bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of transcriptional regulation in the p53 network
Authors: Espinosa Joaquin
Published: June 2021

The tumor suppressor p53 is transcription factor that orchestrates diverse responses to cellular stress via regulation of target genes involved in cell cycle arrest, senescence, autophagy and apoptosis. The p53 transcriptional program varies greatly in a cell type‐ and stimulus‐specific manner, but the mechanisms generating this regulatory diversity are poorly defined.

We have recently performed several genomics studies that revealed novel mechanisms conferring regulatory diversity to the p53 network. These experiments include shRNA screens for gene‐specific p53 cofactors and identification of novel p53‐regulated RNAs via global run‐on deep sequencing. We will present the results of these experiments and discuss new strategies to manipulate the p53 response for therapeutic purposes.
Harnessing Unusual Precursors in Polyketide Biosynthesis
Authors: Thomas Michael G , Park Hyunjun
Published: June 2021

Polyketides biosynthesized by type I polyketide synthases (PKSs) are a large class of natural products that have a wide range of important biological and pharmacological properties. The diversity of activities of these natural products is due to the structural variations afforded by type I PKSs. These enzymes recognize thioesterified carboxylic acid precursors and condense these precursors to generate the polyketide backbone. While PKSs incorporate predominantly malonyl‐CoA and (2S)‐methylmalonyl‐CoA precursors, additional precursors are being identified. We identified two unusual PKS precursors with unique chemical attributes while studying zwittermicin A biosynthesis by Bacillus cereus. These precursors, (2S)‐aminomalonyl‐acyl carrier protein (ACP) and (2R)‐hydroxymalonyl‐ACP, introduce an amino or hydroxyl group, respectively, into the polyketide. Understanding how these precursors are recognized and incorporated into a polyketide backbone will provide a means for reprogramming other PKSs to incorporate these unusual precursors. This will extend the possibilities of polyketide structural variation. Our structure‐function analyses of precursor recognition by PKSs will be discussed
Replication Forks Restarted by Homologous Recombination Display a High Frequency of Errors at Inverted Repeats
Authors: Carr Antony Michael , Murray Johanne Milne
Published: June 2021

Replication restart by HR at the sites of a collapsed replication fork promotes cell survival, but at the expense of increasing the potential for genome rearrangement. We have previously shown that, when a replication fork collapses within a small (~900bp) inverted repeat, homologous recombination is required to restart the collapsed fork. In the majority of cases, the restart event is effective and promotes cell survival. However, the presence of a nearby repeat (homologous sequence) results in an inappropriate template exchange in a few percent of events, producing dicentric and acentric giant palindromic chromosomes.

We also studied the consequence of restarted replication fork progression through a small palindrome. We observed that restarted forks are prone to generate errors when they replicate through small inverted repeats. Thus, two distinct mechanisms can result in gross chromosomal rearrangements: 1. When replication forks collapse within a repeated sequence, incorrect homologous recombination can occur that results in repeat fusion. 2. The restarted replication fork is itself error prone, and is particularly sensitive to making errors when replicating closely spaced inverted repeats.
Positioning the Golgi Apparatus at Microtubule Minus Ends
Authors: Linstedt Adam , Yadav Smita
Published: June 2021

Intracellular motility and organelle positioning depend on the recruitment of molecular motors by specific membrane receptors. Although ER‐to‐Golgi trafficking and Golgi positioning are the best‐known examples of inward motility mediated by the microtubule minus‐end‐directed motor dynein, the dynein membrane receptor is unknown. Work identifying the long sought receptor for dynein in the early secretory pathway will be presented. The findings elucidate a key aspect of organelle positioning and how such positioning is regulated for accurate organelle partitioning during cell division. It also impacts the spatial organization of the secretory pathway and the processes of cell polarity, cell migration and wound healing that depend on this spatial organization.
Effects of prebiotic inclusion and chain length on intestinal barrier, histomorphology, and mRNA abundance in obese C57BL/6J mice
Authors: Cephas Kimberly D. , Mangian Heather F. , Tappenden Kelly A. , Swanson Kelly S.
Published: June 2021

