bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Sphingomyelin Synthesis by Cholesterol
Authors: Kim Yeongho , Burd Christopher G.
Published: May 2022

The most abundant sphingolipid of the plasma membrane (PM), sphingomyelin (SM), binds to cholesterol in a manner dependent on the structure of fatty acids in the SM, and it is a major determinant of the cholesterol content of the PM. Synthesis of SM in the Golgi apparatus produces an mol‐equivalent of diacylglycerol (DAG), a signaling lipid implicated in the fission of secretory vesicles from the Golgi. We speculate that coupled synthesis of SM and DAG in the terminal compartments of the Golgi results in fission of secretory vesicles enriched in SM and integral membrane proteins that favor sphingolipid and cholesterol‐rich membrane. Two classes of SMs are synthesized in the Golgi apparatus that are distinguished by the length of the fatty acyl chain on the ceramide backbone: long chain (LC; ≤C20), and very long chain (VLC; >C20) SMs. Whereas the precursor to LC‐SMs is supplied to the Golgi via non‐vesicular transport of LC‐ceramide from the ER to the Golgi by ceramide transport protein (CERT), it is speculated that VLC‐ceramide is supplied to the Golgi via bulk membrane trafficking pathways. From the Golgi, LC‐SM and VLC‐SM are trafficked to the PM in SM‐rich secretory vesicles that also contain a select cohort of secreted proteins (Deng et al., 2018).

Cholesterol extraction increases sphingomyelin synthesis (Perry and Ridgway, 2006), however, the two classes of SMs are differentially regulated; we discovered that only synthesis of very long chain (>C20) SMs was increased. Furthermore, cholesterol loading reduced the synthesis of VLC‐SM, only. We then asked, what cellular components regulate VLC‐SM synthesis and how do they influence the cholesterol content of the PM and other organelles. Synthesis of VLC‐SM was inhibited by interfering with COPII‐mediated export from the ER and by depletion of ER‐localized ceramide synthase 2, which produces VLC‐ceramide. Reduced VLC‐SM synthesis resulted in the depletion of cholesterol from the PM inner leaflet and a concomitant accumulation in the cytoplasmic leaflets of the membranes of lysosomes and other intracellular organelles. Increased synthesis of VLC‐SMs by cholesterol depletion resulted in increased the level of diacylglycerol (DAG) in the Golgi and the appearance in cytoplasmic vesicles containing TGN46 which is secreted in SM‐rich secretory vesicles. The results suggest that VLC‐SM synthesis is regulated by cholesterol content on the PM and cholesterol depletion‐induced synthesis of VLC‐SM enhances SM‐specific secretory pathway from the Golgi to the PM.
Proteasomes in the Cells of the Ocular Surface in Health and Disease
Authors: Bardag Gorce Fawzia , Wolosin Mario J. , Oliva Joan , Niihara Yutaka
Published: May 2022

Purpose

Limbal stem cell deficiency (LSCD) is associated with corneal conjunctivalization that compromises corneal transparency and causes visual impairment. Previously, we found marked changes in proteasome population in corneas of rabbit with LSCD, and showed immunoproteasome (IPR) presence in central corneas during conjunctivalization compared to healthy rabbit cornea. In the present study, we further map the distribution of proteasomes population in the ocular surface.

Methods

gene expression intensity obtained by microarray analysis of human healthy corneas and conjunctiva previously reported by the group of Dr. Wolosin (Turner H.C. et al 2007) was used to conduct this study. Constitutive proteasome (CPR) subunits (B5, B2, and B1) were compared to the inducible immunoproteasome (IPR) subunits (B5i, B2i, and B1i). The ratio between CPR and IPR subunits was calculated for cornea and for conjunctiva.

Results

The comparison of the ratio B5/B5i, B2/B2i and B1/B1i between cornea and conjunctiva showed a significant high expression of CPR in corneas and a high expression of IPR in conjunctivae. In addition, PA28, the regulatory complex of IPR, and the major histocompatibility complex, class I A and B, were found significantly expressed in conjunctiva. Keratins K3/K12 were specifically expressed in cornea and keratins K4/K13 were specifically expressed in conjunctiva, which could be used to normalize the data analysis.

Conclusion

The results indicated that conjunctivae contain high levels of IPR and that during conjunctivalization, corneas lose CPR to express IPR. The ration CPR/IPR could be used as an indication of conjunctivalization level in case of corneal injuries.
Needs Assessment of Essential Anatomy ‐ A Perspective from Primary Care Residents
Authors: Harmon Derek J. , Hankin Mark H. , Martindale James , Niculescu Iuliana
Published: May 2022

Introduction

. Curricular development begins with identifying a problem, followed by a need assessment of the curricular components. Changes in pedagogical approaches and reductions in curricular time for gross anatomy have been identified as a problem. The objective of this study was to conduct a needs assessment of the most essential anatomy from the perspective of primary care residents.

Methods

. Primary care residents in Family Medicine (FamMed) and General Internal Medicine (IntMed) at Oakland University William Beaumont School of Medicine were recruited for the study. A survey assessed the clinical importance of 907 specific anatomical structures from all anatomical regions on a 1‐4 Likert‐scale (1= “not important and 4 = “essential”). The structures were selected from the bold terms in Gray’s Anatomy for Students, Clinically Oriented Anatomy, and Grant’s Dissector. In the analyses, all 3 and 4 ratings for each structure were summed and divided by the total number of respondents. A structure rated as a 3 or 4 by ≥75.0% of respondents was classified as “essential”, 50.0‐74.9% “more important”, 25.0‐49.9% “less important”, and 0.0‐24.9% “not important”. Kruskal‐Wallis analyses were conducted to assess differences between the two residency groups for each region.

