bluuebunny/crossref_metadata_2025 huggingface dataset:
The Lockable Open‐Source Training Wheel (LOST‐Wheel): A novel device for time‐ and distance‐ limited murine exercise
Authors: Bivona Joseph J. , Poynter Matthew E.
Published: May 2022

Modeling exercise in small animals is more difficult than it may appear. Current methods are either voluntary (wheel running) or forced (treadmill running) protocols. Although commonly used, each have several drawbacks that limit enthusiasm for their adoption. While mice will instinctively run on a wheel, current methods lack the ability to restrain mice from traveling 10‐20 km per day. Forced exercise, while controllable, puts animals into stressful environments in which they are confined and often shocked for “encouragement.” Additionally, both methods require expensive equipment and software, which limit these experiments to well‐funded laboratories. To counter these issues, we developed a non‐invasive mouse running device aimed to reduce handler‐induced stress, provide time and distance based stopping conditions, and enable investigators with limited resources to easily produce and use the device. The Lockable Open‐Source Training‐Wheel (LOST‐Wheel) was designed to be 3D printed on any standard entry‐level printer and assembled using only a few common tools for around 20 USD (Figure 1 A‐C). It features an on‐board screen and is capable of tracking distances, running time, and velocities of mice. The LOST‐Wheel overcomes the largest drawback to voluntary exercise, which is the inability to control when and how long mice run, using a servo driven mechanism that locks and unlocks the running surface according to the protocol of the investigator. While the LOST‐Wheel can be used without a computer connection, we wanted to provide scientists additional data and designed the LOST‐Wheel Logger, an R‐based application that presents milestones and plots on a user‐friendly dashboard (Figure 1D). Seven‐day running experiments confirm distances comparable to other reports (Figure 1E). Using the LOST‐Wheel we implemented a timed running experiment which showed distance‐dependent decreases in serum myostatin as well as IL‐6 gene upregulation in muscle. To make this device accessible, we are releasing the designs, application, and manual in an open‐source format. The implementation of the LOST‐Wheel and future iterations will improve upon existing murine exercise equipment and research.
Reflections on a Plague Academic Year: A Multiyear Comparison of Student Engagement, Investment, and Performance in Undergraduate Physiology Courses During the COVID‐19 Pandemic
Authors: Krauss Nicole E. , Divino Jeffrey N. , Crivello Joseph F. , Redden John M.
Published: May 2022

The ongoing COVID‐19 pandemic continues to alter the landscape of higher education in the United States – even in highly vaccinated states like Connecticut. For example, in Fall 2020, the University of Connecticut operated remotely with most courses being offered in a distance learning format. In Fall 2021, students returned to campus for in person classes. Although there has been a great deal of speculation shared by students and faculty, the long‐term impact of these abrupt transitions on student learning is largely unknown. In the present study, we begin to explore the impact of the pandemic on student engagement, investment, and performance in several undergraduate physiology courses by comparing student data from the Fall 2020 and Fall 2021 semesters. We analyzed the time students spent accessing synchronous and asynchronous learning materials, the frequency of student‐instructor interactions, and scores on matched summative assessments. Furthermore, we explore the impact of course modality on these same metrics by comparing a flipped anatomy and physiology course to other courses offered in a traditional lecture and hybrid format. Our preliminary results indicate that while the overall student investment was comparable despite the instructional format, students engaged more consistently after returning to campus. However, the increased engagement does not correlate with assessment scores or other measures of student success (e.g., DFW rates). Our results contradict a widespread misconception that students were not adequately engaged by online learning during the 2020‐2021 academic year. As pressure mounts to undo student support measures put in place during the pandemic, they may also serve as a warning to academic departments, graduate programs, and other stakeholders that the effects of the pandemic on students are ongoing, despite strong institutional desires for normalcy.
RETRACTED: Fruit‐juice Concentrate of Asian Plum Attenuates Vascular Remodeling Associated with Hypertension
Authors: Okuno Keisuke , Torimoto Keiichi , Kuroda Ryohei , Cicalese Stephanie , Okuno Yoshiharu , Kono Ryohei , Marumoto Shinsuke , Utsunomiya Hirotoshi , Eguchi Satoru
Published: May 2022

A fruit of the Prunus mume is a traditional and familiar food in Asian countries and is most often pickled or used to make wine and juice concentrate. Recently, Bainiku‐ekisu, an infused juice concentrate of Prunus mume is attracting attention as a potential health promoting food. We previously reported that Bainiku‐ekisu (BE) incubation attenuates the activation of growth‐promoting signaling cascade induced by angiotensin II (Ang II) in cultured vascular smooth muscle cells (VSMC). However, whether BE has any effect on an animal model of cardiovascular disease remains unknown. Therefore, this study was designed to elucidate whether BE could attenuate cardiovascular pathology induced by Ang II infusion in mice, representing a model of human hypertension. 8‐ to 10‐week‐old male C57BL6 mice were randomly divided into four groups and infused with Ang II (1 µg/kg/min) for 2 weeks via osmotic mini‐pump or sham‐operated, and given 1% BE containing water (BEW) or normal water (control) for 2 weeks. Blood pressure was evaluated every other day by tail cuff measurement. Cardiac hypertrophy was assessed by heart‐to‐body weight ratio. After 2 weeks, mice were euthanized, and the aorta and heart collected. Extracted aortas and hearts were fixed and used for histological studies to evaluate vascular hypertrophy and perivascular fibrosis. Increase in heart‐to‐body weight ratios were unaltered by BEW. Aortic medial hypertrophy was observed in control mice (n=5) after 2 weeks of Ang II infusion, which was attenuated in BEW group (96 µm vs. 71 µm, respectively, p<0.01). Control mice with angiotensin II infusion showed cardiac perivascular fibrosis, which was attenuated in BEW group (fibrosis index score: 1.02 vs. 0.30, p<0.01). In addition, trend of hypertension attenuation by BEW was observed in Ang II‐infused mice with BEW treatment. We also observed attenuation of DNA synthesis induced by AngII with BE incubation in cultured aortic fibroblasts. In conclusion, BEW prevented Ang II‐induced cardiovascular remodeling such as vascular fibrosis. Potential cardiovascular health benefit to taking BE as a functional food should be further studied.
Gut microbiota depletion with antibiotics enhances fructose induced salt‐sensitive hypertension in normal rats
Authors: Haque Mohammed Z. , Rey Federico , Ortiz Pablo A.
Published: May 2022