Obesity is linked with increased intestinal permeability, which may contribute to low grade inflammation. The objective of this study was to test the effects of prebiotics on intestinal permeability, morphology and gene expression in an obese mouse model. Obese 18‐week old, C57 mice (n=6) were randomized to high‐fat diets containing 5% cellulose, 10% cellulose, 10% scFOS or 10% inulin and fed for 28 d. Distal ileum, cecum and colon samples were collected for Ussing chamber, histomorphology, and RT‐qPCR analyses. Cecal epithelial resistance was greater (p<0.05) in mice fed inulin vs. mice fed cellulose or scFOS. Distal colon epithelial resistance tended to increase (p=0.08) in mice fed scFOS vs. mice fed cellulose or inulin. Ileal and cecal crypt depth was greater (p<0.05) in mice fed scFOS or inulin vs. mice fed cellulose. Ileal MUC2 expression was greater (p<0.05) in mice fed 10% cellulose vs. mice fed 5% cellulose, inulin or scFOS. Distal colon AMPK and ZO‐1 expression was greater (p<0.05) in mice fed scFOS vs. mice fed cellulose or inulin. Ileal and cecal occludin expression was lower (p<0.05) in mice fed inulin or scFOS vs. mice fed 5% cellulose. Colon occludin expression was lower (p<0.05) in mice fed inulin or scFOS vs. mice fed 10% cellulose. These data suggest prebiotics improve intestinal permeability in the obese and involve tight junction proteins, but the effects vary regionally depending on prebiotic properties.

Grant Funding Source: Internal funding
High fat diet‐induced muscle insulin resistance: role of cytokines and local macrophages
Authors: Vandanmagsar Bolormaa , Bermudez Estrellita , Ravussin Eric , Mynatt Randall
Published: June 2021

Obesity is characterized by adipose tissue expansion as well as macrophage infiltration of adipose tissue, resulting in chronic inflammation in adipose tissue, which in turn has a causative role in obesity‐induced insulin resistance. During state of chronic inflammation, levels of inflammatory markers, such as IL‐1b, TNF‐a, IL‐6 increase as a result of macrophage accumulation and activation, specifically a subset of inflammatory F4/80+CD11c+ macrophage/dendritic cells in adipose tissue. It is well recognized that increased production of free fatty acids and cytokine secretion from inflamed fat tissue induce insulin resistance in other insulin‐sensitive organs, such as skeletal muscle and liver. However, whether obesity also results in skeletal muscle inflammation and whether macrophage infiltration in muscle contributes to the development of insulin resistance has not been fully elucidated. We investigated inflammatory cytokine expression and mechanisms involved in the induction of insulin resistance and infiltrated local macrophages and other immune cells in skeletal muscle tissue in diet‐induced obese mice using qRT‐PCR. We found that F4/80+, Cd11b+ macrophages and Cd11c dendritic cells were most abundant immune cells infiltrated into skeletal muscle tissue and increased in response to high fat diet feeding, while mRNA expression of cell markers for CD4+, CD8+ T cells and CD19+ B cells were lower.
Determinants of breastfeeding modes in the prevention of mother‐to‐child HIV transmission (PMTCT) Kesho Bora study
Authors: Bork Kirsten , Cames Cecile , Cournil Amandine
Published: June 2021

Breastfeeding (BF) determinants were investigated in 1028 HIV‐infected African women participating in a 5 site PMTCT study, which provided antiretroviral prevention during pregnancy and postpartum (PP) (a triple antiretroviral prophylaxis up to 6 mo PP or the standard ZDV‐based regimen until 7 d PP). Women were counselled to either breastfeed exclusively up to 6 mo or formula‐feed exclusively for 6 mo. Some 76% ever breastfed, among whom 73% still breastfed at 3 mo (40% exclusively and 24% predominantly). In multiple logistic regression analysis, significant determinants of formula‐feeding were study site, CD4 <200mm3, secondary schooling and emergency C‐section. BF at 3 mo (if breastfed at 1 mo) was independently associated with study site (P<0.001) and neonatal morbidity reported by the mother (OR: 0.60 (0.37–0.985)) but not with report of maternal morbidity (fever or fatigue) during the first mo PP (0.57 (0.31–1.02)), CD4 cell count or schooling (P>0.20 for both). By 3 mo, boys tended to be less breastfed (0.70 (0.46–1.05)) and were less often exclusively breastfed than girls (0.52 (0.34–0.78)). This study suggests that exclusive breastfeeding is shorter among boys of HIV‐positive African women. The importance of neonatal morbidity for breastfeeding duration requires further investigation.

