to lipids. Overall results show transcriptional heterogeneity across adipose tissues depots, especially the unique set of transcripts in CL316, 243 induced beige adipocytes that can serve specific markers.
Investigating the Protective Antibody‐Circumsporozoite Complex of Plasmodium falciparum and its Role in Vaccine Development
Authors: Dobelhoff Katie , Dobelhoff Ben , Fraley James , Suder Karen C. , Replogle Jessica B.
Published: May 2022

Malaria is a parasitic disease associated with high fevers, shaking chills, and flu‐like symptoms. It is typically spread by mosquitoes and caused by infection with Plasmodium falciparum (Pf). Due to its high transmission rate and metastatic properties, malaria is a major public health concern. Insecticide resistance has led to alternative methods of disease management, such as vaccine development. The circumsporozoite protein of Plasmodium falciparum malaria is the foundation for many malaria vaccines, which are designed to block the transmission of Pf. The most promising vaccine candidate is RTS,S, a recombinant protein designed around the central NANP repeat and C‐terminal region of circumsporozoite protein. The repeat region consists of 19 repeating NANP amino acid sequences, allowing multiple antibodies to bind simultaneously. Molecular dynamics simulations and crystallography structures show the importance of the NANP amino acid sequences for binding. The Summit Country Day School MSOE Center for Biomolecular Modeling MAPS Team used 3D modeling and printing technology to examine the structure‐function relationship of the human Fab688 circumsporozoite complex and its associated binding of NANP repeat peptides to protective antibodies. Antibodies that bind to the fully extended peptide are perpendicular to the heavy chain‐light chain interface and rely on the first NANP repeat that follows the only NPDP sequence in the junctional region of Pf circumsporozoite protein, as this arrangement favors free energy contributions and results in superior protection. Junctional peptide antibody protection may relate to the ability to cross‐react with the NANP repeat region. Amino acids Asn11 and Asn13 on the junctional peptide form a hydrogen bond network with Fab668. This web of hydrogen bonds, which connects the residues that form the binding pocket, stabilizes it and contributes to stronger binding.

Fab688 binds to the NANP repeat region with equal or better affinity, shedding light on the affinity and specificity for the junctional region of circumsporozoite. This research lays the framework for future advancements in vaccines by revealing important targets and thereby limiting the spread of infectious diseases.
Identification of alveolar proteins associated with claudin‐18 using BioID
Authors: Ibrahim Yasmin , Lynn K. S. , Smith Prestina , Reed Ryan C. , Koval Michael
Published: May 2022

Tight junctions are protein complexes that form at sites of cell contact that function to regulate paracellular flow of small molecules, water, and ions. Distinct protein components contribute to the barrier function of tight junctions including the claudin family of transmembrane proteins and scaffolding proteins such as Zonula Occludens‐1(ZO‐1). Changes in tight junction morphology from the established smooth cobblestone patterning to a spiked junction are associated with an increase in barrier permeability (leak). Tight junction spike formation is associated with changes in claudin‐18 interacting partners, including ZO‐1. This suggests that protein‐claudin‐18 interactions are essential for tight junction morphology and therefore function. Using rat alveolar epithelial cells (rAECs), we used BioID to investigate the proximal claudin‐18 proteome. BioID uses a promiscuous biotinylating enzyme, BirA, which we fused to claudin‐18 to biotinylate proximal and interacting proteins. To determine that BirA‐claudin‐18 localized to tight junctions, rAECs were transduced with adenovirus to express BirA‐claudin‐18 and stained with claudin‐18 and streptavidin‐Cy3. This showed that the assay was functional and our chimera trafficked to tight junctions and colocalized to proximal proteins. To identify biotinylated proteins, we used tandem mass spectrometry (MS/MS) and identified 3771 protein matches within the BirA‐claudin‐18 sample. Proteins of interest were vetted, and a list of protein candidates were compiled based on peptide spectrum matches (PSMs) and known protein localization. Proteins of interest include retinoic acid and palladin, which are of specific interest because they are associated with a spike like structure found in the testis, the tubulobulbar complex, which we hypothesize behaves as a signaling platform in the testis and is comparable to tight junction spikes found in the lung. These findings show that BioID is a valid method for identification of proteins to claudin‐18, providing a discovery‐based approach to identify novel proteins that play a role in barrier function and tight junction regulation.
Metabolism of the Tyrosine Kinase Inhibitor Masitinib In Vitro
Authors: Latham Bethany , Jackson Klarissa , Vergne Matthew J.
Published: May 2022

