bluuebunny/crossref_metadata_2025 huggingface dataset:
Non‐invasive, Spatiotemporal Characterization of Muscle Activation Patterns from Vagus Nerve Stimulation in Human Subjects
Authors: Feucht Maria , Gupta Anita , Sabau Dragos , Nowak Thomas , Coimbatore Rama , Powley Terry , Ward Matthew
Published: May 2022

Background and Hypothesis

Vagus nerve stimulation (VNS) is used for treatment of epilepsy in over 100,000 patients worldwide and is a potential treatment for inflammatory disorders such as rheumatoid arthritis, diabetes, and sepsis. VNS‐evoked compound nerve action potentials (CNAPs) may have applications as a biomarker of VNS treatment efficacy, but transcutaneous measurement of this activity is obscured by laryngeal muscle artifacts during cervical VNS. Our objective is to develop a more precise understanding of muscle activation patterns that could improve recording and analysis protocols for isolating the vagus nerve (VN) CNAP from EMG artifacts. We hypothesize that analyzing multi‐electrode array (MEA) surface recordings overlying the VN will allow us to characterize this muscle artifact.

Methods

Twelve patients undergoing VNS therapy for epilepsy were enrolled in a clinical study at Indiana University School of Medicine (IRB #2006075899). The anatomical dimensions of the neck were recorded for seven of the twelve subjects. A custom‐made MEA with a grid of 32 soft foam electrodes was placed on the skin overlying the VN on each side of the neck with the short axis parallel to the sternocleidomastoid muscle and the midpoint at the level of the laryngeal prominence. Surface potentials were recorded for approximately 20 minutes at the patient’s established device settings.

Results

VNS‐evoked potentials were visualized in nine subjects (three subjects were excluded due to insufficient data). Two probable muscle artifacts were identified, defined as non‐propagating features in the mean response to n > 600 pulses of VNS. The first had an onset latency of ~1‐3 ms, and the second had a latency of ~7‐10 ms. Both artifacts appeared primarily in the electrodes overlying the laryngeal muscles. In one subject, propagating activity at an approximate velocity of 70‐110 m/s was visualized between 1‐2 ms latency.

Conclusion

The short latency of the first muscle artifact suggests stimulus signal leakage activating the superior laryngeal nerve. The propagating activity visualized in one subject may show the activity of the external branch of the superior laryngeal nerve, which stimulates the cricothyroid muscle to contract, creating the first muscle artifact. The second artifact with longer latency is likely caused by the recurrent laryngeal nerve, which causes contraction of intrinsic laryngeal muscles such as the posterior cricoarytenoids. Previous analyses may have mischaracterized the first muscle artifact as nerve activity [Usami, K., Kawai, K., Sonoo, M., and Saito, N. (2013). Brain Stimul. 6(4), 615‐623]. The use of MEA‐based recordings clarifies our understanding of the VN’s response to VNS, which may lead to better treatment efficacy and the eventual development of personalized VNS therapies for epilepsy and a range of inflammatory disorders.
Pre‐treatment with cinnamon extract alters MCF7 cell morphology and alters expression level of Akt1 signaling protein and SIRT1
Authors: Stockert Amy , Hrivnak William G. , Prak Khanneth
Published: May 2022

One area of cancer research is cancer prevention, specifically the use of polyphenols as anti‐cancer compounds. The proposed anti‐cancer effects of polyphenols include epigenetic changes in the genome that alter expression levels of a variety of genes. Cinnamon extract has high polyphenolic content that has been shown to regulate signaling proteins as well as key transcription factors. Observations suggest that a significant morphological change occurs with cinnamon treatment of MCF‐7 breast cancer cells, suggesting the epigenetic modifications may be occurring which alter the standard cell morphology. This study was aimed at determining if in addition to the morphological changes there were also expression changes in Akt1, FoxO1, and SIRT1, all genes which have been associated with epigenetic changes. Cells were grown to 70‐80% confluence and treated with either cinnamon extract or vehicle for 48‐96 hours, collected by trypsin release for RNA isolation in preparation of the real‐time PCR reactions used to measure relative gene expression levels of Akt1. Initial data suggests cinnamon extract pretreatment has only minor effects of SIRT1 expression but cinnamon extract may alter the activity of SIRT1. Future studies will evaluate the expression levels of FoxO1 and SIRT1 in order to determine if any correlation exists.
Delayed Reperfusion of the Renal Outer Medulla Attenuates Ischemia Reperfusion‐Induced Vascular Congestion
Authors: McLarnon Sarah R. , Sun Jingping , O'Connor Paul M.
Published: May 2022

Vascular congestion, or red blood cell (RBC) trapping, of the renal outer medulla (OM) is a common finding in acute tubular necrosis caused by ischemia reperfusion (IR). Work from our laboratory suggests that vascular congestion originates in the renal venous vasculature of the cortex and OM during the ischemic phase. Following reperfusion, RBCs then fill the OM plexus capillaries as the venous drainage vessels of this region remain blocked. We have also previously reported that pretreatment with low dose lipopolysaccharide (LPS) attenuates IR‐induced vascular congestion, however the mechanisms remain unknown. In the current study, we hypothesized that pretreatment with LPS prevents vascular congestion by delayed reperfusion of blood to the OM following ischemia.