Funding: ANRS, DFID, EDCTP, Trasher Research Fund, GlaxoSmithKline Fund, CDC, NIH, WHO
Television viewing and intake of added sugars related to increased central adiposity in prepubertal children
Authors: Khan Naiman A , Raine Lauren B , Drollette Eric , Scudder Mark , Pontifex Matthew , Castelli Darla M , Hillman Charles H , Donovan Sharon M , Evans Ellen M
Published: June 2021

Central adiposity is related to insulin resistance and may be the most clinically relevant body fat in children as is the case in adults. This study aimed to determine relationships between child's television viewing (TV), aerobic fitness, diet and central adiposity in 172 prepubertal children (8–9 years). Parents reported child's weekday and weekend TV viewing. Maximal oxygen consumption (VO2max) and one 24‐hour recall assessed fitness and diet, respectively. Dual energy X‐ray absorptiometry was used to measure percent fat mass (%Fat) and central adiposity (FM‐abd). Waist circumference to height ratio (WHtR) was also measured. Aerobic fitness was not related to TV, however, it was negatively related to %FM (r=−0.49, p < 0.01) and FM‐abd (r=−0.41, p < 0.01). Weekday and weekend TV was positively related to FM‐abd (r=0.18, p=0.02 and r=0.20, p=0.01). After adjusting for VO2max and total diet energy density (kcals/g), added sugars (g) positively related to FM‐abd (r=0.18, p=0.03). To further assess central adiposity, children were classified as normal (n=129) or obese (n=43) based on the 0.5 cut off for WHtR. Obese children had significantly higher weekday TV (p=0.03) and consumed higher amounts of added sugars (p=0.02). Our findings suggest that central adiposity is related to increased intake of added sugars independent of aerobic fitness and energy density in prepubertal children. (Funding source: NIH HD055352)

Grant Funding Source: NIH HD055352
The gut microbiome of kittens is affected by dietary protein: carbohydrate ratio and correlated with blood metabolite and hormone concentrations
Authors: Hooda Seema , Boler Brittany M Vester , Dowd Scot E , Swanson Kelly S
Published: June 2021

This study was conducted to test the effects of dietary protein: carbohydrate ratio on the composition of the gut microbiota using 454 pyrosequencing and identify associations with blood metabolites of growing kittens. Male domestic shorthair kittens were raised by mothers fed moderate‐protein, moderate‐carbohydrate (MPMC; n=7) or high‐protein, low‐carbohydrate (HPLC; n=7) diets. Fasted blood samples were collected and analyzed for blood metabolites and hormones. Pyrosequencing resulted in a total of 384,588 sequences, with an average of 9,374 sequences per sample. A dual hierarchical clustering dendogram of the most abundant bacterial genera indicated distinct clustering of samples based on protein:carbohydrate ratio. The principal component analysis of fecal bacterial families, blood metabolites, and hormones resulted in 4 distinct clusters. One cluster included blood triglycerides and fecal Clostridiaceae, Eubacteriaceae, Ruminococcaceae, Fusobacteriaceae, and Lachnospiraceae. A second cluster consisted of blood ghrelin and fecal Coriobacteriaceae, Bifidobacteriaceae, and Veillonellaceae. A third cluster consisted of blood glucose, cholesterol, and leptin and fecal Lactobacillaceae. The strong correlations between blood hormones and metabolites and well‐known probiotic bacterial families indicate potential significance as it pertains to satiety and host metabolism.

Grant Funding Source : United States Department of Agriculture‐Cooperative State Research, Education and Extension Service (project ILLU‐538‐396)
Correlation of the Nutrition Metric Profiling Tool
Authors: Magness Allison , McAllaster Michael , McInnis Kathleen , Roberts-Gray Cindy , Sweitzer Sara , Briley Margaret
Published: June 2021

Objectives were to determine the correlation of the Nutrition Metric to other nutrition profiling tools, Guiding Stars, Go‐Slow‐ Whoa, NuVal and WXYfm. A sample of 600 foods was selected from a 24‐month purchase database of 35,000 households (income ≤ $35,000/year). NuVal and Guiding Stars scores were collected from supermarkets that implemented the respective scoring programs and subsequently limited the sample to 174 of the 600 foods. Nutrition Facts panel was used to calculate scores for WXYfm and Nutrition Metric. Foods were sorted by Go‐Slow‐ Whoa categories. Pearson r tests were conducted to test correlation. Nutrition Metric had a positive correlation with all profiling tools for the 174 foods (WXYfm, 0.767; NuVal, 0.739; Go‐Slow‐Whoa, 0.727; Guiding Stars, 0.691 at p<0.01). Nutrition Metric correlated with WXYfm for four MyPlate food groups (grains, 0.766; vegetables, 0.382; fruit, 0.542; protein, 0.744 at p<0.05) while correlations varied across the other profiling tools. Correlation of the Nutrition Metric with other established nutrition profiling tools supports its validity as a tool to profile food purchases in order to provide recommendations for a healthful diet.
School childrenˈs consumption of fat‐free, low added sugar flavored milk
Authors: Yon Bethany A , Johnson Rachel K
Published: June 2021