Masitinib is an orally bioavailable inhibitor of protein kinases such as c‐kit, Lyn, Fyn and MCSFR‐1. These pathways play a key role in cell survival and proliferation of mast cells and microglia. Due to the selectivity of masitinib for these pathways, this drug is under investigation for treating neurodegenerative disorders, inflammatory disorders, as well as pancreatic and prostate cancer. Hepatotoxicity has been observed in some patients taking masitinib during clinical trials and is thought to arise from reactive metabolites formed by cytochrome P450 (CYP) enzymes in the liver. The purpose of this study was to elucidate the metabolism and bioactivation pathways of masitinib using in vitro phenotyping methods to identify the enzymes involved in masitinib metabolic activation. Masitinib (2 μM) was incubated with pooled human liver microsomes in the presence and absence of NADPH. Potassium cyanide was added to trap reactive metabolites as cyano adducts. Metabolites were analyzed using reversed‐phase liquid chromatography ‐ tandem mass spectrometry (LC‐MS/MS). To determine which enzymes are involved in forming masitinib metabolites and reactive metabolites, masitinib was incubated with human liver microsomes in the presence of P450‐selective reversible inhibitors as well as time‐dependent inhibitors. To validate the inhibitor panel findings, masitinib was also incubated with recombinant P450 enzymes. From these assays we found that the primary metabolite, N‐desmethyl masitinib, was primarily formed by CYP2C8 and CYP3A4, with contributions from CYP2D6 and CYP3A5. Other primary metabolites produced by monooxygenation of masitinib, M515a, M515b, and M515d, were formed by CYP3A4 and CYP3A5, while M515c was formed by CYP3A4 with minor contribution from CYP2D6. We also found that reactive metabolite cyano adducts MCN510 and MCN538 were formed by CYP3A4 and CYP3A5. MCN524 and MCN526 were generated by CYP3A4 with contributions from CYP2D6, CYP2C8, and CYP3A5. By elucidating these pathways, we gain further insight into the routes that generate reactive and potentially toxic metabolites of masitinib. Given the polymorphic nature of the enzymes involved, future studies are needed to determine the impact of CYP genetic polymorphisms on masitinib metabolism. This information could ultimately provide evidence to inform precision prescribing for masitinib to ensure safety and efficacy for future patients.
Nematode hexosaminidases – a new biochemical tool for glycan analysis
Authors: Dutkiewicz Zuzanna , Yan Shi , Vanbeselaere Jorick , Paschinger Katharina , B.H. Wilson Iain
Published: May 2022

Hexosaminidases are ubiquitous enzymes with multiple roles in glycoconjugate metabolism as they remove non‐reducing terminal β ‐N‐acetylhexosamine residues from glycans, glycolipids, glycoproteins and glycosaminoglycans. Amongst many functions in non‐invertebrates, hexosaminidases are involved in maturation of N‐glycans.

Previous studies of Caenorhabditis elegans Hexosaminidases (Gutternigg, 2007; Dragosits, 2014) prove that this organism possesses five GH20 family genes: four belong to the subfamily 1: HEX‐2; ‐3; ‐4 and ‐5, one to subfamily 2: HEX‐1. C. elegans HEX‐4 was recently shown to remove terminal GalNAc even from structures in which the underlying GlcNAc was substituted with phosphorylcholine. Now, we have begun to characterize hexosaminidase orthologues from pig parasitic nematodes, specifically Oesophagostomum dentatum, Trichuris suis and Trichinella spiralis. Bioinformatics analyses indicate that T. suis and T. spiralis have only two GH20 genes, one orthologous to HEX‐1 and one paralogous to the HEX‐2‐5, whereas the genome of O. dentatum appears to contain one orthologue each of HEX‐1‐5. Phylogenetic analyses are, however, complicated by incorrect database annotations in most nematode species.