Introduction‐

We reported that 20% fructose in drinking water induced salt‐sensitive hypertension in normal rats. High fructose (HF) or high salt (HS) intake reportedly alter the gut microbiota, which influences a variety of microbial‐regulated bioactive metabolites that can affect blood pressure and renal function. The contribution of the gut microbiota to fructose induced salt‐sensitive hypertension or renal function has not been studied. Aim‐ We hypothesized that gut microbiota depletion with antibiotics (ABX) would change fructose induced salt‐sensitive hypertension, renal excretory function and plasma levels of bacterial metabolites (lactate, β‐hydroxy‐butyrate).

Methods‐

Sprague Dawley rats were treated with a cocktail of antibiotics (ABX group, in g/L 1.0 ampicillin, 1.0 neomycin sulphate, 1.0 metronidazole, and 0.5 vancomycin in drinking water) for 4 weeks followed by a 0.33 fractional maintenance dose for up to 12 weeks) or control diet (no‐ABX). On week 5, all rats were given 20% fructose (HF) in drinking water and fed a diet with normal salt (NS, 0.4% Na) or high salt content (HS, 4% Na). Rats were divided as: Group 1 (HF + NS, no‐ABX, n= 6), Group 2 (HF + NS + ABX, n=6), Group 3 (HF + HS, no‐ABX, n= 8), Group 4 (HF + HS + ABX, n= 8). We trained rats and measured systolic blood pressure (SBP) for 12 weeks by tail‐cuff and collected feces before and after ABX, to measure gut c‐DNA content. On the 12th week, we collected 24‐ hour urine and plasma before the end of the protocol.

Results

‐ Antibiotics induced microbiome depletion (ABX) decreased fecal DNA content by 83%. Baseline SBP (in mmHg) or urine albumin excretion was not affected by ABX. Fructose with normal salt diet did not change SBP with (125±4) or without ABX (124±3). HS significantly increased SBP in rats fed fructose without ABX (from 123±3 to 141±4, p<.05) but the increase was greater in ABX rats (from 124±4 to 155±3, p<.05). Average increase on SBP was 26±2 in HF+HS with ABX, but only 15±2 without ABX (p<.001). In HF + HS groups, ABX decreased plasma lactate from 1657±259 to 905±144 µM (p<.01), and β‐hydroxy‐butyrate from 861±144 vs 220±53 µM (p<.001). Non‐fasted plasma insulin was also decreased by ABX in HF+HS rats (0.77±0.13 to 0.36±0.06 ng/mL, p<.03). The increase in body weight was similar in HF+HS rats with or without ABX.

Conclusion

‐ Chronic antibiotic treatment induced gut microbiota depletion and enhanced fructose induced salt‐sensitive hypertension in normal rats, an effect that was associated with decreased plasma beta‐hydroxy‐butyrate, lactate and insulin. Additional studies should focus on direct renal effects of ABX on salt handling.
Urinary Serpin‐A3 is an Early Predictor of Clinical Response to Therapy in Patients with Proliferative Lupus Nephritis
Authors: Martínez‐Rojas Miguel Angel , Sánchez‐Navarro Andrea , Mejía‐Vilet Juan Manuel , Pérez‐Villalba Rosalba , Uribe Norma , Bobadilla Norma
Published: May 2022

Background

Serpin‐A3 is a regulator of different inflammatory processes, however its role in the kidney is unknown. We previously reported that urinary Serpin‐A3 excretion (uSerpA3) is significantly elevated in patients with active lupus nephritis (LN) and rats with Chronic Kidney Disease (CKD).

Objective

Determine the association between uSerpA3 temporal course during the 1st‐year of treatment and response to therapy in patients with proliferative LN.

Hypothesis

uSerpA3 will correlate with kidney inflammation and will predict poor response to therapy.

Methods

This is an observational longitudinal study including sixty Mexican adults with proliferative LN followed during the 1st‐year after LN‐flare. uSerpA3 at flare and after 3, 6, and 12 months was evaluated semiquantitatively through Western blot; histological evaluation was performed by kidney biopsy. Outcome: Response to therapy 1‐year after the LN‐flare. Relation between uSerpA3 and histopathologic characteristics at flare was evaluated cross‐sectionally; the temporal association between uSerpA3 and response to therapy was analyzed with linear mixed models; prognostic performance for clinical response was evaluated with ROC analysis.