Results

. Fifty adult primary care residents in FamMed and IntMed completed the survey. In total, 16.7% of structures were classified as essential, 58.9% more important, 24.3% less important, and 0.7% not important. FamMed rated structures categorized as skeleton, nerves, fasciae, anatomical spaces, blood vessels, lymphatics and surface anatomy (p<0.0001), and muscles (p=0.002) significantly higher than IntMed, but there was no significant difference in organs (p=0.012). It was notable that 100.0% of cranial nerves were classified as essential, and 94.5% of surface anatomy structures were classified as essential or more important. There were no statistically significant differences between the two groups of residents for any of the anatomical regions.

Conclusion

. Conducting a needs assessment is a crucial early step in curricular development. This is the first study to conduct a needs assessment of anatomy curricular content solely from the perspective of primary care clinicians. Surface anatomy was an area with nearly all structures rated as essential or more important, which reinforces the importance of including surface anatomy in pre‐clinical gross anatomy education.

Significance

. The data from this study provides insight into how clinicians rated specific anatomical structures. Due to the broad variation in medical curricula, the authors suggest that the classifications of importance by these clinicians can serve as a curricular roadmap by anatomy educators for the evaluation, revision, or development of curricula with regard to gross anatomy education in the context of reduced contact hours, new pedagogies, and vertical integration.
Mushroom Tyrosinase Assay is used to Teach Enzyme Kinetics and Inhibition Studies in an Undergraduate Biochemistry Course
Authors: Unnoppet Kylee , Hite Kristopher
Published: May 2022

A spectrophotometric mushroom tyrosinase assay was optimized for use in the undergraduate biochemistry laboratory course at Virginia Tech. The laboratory exercise is designed to teach students about enzyme kinetics and tyrosinase‐inhibitor interactions. The tyrosinase protein structure was first studied using the python based molecular visualization software PyMOL and a PDB ID from the Protein Data Bank. The PDB ID that was used was PY9W and displays a H(2) L(2) tetramer. The H subunits of the complex contain a copper binding site that is coordinated by 3 histidine residues. The structure/function relationship was further studied using an immersive modeling hypercube. The assay uses white button mushrooms as the source of the tyrosinase enzyme, l‐3,4 dihydroxyphenylalanine (L‐DOPA) as the substrate, and kojic acid as the inhibitor. The optimal amounts of enzyme, substrate, and inhibitor are first determined, then a series of experiments are conducted which test varying concentrations of L‐DOPA against varying concentrations of kojic acid. Change in absorbance is measured at 475 nm in a spectrophotometer and the data is plotted to determine the kinetic constants and type of inhibition. Experimental kinetic constants are then compared to previously reported values. Further protein concentration in the tyrosinase extract is determined using Bradford assays. The incorporation of this exercise into the undergraduate biochemistry laboratory course at Virginia Tech will allow students to explore enzyme kinetics and tyrosinase‐inhibitor interactions.
Tissue and Sex‐Specific Changes in RNA Editing During Induced Acute Inflammation
Authors: Nichols Claire E. , Kendrick Kelsey R. , Macander Bridgette M. , Rivas Christian , Ulbricht Randi J.
Published: May 2022

A‐to‐I RNA editing is a process where adenosine (A) nucleotides are deaminated by an editing enzyme, ADAR1, to become inosines (I) in select RNA transcripts. RNA editing can affect the sequence of the encoded protein as well as the regulation of the RNA. ADAR1 also plays a role in regulating innate immunity and is upregulated during inflammation. Current data on the effects of increasing ADAR1 on RNA editing is limited, and most studies are completed only in male mice. We are interested in expanding RNA editing data to include female animals. Lipopolysaccharide (LPS) was used to induce inflammation and increase ADAR1. Organs were dissected four hours after LPS injection and RT‐PCR was used to amplify regions around editing sites of known targets. The amplicons were sequenced and analyzed by measuring the amount of nonedited nucleotides and edited nucleotides at select editing sites. Inflammation was verified in the LPS injected mice through qRT‐PCR of ADAR1 and inflammatory cytokines. Inflammation led to editing levels in male mice that were distinctly different than female mice. There was also a difference in editing between the heart and the brain. This indicates a complex system where RNA editing is regulated in a tissue‐specific and sex‐specific manner. The tissue and sex‐specific changes in editing may provide insight into a mechanism by which tissues adapt to inflammation in unique ways. Overall, this work will uncover information on how infection and inflammation alter the physiology of each organ in a manner unique to each sex.
Structural Landscape of SARS‐CoV‐2 entry & activation of spike glycoprotein & potential interventions
Authors: Vankadari Naveen
Published: May 2022