To test this hypothesis, WKY rats (male, 12 weeks) were pretreated (i.p) with 1mg/kg LPS (n=6) or saline control (n=7) once daily for 3 days. Rats were then anesthetized, and Transonic Laser Doppler probes were inserted in the renal cortex and OM. Doppler flux, as a determinate of regional kidney blood flow was then measured over 10 minutes of baseline, 45 minutes of renal artery clamping and 30 minutes of reperfusion.

We found no differences in baseline blood flow of the cortex (pTREATMENT=0.0543) or OM (pTREATMENT=0.5085) between rats pretreated with low dose LPS or saline control. Following removal of the renal artery clamp, the return of blood flow to the cortex was gradual over the first several minutes of reperfusion (pTIME<0.0001), reaching a plateau following 10 minutes of reperfusion in both treatment groups (pINTERACTION=0.9999) (Fig. A). The reperfusion of blood to the OM, however, rapidly returned to baseline levels within 1 minute of reperfusion in control animals (1 min: 0 to 0.43AU) (Fig. B). In contrast, OM blood flow returned slowly in LPS treated rats (1 min: 0 to ‐0.08AU) and did not return to baseline levels during the 30‐minute reperfusion period (pINTERACTION=0.0127) (Fig. B).

These data indicate that LPS pretreatment, paradoxically, delayed early reperfusion of the renal OM via a regional vasoconstriction response. We speculate that delayed reperfusion of the OM attenuates medullary plexus congestion by allowing time for the RBCs in the shared venous circulation to clear. These findings support the hypothesis that reperfusion of the renal outer medullary plexus before cortical perfusion is restored, is responsible for the development of prolonged vascular congestion. New therapeutics focused on renal hemodynamics to prevent congestion after ischemia may prevent much of the injury associated with acute kidney injury.
Energetic costs of hindlimb‐dominated locomotion in sifakas (Propithecus verreauxi)
Authors: Grider‐Potter Neysa , Zeininger Angel , Schmitt Daniel , Snyder Megan , McGrosky Amanda
Published: May 2022

Human bipedal locomotion is unique, requiring a suite of musculoskeletal adaptations that were acquired gradually throughout hominin evolution. For example, lordotic lumbar spines move the center of mass over the hip joint and resists axial compression, short, laterally‐oriented iliac blades increase the leverage of the hip extensors and abductors, and the adducted hallux facilitates weight transfer during a propulsive toe‐off. Sifakas and other primates occasionally move bipedally in the wild, especially in terrestrial settings, but because they lack specialized anatomy, bipedal locomotion should be less efficient. In this study we test the hypothesis that energy costs differ among sifaka locomotor modes and predict that terrestrial bipedal locomotion is associated with greater energy expenditure than their more habitual mode of arboreal locomotion, vertical clinging and leaping. Bipedal (n=77) and vertical clinging and leaping (n=74) gait cycles were filmed in nine sifakas at the Duke Lemur Center. Locomotor data (stride length, peak body height of the cycle, and velocity) were measured from these videos using ImageJ and used to calculate energy expenditure using published equations. Linear mixed models were used to statistically test the differences in energy expenditure between locomotor modes while accounting for the effects of velocity and repeatedly measuring the same individuals. Results demonstrate that the cost of bipedalism is significantly greater than that of vertical clinging and leaping (p<0.0001). This result supports the idea that the locomotor adaptations in the hominin musculoskeletal system reduce the cost of locomoting bipedally. Furthermore, because sifakas rely on bipedal locomotion in terrestrial settings, these results have conservation implications for understanding the energetic demands of sifaka locomotion in deforested habitats.
Novel mitoNEET ligand NL‐1 improves therapeutic outcomes in an aged rat model of cerebral ischemia/reperfusion injury
Authors: Vijikumar Aruvi , Saralkar Pushkar , Geldenhuys Werner J. , Huber Jason D.
Published: May 2022

Cerebral ischemic stroke is a leading cause of mortality and disability worldwide. Currently, there are a lack of drugs capable of reducing neuronal cell loss after ischemia/reperfusion‐injury after stroke. Previously, we identified mitoNEET, a [2Fe‐2S] redox mitochondrial protein, as a putative drug target for stroke. In this study, we tested the novel mitoNEET ligand, NL‐1, in a preclinical model of ischemic stroke with reperfusion using aged female rats. Using a transient middle cerebral artery occlusion (tMCAO), we induced a 2 h ischemic injury and then evaluated the effects of NL‐1 treatment on ischemic/reperfusion brain injury at 24 and 72 h. Test drugs were administered at time of reperfusion via IV dosing. Results demonstrated that NL‐1 (10 mg/kg) treatment markedly reduced infarct volume and hemispheric swelling in the brain as compared rats treated with vehicle or a lower concentration of NL‐1 (0.25 mg/kg). Surprisingly, the protective effect of NL‐1 was significantly improved when encapsulated in PLGA nanoparticles, where a 40‐fold lesser dose (0.25 mg/kg) of NL‐1 produced an equivalent effect as the 10 mg/kg dose. Evaluation of changes in blood‐brain barrier permeability and oxidative stress using immunohistochemical staining corroborated the protective actions of NL‐1, showing reduced extravasation of IgG and decreased levels of 4‐hydroxynonenal (4‐HNE) in the brains of aged female rats at 72 h after tMCAO with reperfusion. Our studies indicate that targeting mitoNEET following ischemia/reperfusion‐injury is a novel drug target pathway that warrants further investigation.
Novel and Effective: ECG Utilization for MI Confirmation in Mouse Models
Authors: Broughton Philip , Troncoso Miguel , DeLeon‐Pennell Kristine
Published: May 2022

Introduction

Electrocardiography measures the electrical activity of the heart to generate an electrocardiogram (ECG) through a non‐invasive process that involves placing electrodes on strategic points on the body. When an MI occurs, the lack of contractility induces changes to the ECG, visualized by a prolonged QRS wave and amplification of the R wave. Previously, we identified the area under the QRS wave, 1 min after permanent ligation in a mouse model of myocardial infarction (MI) determined surgical success. The purpose of this study was to further demonstrate in a second cohort that the previous ECG parameters identified accurately confirmed that an MI has been administered by left anterior coronary artery ligation in real‐time.