USDA's proposed rule for school meals requires flavored milk (FM) to be fat‐free. The IOM report on competitive foods limits added sugar in FM to 10 gm/8 oz. Milk is an important source of shortfall nutrients. As most elementary children choose FM, it is important to know how well children consume FM meeting these standards. Plate waste studies were conducted in May 2010 and 2011 in 2 southern schools (grades 3–5) using FM meeting both standards. In 2010, FM was USDA compliant (fat‐free). In 2011, added sugar was lowered to meet IOM guidance. FM consumption was highly skewed, thus categorized as a binary variable (0–7oz, >7oz). Excluding unopened cartons (27% in 2010, 20% in 2011), 37% of children in school A (Urban; 97% Hispanic; 88% eligible for free/reduced meals) consumed >7 oz of milk in 2010 compared to 44% in 2011 (p=0.19). School B (Rural; 52% White, 41% Hispanic; 61% eligible for free/reduced meals) changed from plastic bottles to paper cartons in 2011. The number of unopened FM containers increased in 2011 (3% to 16%, p=0.001). Excluding unopened containers, 68% of children consumed >7 oz of milk in 2010 compared to 46% in 2011 (p=0.03). Consistent with earlier research, children drink more milk from plastic bottles. When packaging was consistent, children consumed similar amounts of fat‐free FMs with higher vs lower added sugar.

Grant Funding Source: USDA Hatch and the Dairy Research Institute
CSR1 inhibits XIAP
Authors: Luo Jianhua , Zheng Zhong-Liang , Yu Yan P
Published: June 2021

CSR1 is a tumor suppressor gene located at 8p21, a region that is frequently deleted in a variety of human malignancies. Previous studies indicated that expression of CSR1 induced cell death. In this study, we identified that CSR1 interacted with XIAP through a Yeast Two‐hybrid screening analysis. In vitro and in vivo analyses indicate that the C‐terminus of CSR1 binds with XIAP with high affinity. Through a series of in vitro recombinant protein binding analyses, the XIAP binding motif in CSR1 was determined located at amino acids 513–572. Knocking down of XIAP enhanced the CSR1 induced cell death, while over‐expression of XIAP antagonized CSR1's activity. The binding CSR1 with XIAP enhanced caspase‐9 and caspase‐3 protease activities. Mutant CSR1 that does not bind XIAP contained dramatically reduced cell death induction activity. XIAP mutant that does not interact with caspase‐9 had no impact on CSR1 induced cell death. As a result, this study elucidates a novel mechanism of cell death through relieving XIAP from inhibiting caspase activity by binding with a tumor suppressor.
Pyruvate protects the HT22 neuronal cells treated with rtPA against hypoxia‐reoxygenation damage
Authors: Ryou Myoung-Gwi , Liu Ran , Mallet Robert T , Yang Shao-Hua
Published: June 2021

Recombinant tissue plasminogen activator (rtPA) is the only FDA approved treatment for ischemic stroke, but delayed rtPA treatment may be neurotoxic. Pyruvate has proven neuroprotective in animal models of stroke. We examined pyruvate + rtPA cotreatment of neuronal cells subjected to an in vitro stroke model.

Methods

HT22 cells were exposed to 2 or 6 h hypoxia (0.5% O2) + glucose deprivation (OGD), followed by 24 h reoxygenation. Pyruvate (8 mM) and/or rtPA (10 μg/ml) were applied upon reoxygenation. Cell viability was assessed with Calcein AM. DCF‐DA and mitosox staining detected reactive oxygen species (ROS). Matrix metalloproteinase (MMP) activities were measured by gelzymography; MMP, tissue inhibitor of metalloproteinase 2, HIF‐1α, EPO and actin contents were determined by immunoblot.