The pandemic COVID19 illness caused by SARS‐CoV‐2, which produces pneumonia and lower respiratory tract infections, is a serious public health concern, with frightening mutations causing over 4.5 million deaths globally. Whilst effective immunisation shows promise globally, several antiviral treatments are being clinically evaluated to fill the “therapeutic gap” in treating infected people. Understanding the entire repertoire of diverse host factors engaged by SARS‐CoV‐2 for entry and pathogenicity is required for long‐lasting potential therapeutics or vaccines. Here, using a structural and molecular approach, we show multistage processing of SARS‐CoV‐2 spike‐protein for virion activation, infection, and how mutations influence it. We solved the structures of spike‐protein in complex with different host cell factors (TMPRSS2, Furin, CD26 (DPP4), and NRP1) with functional activity, and these insights into uncovering how viral spike‐protein engages and primed with these multiple host factors, in addition to ACE2, to hijack host cell entry. Furthermore, our COVID19 patient genome sequencing reveals that allele in TMPRSS2 (V160M) and Furin provided protection from COVID19 infection, and its structural mechanism is further addressed and potential drug clinical trials. Additionally, our large‐scale retrospective cohort studies proved Arbidol and derivatives as potential therapies for COVID19, using structural studies, we demonstrated the mechanism of action of Arbidol in disrupting spike function. These findings cognize the complete mechanism of viral spike‐glycoprotein processing/priming that leads to cascading entry into the host cell, paving the door for future vaccine development and identifying key targets. Our comprehensive, multifaceted research reveals the complexity of the SARS‐CoV‐2 spike‐protein and clinical studies aid in therapies.

References: 1. Li, F., et al., Nature Communications 12, 866 (2021). 2. Vankadari, N. & Jacqline, A., Emeging Microbes & Infections, 9(1), 601‐604 (2020). 3. Vankadari, N. Int. Journal of Antimirobial Agents, 56(2),105998 (2020) 4. Andersen, K.G., et al., Nature Medicine 26, 450–452 (2020). 5. Qibin Liu et al., MedRxiv CSH Press (2021), March 22 (2) 6. Ravikanth, V et al., mGENE (2021), 5, e100930. NEJM (Reviewed) 7. Vankadari, N. ACS Journal of Physical Chemistry Letters, 11(16), 6655‐663 (2020).
Is the Use of Oral Contraceptive Beneficial to Female Athletes in the Prevention of Anterior Cruciate Ligament Injury?
Authors: Faragalli Mia , Taylor Kara D. , Welch Abi , Raynes Edilberto
Published: May 2022

Research has shown that women are 2 to 9 times more likely to sustain an anterior cruciate ligament (ACL) injury than men. The increased risk of ACL injuries in females can be attributed to several factors including anatomical, hormonal, biomechanical, and neuromuscular variations. Previous literature found that female hormones such as relaxin, progestin, and estrogen play a role in ligamentous laxity within the female ACL. Oral contraceptives (OC) regulate these hormones by inhibiting the spike of estrogen and progestin during the menstrual cycle. The aims of this study were to investigate the influence of female hormone concentrations on the ACL and to determine if OC can reduce the risk of ACL injuries in female athletes. We hypothesize that OC will have an effect on reducing the incidence of ACL injuries in female athletes. Articles were retrieved from Google Scholar and PubMed databases. A systematic review was conducted and ranked using the Oxford Levels of Evidence. The result revealed there are variations in the ranking of literature based on the Oxford Levels of Evidence. The evidence reveals that OC may have an inhibitory effect on relaxin receptors within the female ACL and could be recommended as a preventive measure for ACL ligamentous injury. Further research is needed to determine the causal effect of oral contraceptives on ACL prevention.
Antioxidant Effects of Pyrazoles in Ischemia/Reperfusion Injury in Cardiomyocytes
Authors: Bess Ezra , Shum Alika , Ara Hosne , Patel Foram , Colvin Chase , Brown Morgan , Horan Sara , Sharma Sudha , Murru Siva , Panchatcharam Manikandan , Miriyala Sumitra
Published: May 2022

Since the turn of the 21st century, cardiovascular diseases (CVDs) have remained the leading cause of death worldwide. In the United States alone, approximately 1.5 million people annually will have a myocardial infarction (MI), or more commonly known as a heart attack. The most common cause of MI is coronary artery disease (CAD), also known as atherosclerosis, where a significant occlusion in the coronary artery leads to an acute mismatch of oxygen supply and demand in the myocardium, leading to myocardial ischemia. Removing the occlusion is critical to salvaging ischemic tissues; however, reperfusion and restoring blood flow to previously anoxic/hypoxic tissues may cause a paradoxical worsening of cardiac cell dysfunction and death. This phenomenon is known as ischemia/reperfusion injury (IRI). Multiple studies have shown that oxidative stress plays a crucial role in the pathophysiology of IRI, and therapeutic studies are now being conducted to find an agent that can counteract the sudden rise in reactive oxygen species (ROS). Pyrazoles are small, heterocyclic molecules found in many biologically active compounds and have been identified as potential therapeutic agents against CVD due to their anti‐inflammatory effects. Most importantly, they are relatively easy to synthesize and can be fine‐tuned to achieve desired electronic and steric effects. Thus, pyrazoles are gaining more attention in the field of drug discovery research. In our present study, the function of pyrazole KT‐01‐14B as an antioxidant was determined by evaluating its effect on the expression of biomarkers indicative of ischemia/reperfusion (I/R)‐induced oxidative stress, which includes manganese‐dependent superoxide dismutase (SOD2), acetylated tubulin, and 4‐hydroxynonenal (4‐HNE) aggresomes. I/R was induced in vivo by using mice models and in vitro by subjecting HL‐1 cardiomyocytes to hypoxia/reoxygenation (H/R). Histologic evaluation of the cardiac tissue in our in vivo models was accomplished using TTC staining, which revealed that pyrazoles significantly reduce infarction size. To measure the mitochondrial superoxide levels in the HL‐1 cardiomyocytes, MitoSOX staining was used, which indicated that superoxide levels were significantly reduced with pyrazole treatment. Further investigation of pyrazole‐treated HL‐1 cardiomyocytes with immunoblotting analysis and immunocytochemistry revealed increased expression of SOD2, increased expression of acetylated tubulin, and decreased 4‐HNE aggresome formation. In addition to studying the effects of pyrazole on the biomarkers, a cell viability assay was performed to determine the effects of pyrazole on cardiomyocyte cell death. These results conclude that the antioxidant function of pyrazoles can prevent cellular damage caused by I/R.
Transcriptome and Mass‐Spectrometry‐Based Lipidome Reveal a Role of PRMT5 in Membrane Transport and Cholesterol Synthesis in White Adipocytes
Authors: Chen Xiyue , Jia Zhihao , Fang Shuyi , Yue Feng , Ferreria Christina R. , Wan Jun , Kuang Shihuan
Published: May 2022