Methods and Results

Permanent coronary artery ligation was performed on WT and CD8atmak1 (CD8‐/‐) mice (n=9/genotype); an ECG was recorded using a surgical monitoring platform and was analyzed on iWorkx LabScribe Data Acquisition and Analysis software. Previous analysis of the first dataset indicated no differences between sexes so both males and females were used in the second cohort. A receiver operating characteristic (ROC) curve was used to link changes in the QRS complex at the 1‐min mark with MI surgical success. By ROC analysis, the area under the QRS complex had an area under the curve (AUC) of 0.94 (p=0.01) indicating a strong association with MI success. To remove any potential effect of surgical platform variability, we normalized the 1 min data to pre‐MI values to determine fold‐change in the area under the QRS complex. ROC analysis of the normalized data also indicated a strong sensitivity and specificity for MI success with an AUC value of 0.90 (p=0.02). Mice in the second cohort were divided into two groups based on a cut‐off point for either the area under the QRS complex (≥0.003 mV/sec) or the normalized data (≥ 3.1 fold change in the QRS complex) to distinguish the two groups. Using the non‐normalized data alone, there was an 84% success rate with 2 false positives and 1 false negative. Analysis using the normalized data alone had an 74% success rate, with 2 false positives and 3 false negatives. None of these false positives or negatives were linked to genotype or sex.

Conclusion

Our data indicates that while changes in the QRS complex 1‐min after ligation may be useful for confirming MI surgical success, there are potential limitations that could skew the data. One possible explanation for the false positives could be variation between ECG boards. To remove this, we normalized post‐MI data to pre‐MI values but, this seemed to decrease effectiveness. It is also important to note that many of the false negatives were due to anomalies within the ECG such as the presence of R‐prime waves. For this reason, users must be consistent when analyzing the ECG data points to minimize variation. In summary, using the ECG data 1 min after ligation had a fairly strong prediction rate for MI surgical success.
Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS‐CoV‐2 Main Protease
Authors: D'Oliviera Angel , Geissler Evan P. , Mugridge Jeffrey S.
Published: May 2022

The SARS‐CoV‐2 main protease (MPro) is responsible for cleaving viral polypeptides to generate functional viral proteins, but like other viral proteases, MPro may also actively target and cleaa

ve endogenous human host proteins during infection. Here we show that a human tRNA methyltransferase, TRMT1, can be recognized and cleaved by SARS‐CoV‐2 MPro. TRMT1 is a SAM‐dependent methyltransferase that installs the N2,N2‐dimethylguanosine (m2,2G) modification onto position G26 of human tRNAs. This abundant m2,2G modification is important for global protein synthesis, cellular redox homeostasis, and has been linked to neurodevelopmental defects. Early in the COVID‐19 pandemic, a host‐virus protein interaction map identified a putative interaction between SARS‐CoV‐2 MPro and human TRMT1. We carried out kinetic assays that show both a short peptide sequence in TRMT1 as well as endogenous, full‐length TRMT1 are actively cleaved by recombinant SARS‐CoV‐2 MPro in vitro. Cleavage of full‐length TRMT1 results in loss of its C‐terminal zinc finger domain thought to be critical for tRNA binding. Furthermore, we determined the co‐crystal structure of the human TRMT1 peptide in complex with SARS‐CoV‐2 MPro, which shows that the bound TRMT1 peptide substrate adopts a unique conformation in the protease active site. These results suggest that tRNA methyltransferase TRMT1 is a viable substrate for SARS‐CoV‐2 MPro, provide new structural insights into the diverse substrate recognition properties of the viral protease, and raise the possibility that TRMT1 cleavage during SARS‐CoV‐2 infection could contribute to downregulation of host protein synthesis or other phenotypes of viral infection.
Genetic Evidence for A Critical Role of Intratubular Angiotensin II AT1aReceptors in The Proximal Tubules of The Kidney in Two‐Kidney, One‐Clip Goldblatt Hypertension in PT‐Agtr1a‐/‐Mice
Authors: Li Xiao C. , Hassan Rumana , Leite Ana P. O. , Dugas Courtney , Sato Ryosuke , Sato Akemi , Zhuo Jia L.
Published: May 2022