Results

OGD lowered cell viability by 50% vs. normoxic control (P <0.05). Although rtPA treatment did not increase post‐OGD cell death, it intensified ROS production and activated MMP. Pyruvate‐induced cytoprotection was not compromised by rtPA cotreatment, and pyruvate dampened rtPA‐induced ROS and MMP. Delayed rtPA treatment intensified cell death 2 h after OGD, but pyruvate salvaged 50% of these cells.

Conclusion

Pyruvate attenuates rtPA‐induced MMP activation and ROS formation, thereby protecting reoxygenated HT22 cells. Pyruvate could be a useful cotreatment with rtPA for ischemic stroke. NIH support: R01NS054687, R01NS054651, R01NS076975, T32AG020494
TRANSCRIPTIONAL REGULATION OF THE METHIONINE ADENOSYLTRANSFERASE 2A GENE DURING HEPATIC STELLATE CELL QUIESCENCE AND ACTIVATION
Authors: Ramani Komal , Tomasi Maria Lauda
Published: June 2021

Objective

Methionine adenosyltransferases (MAT) catalyze the formation of the methyl donor, S‐adenosylmethionine (SAMe). The MAT2A gene encoding the catalytic subunit α2, is induced in de‐differentiated liver particularly in activated hepatic stellate cells (HSCs). MAT2A silencing reduces HSC activation. Our aim is to understand how MAT2A is regulated during HSC quiescence and activation.

Methods

Rosiglitazone (RSG)‐treated HSC cells or bile ducted ligation (BDL)‐activated primary rat HSCs were assayed for MAT2A expression, promoter activity and transcription factor binding.

Results

We identified peroxisome proliferator‐activated receptor (PPAR) response elements (PPREs) in the MAT2A promoter. The PPARγ agonist, RSG, promoted quiescence in HSCs, decreased the expression and promoter activity of MAT2A and enhanced PPARγ binding to MAT2A PPREs. BDL‐mediated liver injury lowered PPARγ interaction with MAT2A PPREs in activated HSCs. Silencing PPARγ increased MAT2A transcription and over‐expressing it had the opposite effect showing that PPARγ negatively controls this gene. Mutation analysis showed that PPREs are required for PPARγ to repress MAT2A in normal HSCs and the loss of PPARγ during HSC activation allows positive regulators like PPARβ to bind to the same PPREs and activate MAT2A transcription.

Conclusion

PPARγ is a negative regulator of MAT2A in normal HSCs. A switch from quiescence to activation abolishes this control and allows PPARβ to up‐regulate MAT2A transcription.

Research support: NIH grants 5K99AA017774‐02 (Komal Ramani), F32AA201502 (Maria Lauda Tomasi).
Endoscopic investigation of the palatopharyngeal muscular anatomy of the great apes: Anatomic models for the study of obstructive sleep apnea etiology
Authors: adar tony , Papaxanthos Michael , Lawson William , Pagano Anthony S , Petit Thierry
Published: June 2021

Laryngeal descent and external basicranial flexion create an upper respiratory tract ideal for spoken language, but predispose humans to obstructive sleep apnea (OSA). OSA may be related to soft palate flaccidity and length, as well as a failure of its paramedian resorption, causing airway collapse. To this end, non‐human hominoids (great apes) were used as anatomic models for a structural configuration divergent from humans. There exist no documented cases of OSA among great apes, likely a result of their upper respiratory tract anatomy. Endoscopic examination was performed on a sample of adult Pan troglodytes (n=1), Pongo pygmaeus (n=1), Gorilla gorilla (n=1), neonate Gorilla gorilla (n=1) and Hylobates sp. (n=1) from La Palmyre Zoo in Les Mathes, France. Great apes exhibited marked soft palate length, which expanded posteriorly in a nearly horizontal orientation—as opposed to the comparatively vertical orientation of humans—to cover a relatively deep nasopharynx. Uvular projection from the palatopharyngeal arch was short compared to humans. The neonates, however, displayed more shallow nasopharynges and vertical soft palate and palatopharyngeal arch orientations, similar to humans. Future studies should investigate whether OSA related to airway constriction at the fauces is prevented in great apes by a greater nasopharyngeal depth and reduced palatopharyngeal and surrounding soft tissue flaccidity.