Adipose tissue (AT) regulates systemic energy homeostasis, and its dysfunction can result in insulin resistance and other metabolic complications. Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetrical demethylation of arginine residues to modulate protein stability and/or function. Besides its well‐studied oncogenic functions, PRMT5 has recently been shown to play a physiological role in AT through poorly understood mechanisms. Here, we combine RNA sequencing and mass‐spectrometry‐based lipidomic analyses of wildtype and Prmt5 knockout (Prmt5AKO) AT to uncover the molecular mechanisms underlying PRMT5 function. We found that Prmt5AKO alters expression of genes related to metabolism and membrane transport. Specifically, Prmt5AKO induces genes enriched in glucose transport and glycolysis pathways, and suppresses genes encoding fatty acid (FA) transporters. This is accompanied by changes in lipid compositions of TAGs, FAs, and phospholipids. The data indicate that Prmt5AKO disrupts fatty acid metabolism while promoting glucose uptake and glycolysis. Prmt5AKO also promotes cholesterol biogenesis, contributing to hyperlipidemia and hepatic steatosis in mice. The omics data together reveal a previously unappreciated role of PRMT5 in membrane transport that affects glucose metabolism and cholesterol synthesis.
Arvanil, a synthetic capsaicin mimetic, synergizes with Irinotecan to trigger enhanced apoptosis in Cisplatin‐resistant Human Lung Cancer
Authors: Moles Emily G. , Friedman Jamie R. , Miles Sarah L. , Brown Kathleen C. , Hopper Kelly J. , Chen Yi Charlie , Dasgupta Piyali
Published: May 2022

Cisplatin‐based combination therapy is the first line treatment for human lung cancer patients. Initially, cisplatin shows excellent therapeutic response inducing remission in about 80% of lung cancer patients; however, the tumor inevitably relapses (within a year) and this relapsed tumor is resistant to cisplatin. Patients with platinum‐resistant or refractory disease have very limited options, as the only standard chemotherapy with an FDA‐approved drug, irinotecan has an objective response rate of approximately 3% and little or no survival benefit. Therefore, agents which improve the therapeutic response (of human lung cancers) towards irinotecan may be useful for the treatment of cisplatin‐resistant human lung cancer. Our published data show that the nutritional compound capsaicin sensitized human lung cancer cells towards the pro‐apoptotic activity of camptothecin (an analog of irinotecan) in cell culture and athymic mouse models. The administration of capsaicin causes stomach cramps, gastrointestinal irritation, gut pain and vomiting in patients. Arvanil is a non‐pungent synthetic capsaicin‐analog which displays potent growth‐suppressive activity in human lung cancer. The present study aims to investigate the combinatorial apoptotic activity of arvanil and irinotecan in cisplatin‐resistant lung cancer. Caspase‐3 activity assays reveal that the combination of irinotecan and arvanil displayed greater apoptotic activity in H69‐CPR human cisplatin‐resistant SCLC cells than the drugs used alone. These experiments were repeated in a second cisplatin‐resistant lung cancer cell line PC9‐CDDP and similar results were obtained. Chou‐Talalay isobologram analysis showed the interaction between irinotecan and arvanil is synergistic in H69‐CPR and PC9‐CDDP cells. The combination of arvanil/irinotecan showed a greater anti‐tumor activity in H69‐CPR tumors xenografted on chicken chorioallantoic membrane (CAM) and in SCID mice. In summary, the results of our studies pave the way for novel combination therapies for the management and treatment of drug resistant lung cancers in patients.
Spherical Invasion Assay: A novel method to measure the pro‐invasive activity of cancer cells
Authors: Sugrue Amanda M. , Nolan Nicholas A. , Richbart Stephen D. , Akers Austin T. , Brown Kathleen C. , Dasgupta Piyali
Published: May 2022