It was previously reported the activation of the renin‐angiotensin system (RAS) in the clipped kidney plays a critical role in the development of two‐kidney, one‐clip Goldblatt hypertension (2K1C). However, most of previous studies used the global angiotensin II (Ang II) type 1a (AT1a) receptor (Agtr1a‐/‐) knockout model or used systemic AT1receptor blockers (ARB) to determine renal mechanisms of 2K1C hypertension. The roles of intratubular AT1 (AT1a) receptors or its downstream target Na+/H+ exchanger 3 (NHE3) in the proximal tubules in the development of 2K1C hypertension has not been determined previously. In the present study, we tested the hypothesis that genetic deletion of AT1 (AT1a) receptors selectively in the proximal tubules attenuates the development of 2K1C hypertension via AT1 (AT1a) receptor‐mediated, Na+/H+ exchanger 3 (NHE3)‐dependent mechanisms. To test the hypothesis, 2K1C Goldblatt hypertension was induced by placing a silver clip, 0.12 mm internal diameter, on the left renal artery for 4 weeks in adult male wild‐type (WT), global Agtr1a‐/‐, proximal tubule (PT)‐specific PT‐Agtr1a‐/‐, or PT‐Nhe3‐/‐ (Na+/H+ exchanger 3) mice, respectively. As expected, systolic blood pressure increased in a time‐dependent manner in wild‐type mice, reaching a maximal response by Week 3 (Basal: 112 ± 2 vs. 2K1C: 149 ± 4 mmHg, n=12, P<0.01). 2K1C hypertension in WT mice was associated with increases in renin mRNA expression in the clipped kidney (Control: 2066 ± 255 vs. Clipped: 3144 ± 569 copies/ng RNA, P<0.01), and decreases in renin mRNA expression in the nonclipped right kidney (1738 ± 341 copies/ng RNA, P<0.05). Plasma Ang II levels were significantly increased in WT mice with 2K1C hypertension (Control: 50.2 ± 7.2 vs. 2K1C: 109.7 ± 17.2 pg/ml, P<0.05). Glomerular and tubulointerstitial fibrotic responses were also significantly increased in the clipped kidney (P<0.01). By comparisons, the development of 2K1C hypertension was entirely prevented in Agtr1a‐/‐ (Basal: 88 ± 4 vs. 2K1C: 92 ± 2 mmHg, n=9, n.s.), or significantly attenuated in PT‐Agtr1a‐/‐ mice (Basal: 101 ± 2 vs. 2K1C: 104 ± 4 mmHg, n=12, n.s.) or in PT‐Nhe3‐/‐ mice (Basal: 103 ± 3 vs. 109 ± 5 mmHg, n.s.). Renin mRNA expression was not different in clipped and nonclipped kidney of Agtr1a‐/‐ mice, but it was decreased in the nonclipped kidney of PT‐Agtr1a‐/‐ mice (P<0.05). Furthermore, glomerular and tubulointerstitial fibrotic responses were also markedly attenuated in the clipped kidney in PT‐Agtr1a‐/‐ mice (P<0.05). We conclude that the results of the present study provide strong evidence for a critical role of AT1a receptors and NHE3 in the proximal tubules of the kidney in the development of 2K1C Goldblatt hypertension in mice.
Protectins Inhibit Estrogen Receptor Negative Breast Cancer Growth in Mice
Authors: Kipper Franciele C. , Kelly Abigail , Rothenberger Eva , Duncan Madeline , Serhan Charles N. , Panigrahy Dipak
Published: May 2022

Cytotoxic breast cancer therapies reduce tumor burden by killing tumor cells, yet simultaneously generates tumor cell debris. Apoptotic and necrotic debris that are not cleared by the immune system create a pro‐inflammatory microenvironment, fueling tumor burden by disrupting the resolution of inflammation. A paradigm shift is emerging in understanding the resolution of inflammation as an active biochemical process with the discovery of novel specialized pro‐resolving lipid autocoid mediators (SPMs), such as resolvins (Rvs) and protectin conjugates in tissue regeneration (PCTRs), as well as endogenous resolution programs. We have previously reported in mice models that resolvins (e.g. RvD1, RvD2, and RvE1) reduce tumor growth, however the role of protectins in cancer remains unknown. To assess whether stimulating the resolution of inflammation would inhibit breast cancer progression, we utilized protectin (PCTR1) to treat established models of estrogen receptor (ER) positive (EO771) and negative breast cancer (4T1). PCTR1 sharply reduced tumor growth in the EO771 and 4T1 breast cancer models at only nanogram concentrations (15 ng/day) without toxicity when administered alone or in combination with chemotherapy (paclitaxel) and/or immunotherapy (anti‐cytotoxic T‐lymphocyte‐associated protein 4, CTLA4). Notably, PCTR1 inhibited tumor growth in combination with immunotherapy (anti‐CTLA4) or immunotherapy plus chemotherapy (paclitaxel) in ER positive (EO771) breast cancer. Also, PCTR1 inhibited ER negative tumor growth (4T1) alone and in combination with paclitaxel and/or anti‐CTLA4. Remarkably, PCTR1 alone or in combination with chemotherapy (paclitaxel) reduced gene expression and protein levels of pro‐angiogenic factor CXCL12/SDF‐1 in the tumor microenvironment. PCTR1 dampened the therapy‐induced cytokine storm, by counter‐regulating levels of TNF‐α, CCL2/MCP‐1, CXCL10, CCL5, CXCL13, and IL‐1β in vivo. Taken together, we demonstrate that PCTR1, a pro‐resolution lipid mediator, inhibits breast cancer progression via debris clearance and counter‐regulation of the cytokine storm. The use of protectins to enhance immunotherapy is a novel approach to inhibit breast cancer progression via resolution of inflammation that remains to be evaluated in humans.
Using digital innovation to support anatomy and surgical education during the pandemic
Authors: Jayanth Aiden , Stone Danya , Dhanda Jagtar , Smith Claire F
Published: May 2022