Results

Among the 60 patients studied, 21 (35%) were class III and 39 (65%) class IV. The class IV LN had higher serum creatinine, lower C3 levels, and higher histological activity and chronicity indexes. The uSerpA3 was higher in class IV than in class III LN (6.98 vs. 2.89 DPI/mg‐creatinine, p=0.01). Furthermore, uSerpA3 had a mild correlation with the histological activity index (r=0.29, p=0.02). There was a significant association between the temporal course of uSerpA3 and the response to therapy. Responders showed a significant drop in uSerpA3 at 6th‐month compared to LN‐flare (p<0.001), while non‐responders persisted with elevated uSerpA3. Moreover, uSerpA3 was significantly lower at flare in responders compared to non‐responders (2.69 vs 6.98 DPI/mg‐creatinine, p<0.05). Furthermore, uSerpA3 was able to identify non‐responders since the 3rd‐month after LN‐flare, (AUC=0.77).

Conclusions

uSerpA3 is an early indicator of kidney inflammation and a potential predictor of long‐term response to therapy in patients with proliferative LN.
Modeling Dehalogenase PceA to Evaluate Pollutant Organohalide Biodegradation
Authors: Lamarre Thomas , Leyendecker Anastasia C. , Moran Elizabeth M. , Replogle Carl J. , Suder Karen C. , Replogle Jessica B.
Published: May 2022

Per‐ and polyfluoroalkyl substances (PFAS) are ubiquitous pollutants found in water and land environments. Environmental buildup of organohalides, such as perchloroethylene (PCE) and trichloroethene (TCE), used in dry cleaning, firefighting foams, and food packaging, pose an increasing public health risk due to their slow degradation. PFAS molecules are now observed in the blood of humans and animals across the world, and their full impact on health is yet to be determined. Studies of PFAS biodegradation have been reported within various microbial organisms. One reductive dehalogenase identified is PceA from Sulfurospirillium multivorans. The Summit Country Day School MSOE Center for Biomolecular Modeling MAPS team used 3D modeling and printing technology to investigate the active site of the reductive dehalogenase PceA. PceA is a homodimer with two independent active sites. Each active site contains a norpseudo‐B12, two 4Fe‐4S clusters as well as three highly conserved amino acids, Tyr246, Arg305, and Asn272. The short distances between the proximal iron‐sulfur cluster and the cobalt ion bound to the corrin ring of norpseudo‐B12 are thought to allow rapid electron transfer for catalysis. The invariant Tyr246 can donate a proton to neutralize the carbanion formed during catalysis. Deprotonated Tyr246 may be stabilized by the guanidinium side chain of Arg305. Additionally, the active sites have a hydrophobic pocket lined with bulky nonpolar amino acids Phe38, Trp56, Tyr102, Trp96, Tyr382 and Trp376. The tightly packed hydrophobic sidechains provide a gate for substrate selection. Understanding PFAS biodegradation and identifying additional reductive dehalogenases is urgent in addressing the pollution that faces our environment and critical in ensuring the health of humans and ecosystems.
Antinociceptive effects of α2/α3‐selective GABAA receptor positive allosteric modulators on opioid‐induced hyperalgesia
Authors: Woodhouse Kristen L. , Cook James M. , Li Jun‐Xu
Published: May 2022

Opioids have been increasingly prescribed to treat chronic pain since the 1980s, despite evidence that long‐term use of opioids may lead to tolerance and pain sensitization called opioid‐induced hyperalgesia (OIH). OIH has been demonstrated in both preclinical models and healthy human volunteers, but is understudied and there is need for novel analgesics capable of mitigating OIH. α2/α3‐selective GABAA receptor positive allosteric modulators (PAMs) act specifically at subunits of the GABAA receptor found to mediate analgesia, and have demonstrated antinociceptive effects in models of chronic inflammatory and neuropathic pain. However, the efficacy of these compounds at relieving opioid‐induced pain hypersensitivity have not yet been investigated. This study systematically examined the antinociceptive effects of α2/α3‐selective GABAA receptor PAMs alone and in combination with acetaminophen in an OIH rat model wherein repeated treatment with the opioid fentanyl induces mechanical hyperalgesia. The von Frey test was used to measure mechanical nociception. Duration of actions of α2/α3‐selective GABAA receptor PAMs (KRM‐II‐81, NS16085, HZ‐166) alone were studied, and combinations of KRM‐II‐81 and acetaminophen were also studied at fixed ratios (1:1, 1:3, 3:1). Dose‐addition analysis was used to assess the antinociceptive interactions between KRM‐II‐81 and acetaminophen. α2/α3‐selective GABAA receptor PAMs were able to fully reverse mechanical sensitivity caused by OIH. Furthermore, KRM‐II‐81/acetaminophen combinations produced additive to supra‐additive interactions depending on the drug mixture ratios. These findings support the idea that α2/α3‐selective GABAA receptor PAMs could serve as novel analgesics for treating OIH, and may interact favorably with other non‐opioid analgesics.
17α‐Estradiol Mitigates the Negative Effects of High‐Fat Feeding in Both Male and Female Mice
Authors: Bubak Matthew P. , Mann Shivani N. , Broomfield Matle E. , Stout Michael B. , Miller Benjamin F.
Published: May 2022