Tumor invasion refers to the process by which cancer cells penetrate the basement membrane into surrounding stroma, neighboring blood vessels and lymph nodes. Tumor invasion is an essential step for metastasis of human cancers. The Boyden Chamber assay is the benchmark technique of measuring the invasion of cancer cells. However, this assay suffers from several caveats. The physiological relevance of transwell inserts (to recapitulate invasion in vivo) is poor. The pore size of the membrane highly influences the number of invaded cells. Another limitation of the Boyden chamber assay is it is very difficult to control the concentration of the chemokine gradient and this may produce aberrant results. All these considerations led us to develop a novel method of measuring tumor invasion namely the SPHERICAL INVASION ASSAY (SIA) in our laboratory. The SIA measures the invasion of human cancer cells as they migrate from the primary Matrigel layer, over the interface and travel into a secondary Matrigel layer. The cells are grown in extracellular matrix (ECM) retain biological characteristics of tumors, such as diffusion gradient of oxygen, nutrients and pH. The growth of the cells inside the ECM allows for complex cell‐cell and cell‐matrix interaction. Most importantly, the results of the SIA correlate with the data obtained in the Boyden Chamber assay, so the SIA can be used to confirm the results obtained in Boyden Chamber assays1. The present protocol examines the effect of the Src Kinase inhibitor PP2 on the invasion of A549 human NSCLC cells. We hope that the SIA will be useful tool for researchers working in the field of tumor microenvironment biology and cancer metastasis.
Activation of a novel α2AAR‐spinophilin‐cofilin axis determines the effect of α2 adrenergic drugs on fear memory reconsolidation
Authors: Saggu Shalini , Chen Yunjia , Wang Hongxia , Jiao Kai , Wang Qin
Published: May 2022

Posttraumatic stress disorder (PTSD) after the pandemic has emerged as a major neuropsychiatric component of the post‐acute COVID‐19 syndrome, yet the current pharmacotherapy for PTSD is limited. The use of adrenergic drugs to treat PTSD has been suggested, yet hindered by conflicting clinical results and a lack of mechanistic understanding of drug actions. Our studies using both genetically modified mice and human induced pluripotent stem cell‐derived neurons reveals a novel α2A adrenergic receptor (α2AAR)‐spinophilin‐cofilin axis in the hippocampus that is critical for regulation of contextual fear memory reconsolidation. We have further found that two a2 ligands, clonidine and guanfacine, exhibit differential abilities in activating this signaling axis to disrupt fear memory reconsolidation. Stimulation of a2AAR with clonidine, but not guanfacine, promotes the interaction of cofilin, an actin binding protein, with the dendritic spine scaffolding protein spinophilin to induce cofilin activation at the synapse. Spinophilin‐dependent regulation of cofilin is required for clonidine‐induced disruption of contextual fear memory reconsolidation. Our results inform the interpretation of differential clinical observations of these two drugs on PTSD, and suggest that clonidine could provide immediate treatment for PTSD symptoms related to the current pandemic. Furthermore, our study indicates modulation of dendritic spine morphology may represent an effective strategy for development of new pharmacotherapies for PTSD.
Prevention at Full Steam ‐ Can Steam Inhalation Stop the Contagion of Sars‐Cov 2?
Authors: Remigio Wilton
Published: May 2022

Covid‐19 vaccination and social barrier efforts worldwide have deescalated the death and morbidity toll due to Sars CoV‐2 infection in most countries. However, intramuscular vaccination leads to a variable antibody response insufficient to provide airway mucosal immunity that prevents contagion and reinfection in asymptomatic carriers. Nasal vaccines could provide this missing piece and are currently being tested with encouraging results, but large‐scale availability and efficacy in humans are unknown. Moreover, previous exposure to the virus would render nasal spray vaccines less effective. Past literature on upper airway infections has shown that water steam inhalation therapy (WSIT) may modulate airway mucosa immunity responses. Airway mucosal immunity in COVID‐19 infection is a critical aspect to consider with WSIT. Based on a small but slowly growing body of evidence, we propose that WSIT may be a relevant home remedy worth revisiting to help halt mass contagion. Here, we review current and past studies on WSIT use in upper respiratory infections (URI). Previous studies reported mixed results on the efficacy of steam therapy on viral respiratory diseases. Safety concerns with steam therapy include possible nosocomial infections associated and burns. There is a paucity of studies with high methodological quality exploring the outcomes of this ancient therapy. Upper airway blood flow increases substantially after 15‐min of steam inhalation with temperatures of 42 ℃. Still, methods to measure bronchial blood flow are lacking, and this may be an essential variable for the analysis of how steam may impact mucosal immune responses. Studies with better methodologies demonstrate that nebulized steam at 80°C for 20 min reduces neutrophils compared to controls in COPD patients indicating an anti‐inflammatory effect. The best evidence for using WSIT for upper airway viral disease shows that 60% of asymptomatic carriers increased viral shedding immediately post steam inhalation, and Rt‐PCR testing confirmed viral negativity at 10‐days post‐treatment. Subjects used steam for a total of 20 minutes per hour with steam temperatures ranging from 55 to 65℃. Efficacy rates decline with research methodologies that use WSIT for less than 20 minutes. In summary, more recent studies using WSIT for URI show encouraging results and may help to disentangle former controversies. Lessons emerging from previous studies indicate that exposure of WSIT less than 20 minutes may be ineffective. Adopting WSIT as an inexpensive public health measure may lead to secondary prevention of airway viral infections, especially in new viral strands that have the potential to become pandemic.
Identification of A‐B, a Novel Chimeric RNA in Colorectal Cancer
Authors: Lynch Sarah , Wu Hao , Li Hui
Published: May 2022