The COVID‐19 pandemic has had a detrimental impact on healthcare education. With restricted access to traditional teaching aids such as the cadaver, educators had to adapt their teaching practise to minimise disruptions to students’ educational process. However, cadaveric dissection is highly regarded as a key component of anatomical and surgical education. This was particularly challenging when teaching was purely online due to pandemic restrictions. To accommodate the shift to online delivery only, the Anatomy Department at Brighton and Sussex Medical School (BSMS) adopted a variety of synchronous and asynchronous teaching methods. As part of this strategy, the department extended its provision of anatomy teaching by live streaming cadaver dissections to first year medical students (n=220). BSMS was the first UK medical school to use this innovative digital tool. A Virtual Reality in Medicine and Surgery (VRiMS) course was also developed to provide training to surgeons worldwide. This work explores the place of cadaver‐based education in a true blended learning provision.

Cadaver dissections were live streamed from the dissection room using operating theatre lights and a 4K camera via Microsoft Teams. These sessions were also interactive in nature, allowing students to ask questions throughout the dissection. Student feedback was collected through module and end of year evaluations. The VRiMS course provided a platform to demonstrate surgical procedures on cadavers using live streaming with virtual reality or 360 cameras. Surgeons were able to choose multiple camera angle perspectives through virtual reality headsets. Course participation data was used to determine the useability of this novel method of teaching and training.

Module evaluation showed that the overall quality of delivery for anatomy practical sessions was rated as good and very good by 86% of the cohort. Moreover, 93% of students found the material to be just right. In the end of year evaluation, students have indicated that live streamed dissection sessions were one of their favourite aspects and has had a positive impact on their time at BSMS. In total, 1354 participants from around the world have watched 923 hours of live broadcasted content across four VRiMS courses to date. The course has seen a 52% increase in participation rate since its launch.

The evaluation findings suggest that a hybrid learning approach with the use of live streaming for anatomical education has had a positive outcome on student satisfaction. Furthermore, VRiMS course participation data indicates that virtual reality has a promising future in surgical training. Live streaming and virtual reality have proved successful in bringing the dissection room to medical students and surgical trainees during unprecedented circumstances. This approach has the potential to prevent the complete loss of interactive cadaver‐based education and a probable solution to cadaver scarcity.
Interactions between the aNT Domains of Human V‐ATPases and Phosphatidylinositol Phosphate Lipids
Authors: Mitra Connie , Kane Patricia M.
Published: May 2022

Each organelle in a eukaryotic cell has a tightly regulated pH important for organelle function and cell growth. This distinct pH defines organelle identity and is maintained principally by vacuolar H+‐ATPases (V‐ATPases). V‐ATPases are highly conserved, ATP driven proton pumps comprised of a cytosolic V1 domain, and an integral membrane bound Vo domain. The cytosolic N‐terminal domain of the Vo a‐subunit is known to modulate the organelle specific regulation and targeting of V‐ATPases. However, the mechanisms for targeting V‐ATPases to distinct membranes and achieving organelle‐specific regulation are incompletely understood. Importantly, loss of function in V‐ATPase a‐subunit isoforms is associated with human diseases such as osteopetrosis, distal renal tubule acidosis, and cutis laxa type‐II. Organelles also have characteristic phosphatidylinositol phosphate (PIP) lipids in the outer leaflet of their membranes. Previous studies have demonstrated that the N‐terminal domain of yeast Vo a‐subunit isoforms, Vph1NT and Stv1NT, interact with distinct PIP lipids in their resident organelle and can affect activity, regulation, and localization of V‐ATPases containing these isoforms. We hypothesize that V‐ATPases and PIP lipids interact with the NT domains of human Vo a‐subunit isoforms, and these interactions regulate activity and targeting of V‐ATPase, thereby impacting pH‐dependent functions of the organelle. The Hua1 isoform resides in lysosomes of many cells and in synaptic vesicles of neurons, and the Hua2 isoform functions in Golgi and endosomes of multiple cell types. We have expressed the N‐terminal domains of human Vo a‐subunit isoforms Hua1NT and Hua2NT in E.coli, then purified and tested their specificity for different PIP lipids in a liposome pelleting assay. Hua1NT shows a preference for liposomes containing PI(3)P or PI(3,5)P2, lipids typically enriched in endosomes and lysosomes, while Hua2NT shows a preference for Golgi‐enriched lipid PI4P. Modeling on existing structures has identified potential PIP binding sites in the HuaNT domains, which were mutagenized and tested for PIP lipid specificity. We have identified PIP‐specific binding sites for both Hua1NT and Hua2NT. We will assess PIP‐specific membrane recruitment of wild‐type and mutant HuaNTs by monitoring their localization when transiently expressed in mammalian cells. Together, these data show that the association between V‐ATPase subunit isoforms and PIPs is preserved in mammalian cells. Defining PIP binding codes on V‐ATPase will improve our understanding of organelle specific pH control and could provide new avenues for controlling V‐ATPase subpopulations.
A Yeast Model System to Study the Human Orthologs of the Ribosome‐Associated Complex
Authors: Marley Amanda , Kim Seongchan , Fandino Pachon Nikole , Zeibekis Theodora , Kitterman Erica , Kelly Christina , Cameron Dale M.
Published: May 2022