17α‐estradiol (17α‐E2) is an endogenous, non‐feminizing enantiomer of 17β‐estradiol. 17α‐E2 supplementation improves the lifespan of male, but not female, mice through metabolic mechanisms. Although the effects of 17α‐E2 on liver and adipose tissue are well described, potential benefits in skeletal muscle are unknown. The objective of this study was to determine if 17α‐E2 can mitigate the negative effects of a high‐fat diet (HFD) on skeletal muscle metabolism. In addition, we sought to determine if there were sex differences in these responses. We hypothesized that 17α‐E2 supplementation would ameliorate the negative effects of a HFD in skeletal muscle of male, but not female mice. To test this hypothesis, wild type male (n=19) and female (n=15) mice began a HFD (45% fat) at 3 months of age. At 9 months of age, half of the males (n=10) and females (n=8) received diets supplemented with 17α‐E2 (14.4ppm) for 3.5 months. Mice underwent weekly assessment of body composition by QMR throughout the 17α‐E2 supplementation, while glucose and insulin tolerance tests were completed 8 weeks into treatment. Leg muscles were harvested and analyzed for inflammatory cytokines via RT‐qPCR, fat infiltration via Oil Red O (ORO) staining, and triglyceride content. 17α‐E2 reduced whole‐body fat mass (p = 0.001, 11.6% ± 0.4%) and whole‐body lean mass (p = 0.003, 3.5% ± 0.2) in males. In females, 17α‐E2 reduced fat mass (p = 0.0001, 33.1% ± 1.1%), but did not alter lean mass (p = 0.99, 0.9% ± 0.2%). There were no changes in the mass of the quadriceps, gastrocnemius, soleus, tibialis anterior or extensor digitorum longus muscles in males or females (p > 0.05). 17α‐E2 improved glucose tolerance in females (p = 0.03, 22301 ± 573 AUC and 33022 ± 1663 AUC) but not in males (p = 0.11, 24856 ± 159 AUC and 29968 ± 466 AUC). 17α‐E2 reduced insulin resistance in males (p = 0.001, 14784 ± 326 AUC and 19220 ± 246 AUC) and females (p = 0.05, 10992 ± 315 AUC and 14644 ± 540 AUC). 17α‐E2 reduced the expression of IL‐6 (p = 0.005), IL‐1α (p = 0.002), and TNFα (p < 0.001) in males, but 17α‐E2 did not change cytokine expression in females (p > 0.05). There was no difference in gastrocnemius ORO staining in the males (p = 0.59) or females (p = 0.68). 17α‐E2 supplementation did not change quadricep triglycerides in males (p = 0.98, 25.6 ± 1.6 mg/g and 27.3 ± 1.7 mg/g). However, females on 17α‐E2 had significantly lower quadricep triglycerides compared to HFD (p = 0.03, 13.6 ± 1.1 mg/g and 26.6 ± 0.7 mg/g). These results suggest that long term 17α‐E2 supplementation ameliorates the effects of a HFD on skeletal muscle in both males and females but in a sex dimorphic manner. Further research is needed to determine how 17α‐E2 supplementation alters skeletal muscle metabolism in both sexes.
The Impact of Varying Caffeine‐Induced Contractile Activity on Metabolic Enzyme Activity
Authors: Schlater Amber , Jowlowsly Sydney , Zupetz Jack , Mayola Nyiel , Butterfield Kylie
Published: May 2022

Exercise intensity is widely known to differentially affect skeletal muscle metabolic approaches, with a general understanding that short duration, high intensity exercise is associated with an increased reliance on anaerobic glycolysis, while long duration, low to moderate intensity exercise is associated with a greater reliance on aerobic processes. In cell culture, caffeine is a commonly used method to pharmaceutically stimulate contractile activity in skeletal muscle cells; upon exposure to caffeine, ryanodine receptors are stimulated to release calcium, thereby allowing crossbridge cycling and subsequent contractile activity to occur. This technique for exercising muscle cells in culture offers a controlled, experimental platform to study the effect of contractile activity on isolated muscle cells; presumably, varying doses of caffeine should mimic varying levels of exercise intensity, though the degree to which caffeine dose impacts metabolic shifts has yet to be fully elucidated. Here, we exposed C2C12 cells, an immortalized line of mouse skeletal muscle cells, to varying levels of caffeine supplementation throughout myotube differentiation in an attempt to recapitulate metabolic signatures consistent with varying exercise intensities. Following standard proliferation protocols, mature myotubes were differentiated for 7 days. On differentiation days 4‐7, experimental cohorts of cells were exposed to either a low dosage (10mM) or a high dosage (40 mM) of caffeine‐supplemented differentiation media for 20 minutes daily. All cells were harvested for protein on differentiation day seven. Protein concentration across cohorts was measured using a Bradford protein assay. Metabolic changes were characterized by running functional enzyme assays for lactate dehydrogenase (LDH) and citrate synthase (CS) to compare anaerobic and aerobic changes, respectively. Preliminary data suggest that, relative to controls, cells exposed to 10mM caffeine have a decrease in LDH activity, and a trend toward increased CS activity. Conversely, cells exposed to 40mM caffeine show no change in LDH and a trend toward decreased CS activity. Collectively, these preliminary data suggest that C2C12 cells exposed to low doses of caffeine simulating low intensity exercise may have less reliance on anaerobic metabolism with a potential greater reliance on aerobic metabolism, while those exposed to high doses of caffeine simulating high intensity exercise may have a lesser reliance on aerobic metabolism. While more research needs to be done to characterize the precise impact of varying caffeine dosages on C2C12 cells, these initial findings are consistent with previously established trends in exercise intensity and metabolism.
Enhancing Membrane Repair in Neuronal Cell Types as a Potential Therapeutic for Neurodegenerative Diseases
Authors: Bulgart Hannah R. , Madalena Kathryn M. , Mital Rohan , McElhanon Kevin E. , Kwiatkowski Thomas A. , Rose Aubrey L. , Lerch Jessica K. , Lopez Perez Miguel A. , Paleo Brian J. , Weisleder Noah L.
Published: May 2022