Colorectal cancer (CRC) is the third leading cause of cancer‐related deaths worldwide, affecting more than one hundred thousand men and women in the United States every year. With the incidence of early‐onset (<50 years old) CRC increasing at an alarming rate, there is an urgent and unmet need for early biomarker discovery and the identification of novel therapeutic targets in CRC. Chimeric RNAs and their protein products are well‐established as ideal biomarkers and drug targets for malignant cancers, including BCR‐ABL in chronic myelogenous leukemia and EML4‐ALK in lung cancer. Using RNA‐seq data from The Cancer Genome Atlas (TCGA) database, we have identified a chimeric RNA, denoted as A‐B, present in 45.2% of CRC samples and just 17.6% of adjacent normal margin tissue, but absent in non‐cancer colon tissues. We determined that this chimeric RNA results from cis‐splicing between adjacent genes (cis‐SAGe), and includes a splice variant of the 5’ gene, altering the reading frame of the 3’ gene. This chimeric RNA encodes a novel transmembrane protein, with the alternate reading frame on the extracellular surface, making it an ideal target for antibody therapeutics. After generating an antibody to the chimeric protein and validating its use in immunoblotting, immunoprecipitation, and immunohistochemistry, we found this novel protein is present in patient CRC samples in a distinct, punctate pattern. Overexpression of this chimeric RNA resulted in increased cell proliferation, while siRNA mediated knockdown led to a reduction in cell proliferation. We are currently investigating the function of this chimeric RNA and its biomarker potential in clinical CRC samples.
A Bifidogenic Prebiotic Supplement Beneficially Alters Gut Microbial Composition, Activity, and Plasma Metabolites in Healthy Individuals Consuming Low Fiber Diets
Authors: Kang Jea Woo , Tang Xinyu , Zivkovic Angela M.
Published: May 2022

A randomized order, double‐blinded, placebo‐controlled cross‐over intervention study was conducted to test the effectiveness of a prebiotic supplement in healthy participants consuming a diet low in fiber. This clinical trial was registered at clinicaltrials.gov as NCT03785860.

Fecal microbial DNA was extracted and sequenced with shallow shotgun sequencing for metagenomics. Plasma metabolites were analyzed with untargeted liquid chromatography–mass spectrometry for metabolomics. Statistical analyses were performed with the package edgeR in R version 4.0.2. For each outcome, linear mixed models were used with fixed effects of treatment, treatment order, and their interaction and random effect of individual participants.

From the metagenomic analysis, the relative abundance of the genus Bifidobacterium and several bifidobacterial species increased after prebiotic supplementation compared to the placebo. Additionally, gene counts of genes sacA, xfp, xpk associated with prebiotic utilization significantly increased after the prebiotic supplement. Other genes that increased in counts were poxB and ackA, which are related to acetate metabolism. Changes in bifidobacteria abundance were positively correlated with changes in genes related to fructose and choline metabolism, and negatively correlated with a gene that encodes for acetate kinase.

Plasma concentrations of indolepropionate significantly increased whereas choline and several acylcholines such as stearoylcholine, dihomo‐linolenoyl‐choline, linoleoylcholine, arachidonoylcholine, palmitoylcholine, and oleoylcholine significantly decreased after the prebiotic compared to the placebo arm.

The results of this study demonstrate that a convenient, easy to use bifidogenic prebiotic supplement beneficially altered gut microbial composition and activity, which was reflected in changes in plasma metabolites in healthy individuals consuming low fiber diets.
Changes in brain interstitial pH induced by hypoxia versus asphyxia in normothermic or hypothermic newborn pigs: implications for the preclinical study of neonatal hypoxic‐ischemic encephalopathy
Authors: Domoki Ferenc , Tóth‐Szűki Valéria , Kovács Viktória , Remzső Gábor
Published: May 2022

Brain interstitial pH (pHbrain) alterations contribute to neuronal injury in neonatal hypoxic‐ischemic encephalopathy (HIE), the severe neurodevelopmental consequence of birth asphyxia affecting millions of neonates each year. In various HIE animal models, including the established large‑animal piglet model, normocapnic hypoxia or asphyxia are both employed as insults to elicit HIE. However, hypoxia or asphyxia may trigger neuronal injury by distinct pathomechanisms as suggested by a recent study reporting that hypoxia triggered an alkalotic while asphyxia an acidotic shift in pHbrain in hypothermic P6 rat pups (Acta Physiol 229:e13467, 2020). We have recently shown that asphyxia results in a profound cerebral acidosis that was restored in the first hour of reventilation then remained stable throughout the 24h observation period in normothermic piglets, but information on the effect of normocapnic hypoxia on pHbrain and the potential confounding effect of body temperature alteration in piglets was unknown. Anesthetized, mechanically ventilated P1 piglets (n=21) were equipped with an open cranial window to determine pHbrain using pH‐selective microelectrodes inserted into the parietal cortex. Asphyxia or hypoxia was induced by swithching the ventilation to a 6%O2‐ 20%CO2 gas mixture or to 6%O2 for 20 min, respectively in either normothermic (38.5 °C) or hypothermic (33.5 °C) pigs (n=5‐6 in each group) followed by 60 min reoxygenation/reventilation. Both arterial blood pH (pHa) and pHbrain (Figure 1) showed an acidotic shift during the insult in all four experimental conditions. Cerebral acidosis was similar in normothermic animals exposed to either asphyxia or hypoxia, despite the shift in pHa was 0.46 (0.36‐0.56) pH units larger in the asphyxiated animals (Figure 2). In hypothermic conditions, however, the acidotic pHbrain shift was greatly attenuated only in the hypoxia but not in the asphyxia group. Our findings suggest that using hypoxia or asphyxia insults result in similar cerebral but not systemic (blood) acidosis in normothermic piglets. However, the degree of developing cerebral acidosis to hypoxia is greatly attenuated in hypothermic animals, although an alkalotic shift reported in rats could not be observed.
3’ Terminal end‐labeling of structured RNA by Klenow DNA polymerase
Authors: Mwangi Mary N. , Baird Nathan N.
Published: May 2022