Prions are a class of misfolded proteins that form protein aggregates that are infectious and can self‐propagate, causing human diseases like Creutzfeldt‐Jakob. Additionally, many other diseases are known to involve protein misfolding and formation of amyloid aggregates as a characteristic of their pathology. To suppress the formation of misfolded proteins, cells have evolved various quality control systems, including chaperone proteins that can help protect nascent polypeptide chains from misfolding. Saccharomyces cerevisiae, also known as brewer’s yeast, has been used as a model organism to study protein misfolding and prion formation, including the yeast [PSI+] prion formed by the misfolding of the translation termination factor Sup35. The ribosome‐associated complex (RAC) is a chaperone complex composed of two subcomponents, in yeast called Zuo1 and Ssz1. Previous work from our lab and others has shown that the presence of the RAC on ribosomes decreases the frequency of the formation of the [PSI+] prion and reduces the toxicity of aggregation‐prone polypeptides. The human ortholog of yeast RAC is composed of Mpp11 and Hsp70L1. Previous studies have shown that human RAC can rescue the growth defect associated with deletion of the yeast RAC components. We therefore hypothesized that the function of the RAC complex, including its ability to suppress prion formation and toxicity of aggregation‐prone proteins, may be conserved from yeast to humans. We transformed yeast lacking the RAC components Zuo1 and Ssz1 with plasmids expressing the human orthologs of these chaperones. Growth assays confirmed previous studies showing that the human RAC components partially restore growth to yeast lacking endogenous RAC. In addition, like the yeast counterparts, human RAC reduces the formation of the [PSI+] prion. This result highlights the potential of the yeast model system to investigate the roles of human chaperones in modulating protein folding and misfolding. To further investigate the potential of this yeast model system, we have studied the ability of the human RAC components to counter the toxicity of several human disease‐associated proteins expressed in yeast, including those with polyQ repeats, the amyloid‐β peptide, and □‐synuclein, which are associated with Huntington’s, Alzheimer’s, and Parkinson’s Diseases in humans, respectively. The results provide insights on the use of S. cerevisiaein studying human diseases involving protein misfolding.
Using A Community of Inquiry in the Design of Histology Curriculum for First Year Medical Students
Authors: McIntosh Leslie P. , Koch Douglas , Claeson Kerin , D'Angelo Marina
Published: May 2022

Histology faculty in the Doctor of Osteopathy (DO) program at the Philadelphia College of Osteopathic Medicine revised the delivery methodology of first‐year histology content in order to improve student performance, increase social interaction among students and faculty, shift to an all‐distance learning environment, and align curricula across our three campuses. This abstract describes an Action Research approach to implementing a Community of Inquiry (CoI) model of learning during a time of unprecedented change at both the program and the global level. An Action Research approach is a pragmatic methodology for improving medical education practice by incorporating change in a collaborative, reflective, cyclical, planned, and practical manner. We chose the CoI model of learning as it is a collaborative‐constructivist approach to instruction and well suited for online and team‐based learning. This model encompasses three prongs to delivering content: a teaching, social, and cognitive presence. In our curriculum, the teaching presence is composed of curated content in the form of self‐guided learning activities (videos, text, assessments) delivered through the Tophat learning platform and organized to support retrieval, interleaving, and spaced learning; the social presence involves student interactions online and in the classroom through discussion boards and team‐based learning activities (independent and faculty‐led); and the cognitive presence takes place with students interacting with learning modules, posting and responding to discussion threads, taking assessments, receiving feedback from assessments and engaging in problem solving exercises during the small‐group learning sessions. Within this model, students are encouraged to take responsibility for self‐directed learning, allowing for personalized investment in life‐long learning. CoI provides a platform to implement the best evidence‐based practices for histology instruction across three campuses with varying student population sizes and educational needs. Early data suggests an increase in student engagement and an improvement in student performance. We believe our model provides insight into effective curricular design for the ever‐changing face of undergraduate medical education.
Investigating the chromatin‐modulated transcriptional response of cancer cells to drug treatment
Authors: Vélez Velázquez Jaleisha J. , Frederick Jane , Backman Vadim
Published: May 2022

Macromolecular crowding within the nucleus contributes to heterogeneity in chromatin packing and thus plays an important role in the regulation of gene expression. Nuclear crowding models predict that a change in chromatin structure will affect gene accessibility and transcription, which may have important functional consequences in cellular stress response. Since these studies have focused on ovarian adenocarcinoma, we aim to determine whether this theory can be extended to other types of cancer. Using differential gene expression analysis on bulk RNA‐sequencing data and partial wave spectroscopic (PWS) microscopy imaging of colon and mutant ovarian cancer cell lines, we see that the impact of crowding and chromatin packing on transcription can be extended to other cancer types. Comparison of these results with predictions from the chromatin packing‐macromolecular crowding model (CPMC), which relates physical changes in chromatin structure to changes in gene expression patterns, further showed that the complex bidirectional response of transcription to crowding is not cell‐line specific. Additionally, we show that the transcriptional response changes when the cellular stressor is a chemotherapeutic agent rather than a benign ion modulating compound. The results have important implications in understanding the role of chromatin structure in cellular stress response and drug resistance in cancer.
Biochemical Characterization of SARS‐CoV‐2 Spike RBD Mutations and Their Impact on ACE2 Receptor Binding
Authors: Hoter Abdullah , Naim Hassan Y.
Published: May 2022