Cell membrane repair is an essential cellular process to repair damage to the membrane and maintain the integrity of the cell membrane. Impaired membrane resealing kinetics can potentially lead to neurodegenerative diseases. Enhancing membrane repair via therapeutic intervention could minimize neurodegeneration in neuronal disease states. Recombinant human MG53 (rhMG53) and poloxamers have been an effective therapeutic to enhance membrane repair in muscle and non‐muscle cell types. Here, we aimed to assess if the membrane repair response is active and the efficacy of enhancing membrane resealing in neuronal cells. To assess if neuronal cells have an active membrane repair response, mouse neuroblastoma N2a cells were transfected with GFP or GFP‐MG53. MG53 localization was tracked following multiphoton infrared laser injury. N2a cells were transfected with GFP or GFP‐MG53, and subjected to multiphoton infrared laser injury to investigate if MG53 expression enhances membrane resealing following damage. An infrared laser was used to damage the cell membrane of N2a cells and primary mouse dorsal root ganglion neurons (DRG) in the presence of FM4‐64 dye. To test the efficacy of therapeutics, N2as and DRGs were treated with 1μM rhMG53 or 100μM Poloxamer 188 (P188) and challenged with the multiphoton laser injury. Lastly, the therapeutic potential of rhMG53 was tested in vivo with a sciatic nerve crush injury model. C57Bl/6 mice sciatic nerves were mechanically crushed for 10 seconds and then injected with 1μL rhMG53 (1mg/mL) or saline. The sciatic nerves were harvested and immunostained with an axonal regeneration marker, SCG10. SCG10 staining intensity was measured to calculate the regeneration index. Overall, we observed MG53 trafficking to the injury site which indicates there is an activate membrane repair mechanism within neuronal cells. Therapeutic levels of rhMG53 and P188 increased membrane resealing in both N2as and DRGs. Lastly, rhMG53‐treated sciatic nerves displayed a two‐fold increase in regenerative capacity following sciatic nerve crush injury compared to the saline vehicle control. These results illustrate an active membrane repair process in neuronal cells and potential for therapeutic intervention in the context of neuronal diseases, specifically neurodegenerative diseases.
A Novel Connection Between [PSI+] Prion Formation and RNA Splicing in Saccharomyces cerevisiae
Authors: Jones Elizabeth A. , Riccitelli Audrey N. , Cameron Dale M. , Kress Tracy L.
Published: May 2022

Relatively new studies on prions in Saccharomyces cerevisiae show that some protein prion conformations may be beneficial to cell survival in response to environmental stress. Sup35, a translation termination release factor, forms the prion [PSI+]. When [PSI+] forms, translation termination efficiency decreases, which has been shown to result in the alteration of the abundance of cellular proteins, including splicing factor abundance. Therefore, we are testing the hypothesis that [PSI+] prion formation alters splicing efficiency in yeast. First, we employed quantitative RT‐PCR and revealed that splicing efficiency improves upon [PSI+] prion formation. The proposed mechanisms for the effect of [PSI+] on protein abundance include nonsense suppression and non‐stop decay. Non‐stop decay would lead to degradation of the mRNA encoding a splicing protein and therefore would reduce splicing factor protein levels. Non‐sense suppression, a result of translation readthrough, generates a C‐terminally extended protein, which could alter the function of splicing factors, or alter protein levels. We are initiating RT‐PCR and Western Blot analyses to test the contribution of the two mechanisms on splicing factor abundance. To further test the relationship between RNA splicing and [PSI+] prion formation, we used knockout PCR to create splicing factor deletion mutations in a [psi‐] strain, as well as a weak [PSI+]SC37 strain or a strong [PSI+]Str strain. We found that the [PSI+]SC37 conformation can suppress the growth defect in a yeast strain with a deletion of the gene encoding SNU66, a component of the tri‐snRNP. Similarly, the [PSI+]SC37conformation can suppress the growth defect in a yeast strain with a deletion of the MUD2gene, which encodes a gene important for commitment complex formation. In addition, we found that [PSI+]SC37can rescue the splicing defect observed in a mud2Δstrain, consistent with the suppressive growth phenotype. Together, the results show that [PSI+] formation impacts RNA splicing in S. cerevisiae and our future work will focus on determining the mechanisms that underlie this effect.
Survey and Analysis of Virtual vs. In‐Person Biology Teaching Labs
Authors: Renna Lauren , Kumbaraci Nuran
Published: May 2022