RNA labeling is a key step in studying RNA localization, folding, and structural dynamics using fluorescence based assays. Current methods of fluorophore attachment to long (>100 nucleotides) and structured RNAs are hampered by low labeling yields and limitations in attachment position. These challenges have led to an interest in developing alternative methods that would enable the site‐specific labeling of long and structured RNAs. To this end, we have developed a site‐specific, chemo‐enzymatic method of fluorophore incorporation to RNAs. This novel method uses Klenow enzyme, a DNA polymerase, to add modified nucleotides to RNAs 3’ end in a templated manner. To facilitate this modified nucleotide addition in structured RNAs, we designed helper DNA oligonucleotides to disrupt any pre‐existing RNA intramolecular structure. Using this method, we have achieved efficient 3’ terminal end‐labeling of chemically synthesized and in‐vitro transcribed RNAs of different lengths with cyanine dyes.

This method will be routinely used in fluorescence‐based assays that investigate the structure‐function relationships of RNAs involved in cancer and HIV progression.
Effect of prolonged lipopolysaccharide exposure on lung function and health
Authors: Putz Nathan , Gibson Dustin , Jesse Jordan , Newcomb Dawn , Bastarache Julie
Published: May 2022

Rationale

Patients with Chronic Obstructive Pulmonary Disease often have persistent inflammation of the airways that lead to symptoms of chronic bronchitis and airway obstruction. While it is known that long term exposure to cigarette smoke can cause persistent airways inflammation, it is not clear if this is a direct result of tobacco exposure or if it is a result of chronic inflammation. We hypothesized that a recurrent and prolonged inflammatory insult would result in alterations in lung mechanics characterized by airway obstruction.

Methods

. To test the independent effects of recurrent and persistent lung inflammation on lung mechanics, C57Bl/6 8 week old male mice were treated weekly with PBS or lipopolysaccharide (LPS) via direct intratracheal injection. Monthly, we evaluated lung function analysis including: airway resistance and compliance, tissue damping, pressure volume loops, and forced expiratory volume using the flexiVent system.

Results

. Airway resistance at month 1 was 0.5543±0.017 PBS vs 0.552±0.009 cmH2O.s/mL LPS (p=0.999), month 2 0.5177±0.015 PBS vs 0.576±0.017 LPS (p=0.999), month 3 0.461± 0.03 PBS vs 0.47±0.038 cmH2O.s/mL LPS (p=0.999), month 5 0.43±0.015 PBS vs 0.468±0.026 cmH2O.s/mL LPS (p=0.999), month 6 PBS 0.408± 0.06 vs LPS 0.6465±0.18 cmH2O.s/mL (p=0.999), and month 7 PBS 0.384 ± 0.023 vs LPS 0.3498 ± 0.015 cmH2O.s/mL (p=0.999). Forced expiratory volume 0.1 (FEV 0.1) at month 1 was 0.4±0.08 PBS vs 0.803±0.107 mL LPS (p=0.999), month 2 0.2115±0.01 PBS vs 0.184±0.03 mL LPS (p=0.999), month 3 1.071±0.09 PBS vs 1.098±0.06 mL LPS (p=0.999), month 5 0.853±0.243 vs 0.756±0.21 mL LPS (p=0.999), month 6 2.671±1.06 PBS vs 1.669±0.87 mL LPS (p=0.999), month 7 0.9049±0.27 PBS vs 1.127±0.152 mL LPS (p=0.999).

Conclusion

It is well known that chronic smoking can have a significant impact of lung function. Through this project we aimed to discover whether chronic inflammation alone would alter lung function. Interestingly even after weekly direct intratracheal injections of LPS there were no significant changes in lung function compared to PBS treated mice. We have shown here that chronic inflammation alone was not sufficient to alter lung function in mice, and that the decrease in lung function seen in chronic smokers is attributable to numerous factors and not inflammation alone.
Surgical Skills in General Surgery Residency Program: Current Trends and Future Directions
Authors: Marek Mason , Balta Joy Y.
Published: May 2022

Today, general surgery residents (GSR) face unorthodox challenges due to variation in caseloads and the implementation of duty hour restrictions. It has been stated that the most concerning change in the education of GSRs was the downward trend in weekly operative cases. The integration of surgical skills labs utilizing human body donors could prove to be beneficial for education. The use of human body donors for anatomy education is not a novel idea, however research needs to be done to better understand pros and cons of different preservation methods for surgical skill training. Traditionally formaldehyde is used as the main preservative however soft‐preservation techniques (such as alcohol and Thiel) and fresh‐frozen methods have been gaining popularity as an educational tool for professional students and surgical residents due to the many lifelike qualities the tissue retains during preservation. Studies using soft preserved donors have investigated skill confidence of GSR which all showed an increase in confidence for surgical skills for all PGY residents. Some of these skills include: surgical airway, tracheoesophageal injuries, thoracotomy‐cardiac injury, solid abdominal organ injury, and more. Integration of human body donors into GSR programs has proven to be advantageous for development of surgical skills and confidence in the capabilities of surgical residents. In an effort to improve anatomy education and surgical skill development for general surgery residents additional research should be done. Future studies need to compare the various educational anatomical models and preservation methods could prove to be advantageous.
In‐Vitro Combo Treatments of Human Melanoma (1205Lu) Cell line with Curcumin, Vitamins and Steroids
Authors: Ramaraj Pandurangan
Published: May 2022