Infection of mammalian cells by SARS‐CoV‐2 coronavirus requires primary interaction between the receptor binding domain (RBD) of viral Spike protein and the host cell surface receptor angiotensin converting enzyme 2 (ACE2) glycoprotein. Several mutations in the RBD of SARS‐CoV‐2 spike protein have been reported for several variants and resulted in wide spread of the COVID pandemic. For instance, the double mutations L452R and E484Q present in the Indian B.1.617 variant have been suggested to cause evasion of host immune response. The common RBD mutations N501Y and E484K were found to enhance the interaction with ACE2 receptor. In the current study, we analyzed the biosynthesis and secretion of the RBD double mutant L452R and E484Q in comparison to the wild‐type RBD and the individual mutations N501 and E484K in mammalian cells. Moreover, we evaluated the interaction of those variants with ACE2 by means of expression of the S protein and co‐immunoprecipitation with ACE2. Our results revealed that the double RBD mutations L452R and E484Q resulted in a higher expression level and secretion of Spike S1 protein as compared with other mutations. In addition, an increased interaction of these mutant forms with ACE2 in Calu3 cells was observed. Altogether, our findings highlight the impact of continuous S1 mutations on the pathogenicity of SARS‐CoV‐2 and provides further biochemical evidence for the dominance and high transmissibility of the double indian mutations
Diabetic Triple Negative Breast Cancer: Polyamine Enzymes, Ornithine decarboxylase and Spermine oxidase as Potential Targets
Authors: Tofilau Hadassha M. , Casero Robert , Chandra Surabhi
Published: May 2022

Breast cancer (BC) is the second leading cause of death in women. Co‐existence of diabetes and BC, termed diabetic BC, can be lethal, leading to higher mortality (15‐40%) than BC alone. In triple negative breast cancer (TNBC) patients, chance of TNBC recurrence is about 3 months, and chance of mortality can be as high as 75%. TNBC is highly refractive, heterogenous, and resistant to most chemotherapies. Polyamines, which are ubiquitous in most cells, and play a role in cell growth, have been shown to be elevated in cancer, though their role in diabetic BC has not been explored. We hypothesized that high glucose/diabetic conditions increase TNBC cell proliferation through modification of the enzymes (ornithine decarboxylase and spermine oxidase) in the polyamine pathway. The objective was to determine potential targets in the polyamine pathway to mitigate TNBC advancement in diabetic conditions. For this purpose, MDA‐MB‐231 (TNBC) and MCF10‐A (normal breast epithelial) cell lines were used. Both were treated with low (5mM) and high glucose (25mM) concentrations and effects on polyamine levels, polyamine enzyme, and cell proliferation were monitored. Polyamine levels were assayed using reverse phase high performance liquid chromatography. Cell proliferation was assessed using a fluorescence‐based assay, and polyamine enzyme expression was monitored at the mRNA and protein level using RT‐PCR and Western Blots. Samples were sent to Johns Hopkins University for polyamine enzyme activity analysis. Further, an approved inhibitor of polyamine synthesis, alpha‐difluoromethylornithine (a‐DFMO, 5mM) was used in combination with glucose treatments. Under diabetic/high glucose conditions, putrescine levels were elevated in MDA‐MB‐231 cells (135%), which correlated with higher cell proliferation (123%). Polyamine enzyme activity of ODC increased (from ~75 pMolCO2/hr/mg protein to ~150 pMolCO2/hr/mg protein) and SMOX decreased (from ~0.4 pMolH202/min/mg protein to ~0.06 pMolH202/min/mg protein) in concert with changes observed at protein levels with high glucose exposure. DFMO was effective in reducing polyamine levels and cell proliferation, however re‐supplementation of polyamines (spermidine and spermine) restored cell proliferation to that, indicating that DFMO is only cytostatic. MCF‐10A cells showed marked elevation in cell proliferation with high glucose. DFMO prevented this increase; however no apparent change was observed in polyamine levels. The polyamine biosynthetic enzymes (ornithine decarboxylase and spermine oxidase) play an active role in increasing cell proliferation under high glucose conditions in TNBC. Since DFMO caused only cytostatic effects, there is a need for a more targeted approach to regulate polyamine levels and mitigate TNBC growth under diabetic conditions.
Effect of di‐ethylhexyl phthalate (DEHP) on androgen related proteins in JEG‐3 placental cells
Authors: Kruger Laken , Prasad Bhagwat
Published: May 2022

The placenta is a critical fetal endocrine organ that plays an important role in fetal development, xenobiotic detoxification, and sex steroid homeostasis. Although the importance of the placenta in endocrine homeostasis is known, the effect of endocrine disruptors, like phthalates, on placental endocrine function needs further exploration. A common phthalate that expectant mothers can be exposed to is di‐ethylhexyl phthalate (DEHP) which is used to make flexible plastics. We aim to determine the effect of DEHP on the androgen related proteome in JEG‐3 placental cell model.

JEG‐3 cells were seeded in 6‐well plates at 100,000 cells per well and treated with 0 (vehicle control), 1, 10, and 100 mM DEHP for 72 hours. The cells were harvested, homogenized, and protein sample was digested using an optimized protocol. Global proteomics data were acquired using a Q‐Exactive HF Quadrupole‐Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). The data were processed with MaxQuant (version 1.6.8.0) using the Andromeda search engine to search the high‐resolution MS/MS spectra against the human proteome database curated from Uniprot. Label‐free quantification (LFQ) intensities of androgen related proteins were compared across control and treatment groups.