In March 2020, due to the COVID‐19 pandemic lockdown, teaching labs at Stevens changed teaching styles from in‐person to online‐virtual in the middle of the semester. Virtual labs then became the new norm in the following semesters. The impact of the transition from in‐person to virtual biology lab teaching environments on college students is unknown. The goal of this study is to assess the effectiveness of biology teaching labs utilizing different educational programs and platforms. In this study, an anonymous opinion survey was conducted among Stevens students who took in‐person and online‐virtual biology teaching labs during the last two years. 50 undergraduate students responded to an online survey and answered questions regarding their lab experience, preferences, likes and dislikes, and knowledge retention. The collected data were analyzed to determine if there was a statistically significant difference between in‐person and online‐virtual teaching labs. Among this group of 50 students, 74% of students stated they preferred in‐person labs over virtual labs. 60% of students explained how they appreciated the hands‐on experience in‐person labs provided them with and felt as though they retained the lessons learned in biology labs better through interacting with the equipment and materials hands on. However, 44% of the participants also noted some slight issues they have with in‐person labs including a small room for error, increased difficulty, and longer completion times. For the students that preferred virtual labs, the main reason was based on time. Students who preferred virtual labs admired the independence virtual labs allowed them to have including allowing them to work at their own pace. It is evident from the results of this study that students prefer in‐person labs over virtual and they value the interactive and experiential aspects of in‐person labs. For 3 out of the 4 specific labs surveyed, students that took the virtual version of the lab were more likely to answer correctly than students who took in‐person labs. However, for each of the 4 labs, the percentages for answering correctly differed no more than 15%. For the lab in which more in‐person students answered correctly, 78% of the questions were answered correctly by in‐person students and only 63% of the questions were answered correctly by virtual students. Although the analyzed data shows that students tended to retain more knowledge from virtual labs versus in‐person labs, 13 students stated they felt as though they learned more from in‐person labs. These results will ultimately allow alterations to be made to biology teaching labs based on student feedback and overall lab effectiveness in order to better suit the learning needs of the students both during and after the pandemic. This opinion survey received Stevens IRB Approval for Protocol No: 2021‐011(N) and extension 2021‐011(22‐COAS1) on 10/25/2021 and conducted in October 2021.
Renal inflammatory signaling alters cardiomyocyte kinetics and lead to HFpEF in CKD: A mechanistic proof of concept study
Authors: Chade Alejandro R. , Mohamed Tamer M. , Eirin Alfonso
Published: May 2022

Background

Chronic kidney disease (CKD) carries a higher cardiovascular mortality and is a prominent risk factor for development of HFpEF. However, the pathophysiology of HFpEF in CKD has not been established. We have recently shown that experimental CKD‐HFpEF is associated with increased renal release of inflammatory cytokines, particularly tumor necrosis factor (TNF)‐α and interleukin (IL)‐6, to the systemic circulation. Using a unique physiological biomimetic heart culture system, we test whether inflammatory cytokines of kidney origin contribute to the development of HFpEF in CKD.

Methods

Pigs (4 each) were assigned to sham or CKD (bilateral renovascular disease and dyslipidemia) groups and followed for 14 weeks. Circulating levels of TNF‐α and IL‐6 were quantified (ELISA). In a biomimetic heart culture system, pig heart slices were exposed to plasma from normal or CKD pigs, or from normal pigs enriched with TNF‐α and IL‐6 (matching CKD concentration) with or without cytokine neutralizing antibodies (NA). Contractility and relaxation kinetics in vitro were determined in a dose/time dependent manner.

Results

CKD‐HFpEF pigs exhibited elevated systemic levels of TNF‐α and IL‐6 (Figure 1A), associated with renal dysfunction, cardiac hypertrophy and fibrosis, and diastolic dysfunction. Plasma was collected from all animals. Then, pig heart slices were exposed to CKD‐HFpEF, normal, or normal plasma enriched with cytokines, showing impaired contractility and speed of relaxation that improved after TNF‐α and IL‐6 neutralization (Figure 1B).

Conclusions

Our observations implicate circulating inflammatory mediators released by dysfunctional kidneys in imposing damage to the remote myocardium, supporting a role of inflammatory cytokines of renal origin in renal‐cardio pathophysiology in CKD‐HFpEF.
Structural Analysis of the Interaction of Pyrazole and Benzimidazole Core Compounds with PfATP4
Authors: Nepal Binod , Morrisey Joanne , Brand Stephen , Vaidya Akhil , Kortagere Sandhya
Published: May 2022

P‐type cation ATPase in Plasmodium falciparum(PfATP4) serves as one of the important emerging drug targets for the treatment of the malaria. PfATP4 maintains sodium homeostasis in the parasite and its inhibition causes the death of the parasite through a mechanism that mimics premature schizogony. Over the past few years, we and others have identified ~20 different chemotypes of molecules that can disrupt sodium homeostasis via PfATP4. We have developed two series of potent PfATP4 inhibitors with a pyrazole and/or benzimidazole core and tested them on wild type and lab resistant strains of P. falciparum. However, due to lack of PfATP4 crystal structure, we have limited understanding of the structure‐activity relationship among the analogs of these molecules and the mechanism of inhibition of PfATP4 which has hindered the rational optimization of the discovered inhibitors. In this study, we modeled the E1 and E2 states of PfATP4 using homology modeling with the corresponding state templates from the mammalian Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (SERCA). Models of PfATP4 were refined using molecular dynamics simulations and ~200 pyrazole and benzimidazole compounds were docked to the binding site using GOLD molecular docking software. Results from the docking study suggests that the compounds preferred mode of binding is to the proton bound E2 state of PfATP4. The docking scores significantly correlated with IC50 values with a correlation co‐efficient > 0.7. Further MD simulations of the docked complexes suggested residues GLN172, GLN351, SER175 and ASN207 play a key role in ligand binding which may have a role in the development of resistance to PfATP4 inhibitors which needs further experimental validation.
Adaptable online discussion format to increase student interest, peer interactions, and information literacy
Authors: Wilson Kristy J.
Published: May 2022

Online instruction poses several challenges including creating opportunities for students to interact and get to know each other. I will discuss an adaptable online discussion format that has students explore the real‐world implications of biochemistry and molecular biology to provide course context to what they are learning and allow students to build the classroom community. The discussion format utilizes current events and elements of students' daily lives. First, students explore instructor provided information on a topic, and then reflecting on what else they would like to learn by identifying possible search terms. Then students locate information related to the topic and write their post to communicate what they have learned. This format allows students to explore and share their interests leading to a large diversity of connected topics. Students then read and comment on others posts and develop an understanding of other students’ interests. Student response to discussion format has been overwhelmingly positive in a variety of classes including biochemistry, molecular genetics, and a senior seminar course. Survey results in biochemistry indicate that students appreciated “understanding other’s points of view” and “connecting…learning to actual research going on.” Students reported in a Likert scale survey items that the discussion format increased interest and interaction with peers.
Investigating the Roles of NAC and RAC in Suppressing Prion Formation in Saccharomyces cerevisiae
Authors: Kitterman Erica , Kim Seongchan , Yilanjian Mark , Kelly Christina , Cameron Dale
Published: May 2022