Our previous work on combo treatments of human melanoma (BLM) cell line pointed to the two combos A+RU and Cur+P+RU as the best combination to decrease cell growth, a basal secretion profile of pro‐inflammatory cytokines and a decrease in the major pro‐inflammatory cytokine IL‐8 secretion by the cells. In order to confirm these findings, experiments were repeated on the 1205Lu cell line. Cells were incubated in a 96 well plate along with a combination of compounds for 48 hrs. After 48 hrs, cell growth was assayed using MTT and supernatants were collected for Elisarray and IL‐8 quantitation by Elisa. Single or solo compound treatment resulted in cell growth in the range of 37 to 59% compared to untreated control 1205Lu cell growth at 100%. A double combo of compounds such as Cur+D3, Cur+P, Cur+RU, D3+P, D3+RU, P+RU, A+D3, A+P, A+RU resulted in cell growth in the range of 17 to 37%. A triple combo of compounds such as CUR+P+RU, Cur+A+D3, Cur+D3+RU resulted in cell growth in the range of 19 to 30%. Elisarray of the supernatants from various treatments pointed to A+RU, A+D3, Cur+D3+RU, Cur+P+RU as the combos having a basal secretion profile of pro‐inflammatory cytokines. Quantitation of IL‐8 by Elisa suggested A+RU and Cur+P+RU as the two combos having a minimal amount of IL‐8 secretion 17.5 pg/ml and 8.0 pg/ml respectively.

Conclusion

These experiments suggested that the combination of compounds really decreased cell growth and IL‐8 secretion. Two combos A+RU and Cur+P+RU were found to work well on both (BLM and 1205Lu) cell lines. These experiments will be repeated on other human melanoma cell lines for the confirmation of the above findings.
A Novel Approach to Assess Podocyte Damage at Single Cell Level
Authors: Chen He , Padvitski Tsimafei , Özel Cem , Brinkkötter Paul , Benzing Thomas , Beyer Andreas , Kann Martin
Published: May 2022

Diseases affecting podocytes in kidney glomeruli are heterogeneous with respect to triggers, molecular mechanisms, and speed of progression. Frequently, healthy and diseased podocytes are present in parallel. Single cell technology is ideally suited to capture such heterogeneity including temporal resolution, which can be achieved by trajectory analysis. However, trajectories require large numbers of cells, which are difficult to obtain for rare cell populations such as podocytes. Here, we propose a novel analysis approach for single‐cell data in rare cell types, which relies on a podocyte disease score (PDS) to monitor the health status of individual cells. PDS is based on integrative meta‐analysis of published and our own bulk and single‐cell transcriptomic data of glomeruli and primary podocytes obtained from a multitude (n=37) of mouse models featuring podocyte damage. Among others, podocyte damage triggers integrated in the analysis include diabetes, ageing, mutations in slit diaphragm proteins, alterations of transcription factors, and adriamycin‐induced podocytopathy. In a rank‐based approach, a geneset was derived, whose expression quantitatively predicts podocyte damage irrespective of the trigger and at single‐cell resolution. Our PDS is robust, correlates with albuminuria in mice and humans, and allows for aligning disease states across independent and novel datasets. Applying PDS to published and novel glomerular single‐nucleus RNAseq data from mouse models recapitulated established molecular pathways relevant to the models, and identified common and model‐specific pathways related to podocyte damage. As such, Eph‐ephrin signaling was identified as a pathway that is universally altered upon podocyte damage at transcriptional level, and was confirmed by STED microscopy on the protein level in two mouse models. In conclusion, PDS is a valid and versatile method to assess podocyte damage at single‐cell level and allows to characterize cellular processes in podocytes in mice and humans.
Integrating Clinical Education into the Anatomy Curriculum; The Advantages of Soft Preservation Techniques
Authors: Fair Jarrod D. , Boucher Laura C. , Agnor Celine , Balta Joy Y.
Published: May 2022

Practitioners and educators continually evaluate alternative teaching methodologies to increase the learning of anatomical and clinical based skills. Traditionally, anatomy and clinical skills are taught separately to students in both physical and occupational therapy curricula. Formaldehyde preservation is seen as the primary embalming methodology within anatomical education but has been described as lacking when utilized within educational institutions. This study aimed to evaluate the integration of clinical skills into anatomical education using an alternative preservation technique called Imperial College London‐ Soft Preservation (ICL‐SP).

Physical and occupational therapy completing the Musculoskeletal Anatomy course were presented with donors preserved with either formaldehyde or ICP‐SP. The students were tasked with traditional dissection and with performing clinical tests on the different donors followed by the administration of a questionnaire. The inclusion of ICL‐SP donors showed there was a better model for the representation of the vascular system and the nervous system without showing significant deficits in any other categories when compared to formaldehyde. The integration of clinical skills and anatomical curriculum presented increased student perceived understanding of associated anatomy (p < 0.05), gave better understanding of how anatomy is important to their professions (p < 0.05) and increased motivation to learn anatomy (p < 0.05). Students noted ICL‐SP was more beneficial forclinical skill procedures (Lachman's test: p < 0.05; Valgus Stress Test of the Elbow: p < 0.05). In conclusion, the integration of clinical skills into anatomical education can potentially provide clinicians with higher confidence performing clinical skills and may be complemented by the utilization of the new ICL‐SP methodology. Ethical approval was granted by the Ohio State University Institutional Review Board under log number 2021E0469.