Out of the total 2368‐ 2530 proteins detected, 2085 proteins were common in control and all treatment groups. The control group had 49 unique proteins and DEHP treatment showed 63, 42, and 119 unique proteins to 1, 10 and 100 mM, respectively. ABCG2 was uniquely identified in the higher DEHP treatment group. DEHP treatment elevated the levels of aromatase (CYP19A1) and aldo‐keto reductase 1B1 (AKR1B1) in a dose‐dependent manner with a 291% and 59% increase in the 100 mM treatment, respectively. Steryl‐sulfatase (STS), estradiol 17‐beta‐dehydrogenase 11, and aldehyde dehydrogenase 3A2levels were unaltered by the DEHP treatments.

Determining how environmental endocrine disruptors, like DEHP, affects the placental endocrine pathways is vital to mitigating the toxicity. The elevation of aromatase with DEHP treatment may lead to estrogenic effects as CYP19A1 converts androstenedione and testosterone to estrogens, resulting in hormone imbalance which can cause adverse fetal outcomes both physically and neurodevelopmentally.
Three‐Dimensional Visualization and Measurement of the Liver and Gallbladder and Their Spatial Orientation
Authors: Cui Dongmei , Yang Gongchao , Garza Andrew W. , Meyer Edgar R. , Sinning Allan R.
Published: May 2022

The liver and gallbladder are part of the hepatobiliary system and they are common sites for many diseases. The acquisition of understanding of and familiarization with the anatomical relationship of the liver and gallbladder may help medical students and clinicians understand the anatomical relationships and relevant to diagnosing and surgical treatment diseases. In this study, the curvature angle and length of the liver and gallbladder have been measured using two‐dimensional (2D) and three‐dimensional (3D) measurements. The models were created using semi‐auto combined rendering methods from computed tomography angiography (CTA) images. The visualizations of the liver and gallbladder can be viewed in 360 degrees via stereoscopic presentation in virtual space, and the spatial orientations of these organs can be viewed via zoom free rotation features. Images of xy (transverse), xz(coronal) and yz(sagittal) views were captured to display the models anatomical relationship. The measurement of the models, in turn, are used to compare the size and shape of the liver and gallbladder in 2D and 3D curvature angle and length. These visualization and measurements might have the potential benefits of helping students and clinicians to better understand anatomical knowledge in medical education and clinical training. In addition, these stereoscopic visualizations and measurements might enhance surgeon’s abilities to perform diagnostics and procedures whose successful completion is dependent upon advanced knowledge of the anatomical relationships between the liver and gallbladder.
A Computational Study of the Dissociation of CaS Nanoclusters in the Extracellular Fluid of Cancer Cells: the Effect of pH in the Structure and Stability of Protonated CaS Nanoclusters
Authors: Soto Acevedo Angeliz A. , Rodríguez Martínez Olga M. , Colón Colón Carolina C. , Castro Rosario Miguel E.
Published: May 2022

The acidic pH found in the extracellular fluid of many cancer tumors is an important difference from the slightly basic pH found in the corresponding benign cells. Contrary to common anti‐cancer drugs that have significant side effects in healthy cells, calcium sulfide (CaS) nanoclusters have shown to induce apoptosis selectively in human lung, skin, and breast cancer cell lines in vitro. We hypothesize that CaS nanostructures selectively dissociate in the characteristic acidic extracellular environments of cancer cells into free Ca2+ ions and sulfides such as H2S, HS‐ and S2‐, which can be proved via bench and computational experiments. Theoretical calculations at the DFT/B3LYP/DGVZVP level of theory of bare and protonated (CaS)n clusters (n = 1 to 4) were performed to address this hypothesis using the Gaussian 16W and GaussView 6.0.16 programs by varying the number of protons in a CaS cluster to assess cluster stability and reaction pathways in both acidic and neutral media. The total cluster energy decreases with the number of monomer units (n) in the (CaS)n clusters. The CaS monomer and dimer are more stable in water than in their corresponding standard state by about 336kJ/mol and 390kJ/mol, respectively. A single protonation of a sulfur atom in the solvated monomer results in an increase in the Ca‐S bond length from 2.55Å to 2.73Å, indicative of increasing drive to dissociate. It requires protonation of a sulfur atom by two hydrogen atoms to dissociate the cluster into free Ca2+ and H2S. Water facilitates the dissociation of the clusters when the sulfur atom is bonded by two protons. The products of the reaction of the solvated (CaSH2)2+ is more stable than the corresponding gas phase cluster ion by a significant 1479 kJ/mole. These results are consistent with the dissociation of CaS clusters to free Ca2+ and H2S in acidic media like the one found in the extracellular fluid of malignant tumors. Ca2+ ions are known to cause cell damage in cytosolic concentrations higher than 500nM. The biochemistry of H2S is still under study by several groups worldwide but it is generally accepted that it can be genotoxic and induce apoptosis. Therefore, CaS nanoclusters are promising drugs to selectively inhibit malignant tumor growth with little impact on benign tissue. The results are discussed in terms of the basic requirements of a (CaS)n‐based formulation for cancer treatment.