The correct three‐dimensional folding of a polypeptide is essential for its correct structure and function. The appearance of misfolded proteins can be found in relation to many disease processes. Prions are a class of infectious misfolded proteins, which are characterized by their ability to self‐propagate. In order to help ensure correct protein folding for the survival of the cell, several chaperone quality control complexes interact with nascent polypeptides as they are being synthesized by the ribosome. In yeast, two important ribosome‐tethered chaperone complexes exist, the Ribosome‐Associated Complex (RAC), composed of two subunits, Hsp70 chaperone Ssz1 and Hsp40 chaperone Zuo1, and the Nascent Polypeptide Associated Complex (NAC), a heterodimeric complex composed of an α subunit, Egd2, and one of the two β subunits, Btt1 or Egd1. Previous research by our lab and others has shown that cells lacking Zuo1 and thus, lacking functional RAC, show an increase in prion formation of the [PSI+] prion, a misfolded form of the Sup35 translation termination factor protein. In addition to its involvement in protein folding in the RAC, Zuo1 serves as a transcriptional co‐activator for genes in the pleiotropic drug resistance (PDR) response, which aids yeast survival in response to environmental stressors such as drugs or toxins. Environmental stress can also trigger prion formation in yeast. An overexpression screen for factors that increase yeast survival in the presence of the antibiotic cycloheximide, a compound known to result in PDR activation, revealed Btt1 to increase cell survival in a Zuo1 dependent manner. To investigate the interactions between RAC and NAC, as well as the role these complexes play in modulating prion formation during periods of cellular stress, we conducted prion formation assays and growth assays to assess how the complexes work together and separately in relation to the formation of the [PSI+] prion.
A Flavonoid Compound 7, 4 Dihydroxy Flavone as a Potential Therapeutic for the Treatment and Management of EoE
Authors: Maskey Anish R. , Wang Zhen Zhen , Chen Xin , Dunkin David , Soffer Gary , Yuan Qian , Li Xiu‐Min R.
Published: May 2022

Eosinophilic Esophagitis (EoE) is a chronic condition characterized by esophageal dysfunction including dysphagia, abdominal pain and eosinophilic inflammation (>15/HPF) of the esophagus. At present, the most commonly used treatment for the management of EoE are steroids, both systemic as well as tropical, and dietary elimination. The advantage of the systemic steroids include ease of administration, rapid response, and very high response rate. However, poor solubility and the adverse effect related to prolonged steroid use are well documented. Moreover, there is a high relapse rate of EoE after the treatment is discontinued which limit the use of oral steroids as a maintenance therapy. Similarly, Ranitidine (Zantac), histamin‐2 blocker, used to treat gastrointestinal reflux and heart burns, has been recently withdrawn from the market by FDA due to increased risk of cancer. Digestive Tea has been used at the Traditional Chinese Medicine (TCM) practice to improve gastrointestinal (GI) symptoms such as stomach pain, constipation and abnormal bowel movement. Different versions of Digestion Teas are further developed to address specific clinical GI symptoms. Compound 7, 4 Dihydroxy flavone (DHF) is a flavonoid purified from Glycyrrhiza uralensis from Digestive Tea. Previously our group has shown inhibition of Eotaxin, Th2 cytokines and IgE in‐vitrousing DHF. The objective of this study is to reveal potential mechanisms underlying DHF improving symptoms related to EoE using computational modeling, including target mining, gene ontology enrichment, pathway analyses, protein‐protein interaction analyses, and in silico molecular docking. Based on the pathway analyses, the most important targets identified were TNF‐ α, IL‐6, IL1β, MAPK1, MAPK3, AKT1, CASP3 and CCND in AGE‐RAGE pathway. We further validated the targets of our computational modeling in human esophagus biopsy sample by ELISA and qRT‐PCR. We found significant reduction in levels of TNF‐α (p<0.001), IL‐6 (p<0.001), IL‐8 (p<0.001) and IL1‐β (p<0.05) in the supernatant of biopsy sample cultured with DHF. Moreover, gene expression profile showed significant reduction in level of TNF‐ α (p<0.01), IL1‐β (p<0.05), IL‐6 (p<0.01) and CCND (p<0.05) in the biopsy sample cultured with DHF. Furthermore, our molecular docking analysis revealed that DHF can directly bind TNF‐ α with free binding energies of ‐7.7 kcal/mol. Taken together, the current study may lead to development of DHF as an active therapeutic candidate in treatment of EoE.
Investigating a novel role of LARP along the algal TOR pathway
Authors: Slone Danielle R. , Smythers Amanda L. , Hicks Leslie M. , Kolling Derrick R.
Published: May 2022

Over the last few decades, global warming has become an increasingly desperate rallying cry for the need to find and optimize efficient renewable energy sources.1 Microalgae are considered apromising source of energy because they contain a high concentration of lipids, but due to inefficiencies in their lipid production, microalgae cannot produce enough biofuel to replace current fuel needs.2

Chlamydomonas reinhardtiiis a unicellular green alga that is a great candidate for manipulation and an excellent model organism used to better understand algal metabolic pathways and improve biofuels. Our previous research has shown that inhibiting the target of rapamycin (TOR) kinase of C. reinhardtii with AZD8055 causes the growth of the cell cultures to stagnate, the photosynthetic efficiency to decrease, as well as a direct link between oxidative signaling and the upregulation of TAGs.3 Additionally, our work has indicated that La‐related protein 1 (LARP1) is a TOR target and involved in propagating the intracellular response following inhibition. In mammalian systems, LARP1 interacts directly with mTOR and regulates protein synthesis by binding and stabilizing mRNA before recruiting polysomes to initiate translation. LARP1 is essential in mammalian systems, and deficiencies lead to inhibited growth, defects, and increased apoptosis. However, LARP1 deficiency in C. reinhardtii does not cause the same phenotype, indicating a distinct uncharacterized role in phototrophic organisms.