In terms of in vitro characterisation, the subfamily 1 GH20 enzyme from T. suis was expressed in yeast and observed to favor pNP‐β‐GalNAc with a pH optimum of 6.5; however, it can remove a single GlcNAc from the a1,3‐mannose of the N‐glycan substrate GnGn‐PA, as well as both GalNAc residues from a biantennary LacdiNAc‐containing structure. Thus, this enzyme appears to have the properties akin to both HEX‐2 and HEX‐4 from C. elegans. Homology‐based 3D‐structure prediction of hexosaminidases from C. elegans and T. suis showed no significant differences in the substrate binding pocket.

In summary, nematode hexosaminidases have distinct substrate specificities and different numbers of homologues per species exist. These nematodes enzymes are not only interesting due to their roles in glycan biosynthesis; due to their specificities, they are useful as a complement to mass‐based glycomic workflows.
MOTS‐c protein expression increases following aerobic exercise training and remains elevated during detraining
Authors: Pullman Beau R. , Hyatt Jon‐Philippe K.
Published: May 2022

Mitochondrial open reading frame of the 12S rRNA‐c (MOTS‐c) is a mitochondrial‐derived peptide that is released into the circulation following intense physical activity and, purportedly, has systemic impacts on energy metabolism and insulin sensitivity. Exogenous administration of MOTS‐c in rodent models enhances physical activity / endurance and reduces fat mass in aged mice through the regulation of, in part, the glycolysis / pentose phosphate pathway and amino acid metabolism. Considering that mitochondrial volume and mitochondrial DNA copy number increase following chronic aerobic exercise training, we hypothesized that MOTS‐c expression also would be elevated in exercise‐trained skeletal muscle. MOTS‐c protein expression from phenotypically mixed plantaris muscles of adult female Sprague Dawley rats were compared from six conditions: sedentary (SED; n=11), voluntary exercise training for four (EX4; n=9), six (EX6; n=6), or eight weeks (EX8; n=8), and detraining after four (DETR4) or six (DETR6) weeks, which followed four or six weeks of exercise training, respectively. Total running distances were ~551±61, 371±94, 560±71, 321±44, and 519±27 km for EX4, EX6, EX8, DETR4 and DETR6, respectively. DETR4 rat running distance was significantly lower than EX4, EX8, and DETR6 rats (p<0.01). Total muscle protein homogenate was isolated from mid‐belly muscle chunks and separated using 15% SDS‐PAGE gels (10 μg/sample). Following transfer, membranes were incubated with a custom rabbit anti‐rat MOTS‐c polyclonal antibody, developed, scanned, stripped and re‐probed for GAPDH as a loading control. MOTS‐c protein was significantly elevated from SED (set at 100%) to 188±61, 158±37, 212±27, 168±29, 223±36% in EX4, EX6, EX8, DETR4 and DETR6, respectively (p<0.05). Furthermore, preliminary findings indicate that MOTS‐c is present exclusively within the cytoplasm of skeletal muscle and not compartmentalized within the nuclei or mitochondria. Given that long‐term aerobic exercise augments mitochondrial volume within skeletal muscle, our data suggest that the observed increase in MOTS‐c protein in the present study parallels this mitochondrial adaptation to aerobic physical activity. Taken together, these findings indicate that a more aerobically‐trained individual would have an enhanced MOTS‐c presence within skeletal muscle and, therefore, a greater potential to regulate energy and insulin sensitivity when MOTS‐c is released systemically; additionally, the potential benefits of MOTS‐c are sustained during a period of physical inactivity (e.g., detraining) following exercise training.