This study seeks to understand this role by using a LARP1 knockout mutant to compare the physiological changes following inhibition with the parent strain. Both strains were assessed for biochemical composition, cell size, and photosynthetic electron fluxes. Together, these data support LARP1 as a regulator of protein degradation along the TOR pathway. The differential delineation of metabolic processes in LARP1 knockouts provides a framework for further TOR pathway characterization in phototrophic eukaryotes.

Literature References

1 IPCC, 2014: Climate Change 2014: Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, R.K. Pachauri and L.A. Meyer (eds.)]. IPCC, Geneva, Switzerland, 151 pp.

2 Sharma, Kalpesh K., Holger Schuhmann, and Peer M. Schenk.. High Lipid Induction in Microalgae for Biodiesel Production. 2012. Energies. 5:1532‐1553.

3 Ford, M. M., Smythers, A. L., McConnell, E. W., Lowery, S. C., Kolling, D. R. J., & Hicks, L. M. (2019). Inhibition of TOR in Chlamydomonas reinhardtii leads to rapid cysteine oxidation reflecting sustained physiological changes. Cells, 8(10), 1171.
Characterization and Modeling of Conserved RNA Elements Within the Human Coronavirus OC43 5’ UTR
Authors: MacKeown Matt , Davila‐Caulderon Jesse , Ford Will , Luo Le , Chiu Liang‐Yuan , Tolbert Blanton
Published: May 2022

Recent global events have highlighted the need for the rapid characterization of viral components used for replication which may serve as potential drug interaction sites. Structural RNA elements serve as a scaffold for various cellular and viral proteins to assemble, providing opportunities for small molecules to act as potential drug targets. Here, we report the rapid identification and characterization of four conserved RNA elements of the 5’‐untranslated region (5’‐UTR) of human coronavirus OC43 (HCoV‐OC43) genome using a combination of experimental and computational methods. Evolutionary trace analysis (ETA) was used to determine highly conserved regions within the 5’‐UTR. The hydrogen‐bonding patterns were studied using chemical probing with dimethyl sulfate (DMS) and NMR‐based experimentation. In order to refine the de novo structures generated using AMBER force fields, semi‐empirical models were generated using small‐angle X‐ray scattering (SAXS) data, as well as experimental data collected with NMR and chemical probing. These models were then used to visualize the evolutionary trace and DMS reactivity data in a site‐specific manner throughout the 5’‐UTR of OC43. Analysis of these results provides insight and a structural basis for the evolutionary importance of cis‐acting RNA elements within the HCoV‐OC43 5’‐UTR. The data presented herein provide a foundation for further biophysical characterization of small molecule interactions with effects on viral processes.
Fatty Acid‐Binding Proteins Respond Differently When Interacting with Endogenous Cannabinoids under High Pressure
Authors: Rosario Irving , Azad Roksana , Lai May Poh , Gagne Donald , Wang Hsin , Katz Francine S. , Gardner Kevin H. , Stark Ruth E.
Published: May 2022

Liver‐type and intestinal fatty acid‐binding proteins (LFABP and IFABP, respectively) are both located in enterocytes, but their different functions are demonstrated by phenotypic differences observed in knockout mice for either protein. Though the two proteins are structurally similar, they differ significantly in their ligand‐binding cavities. The region of a protein with the greatest conformational variability is often found in the area surrounding the ligand‐binding cavity to enable an induced fit to a specific substrate. We used solution NMR spectroscopy while applying increasing pressure to investigate the interaction of these two proteins with ligand, in the hopes of capturing transient intermediate conformations of the protein and to identify potential on‐pathway states for substrate binding that would not otherwise be seen under standard conditions.

Our initial experiments showed apo‐ and ligand‐bound IFABP remains stable under increasing pressure, up to 2500 bar. In contrast, apo‐LFABP and LFABP with anandamide completely unfolds at 1750 bar. Interestingly, the presence of oleate and arachidonic acid stabilizes LFABP, making it resistant to unfolding at high pressure. Our results show that this stabilization is ligand specific, correlating with the ligand binding affinities we determined previously. This trend suggests that specific protein/ligand interactions within the cavity are required for stability.

To make a more detailed analysis of which residues are involved in binding, we generated plots of the pressure‐dependence of backbone 15N and 1H chemical shifts of LFABP and IFABP in either apo‐ or holo‐ form. Two regions of the protein were most responsive to changes in pressure as assessed by the nonlinear chemical shifts changes: i) the conformationally flexible portal and ii) a region of the ligand‐binding cavity we had previously reported as involved in the specificity of binding endocannabinoid ligands. This finding is consistent with the requirement of the conformational variability in these regions for ligand binding. We are currently performing a comprehensive analysis of these effects for the interaction of both proteins with a range of structurally related endogenous cannabinoids.