Grant Funding Source : alumni association
Functional imaging of signal propagation in native and engineered myocardium
Authors: Sarvazyan Narine , Asfour Huda , Kay Matthew W
Published: June 2021

There are both similarities and differences between imaging of calcium and electrical activity from excised hearts and engineered tissue constructs. We compare the two and consider critical methodological issues that can affect interpretation of optical mapping data from hearts implanted with cardiac tissue constructs. Dual imaging modalities, including mapping of epicardial electrical activity with tissue metabolic state, are also considered. Such modalities are important to reveal differences between metabolic demands of native and surgically implanted engineered myocardium. The mismatch between the two can lead to graft‐induced arrhythmias, especially in settings of intermittent flow. The differences between native and engineered myocardium can become more apparent when they are studied using physiologically relevant conditions, i.e. when cardiac muscle contracts against a mechanical load. This requires performing experiments using a bi‐ventricular working heart preparation, instead of a Langendorffâ€”based setup. Additional post‐processing steps, such as wavelet‐based analysis, can be then used to minimize effects of motion on optical signals. Lastly, we discuss feasibility of shipping engineered cardiac constructs for implantation and/or high‐resolution optical mapping at a different institution (supported by NIH HL095828 Award).
Modulation of secretion in Drosophila by amino‐acid starvation
Authors: Rabouille Catherine
Published: June 2021

Secretion is mediated by the secretory pathway that have a basic functional organisation very similar in Drosophila as in mammalian cells.

Here, I will discuss evidence showing that secretion is be modulated by physiological conditions. Using Drosophila S2 cells, we have shown that amino‐acid starvation stimulates a pathway that inhibits secretion. This pathway relies on the atypical MAP Kinase ERK7 that we found is stabilised by amino acid starvation, leading in turn to the modification of Sec16, a key protein required for the functional organisation of the ER exit sites that are critical for secretion. Once modified, Sec16 no longer associates to ER membrane, ER exit site biogenesis is inhibited, and so is secretion as a whole. Furthermore, this pathway is independent of the TOR complex 1.

These results indicate that secretion inhibition under amino‐acid starvation is an active mechanism requiring ERK7, not only a passive response to the absence of growth factors. Furthermore, they show that in the absence of nutrient, cytoplasm growth is inhibited via TORC1 inhibition whereas plasma membrane growth via secretion is inhibited independently.
Inflammation and Insulin Resistance
Authors: Olefsky Jerrold M
Published: June 2021

Current evidence indicates that chronic activation of the proinflammatory state may be an underlying mechanism for insulin resistance. Together, these findings suggest that increased macrophage activation can be an important contributor to the pathogenesis of cellular insulin resistance. To assess this concept, we have used genetic deletion of key molecules controlling the inflammatory pathway, in mouse KO studies. Macrophage specific deletion of IKKβ or JNK1 disables the cytokine stimulated NFκB or JNK1/AP2 proinflammatory pathways leading to decreased tissue inflammatory markers, enhanced insulin sensitivity, and protection from the effects of high fat diet to cause insulin resistance. The enhanced insulin sensitivity and protection against insulin resistance were manifested in liver, muscle, and adipose tissue, indicating that inhibition of the inflammatory pathway within macrophages leads to a global state of heightened insulin sensitivity. Taking another approach to this issue, omega‐3 fatty acids (ω‐3 FAs), DHA and EPA, exert anti‐inflammatory effects, but the mechanisms are poorly understood. We found show that the G protein‐coupled receptor 120 (GPR120) functions as an ω‐3 FA receptor/sensor. Stimulation of GPR120 with ω‐3 FAs or a chemical agonist causes broad anti‐inflammatory effects in monocytic RAW 264.7 cells and in primary intraperitoneal macrophages. All of these effects are abrogated by GPR120 knockdown. Since chronic macrophage‐mediated tissue inflammation is a key mechanism for insulin resistance in obesity, we fed obese WT and GPR120 knockout mice a high fat diet with or without ω‐3 FA supplementation. The ω‐3 FA treatment inhibited inflammation and enhanced systemic insulin sensitivity in WT mice, but was without effect in GPR120 knockout mice. Thus, GPR120 is a functional ω‐3 FA receptor/sensor and mediates potent insulin sensitizing and anti‐diabetic effects in vivo by repressing macrophage‐induced tissue inflammation.
Designing a laboratory‐based microscopic anatomy class for undergraduates
Authors: Smith Sally Kathryn Sommers
Published: June 2021

Undergraduate students who are preparing for dental school at Wellesley College petitioned for training in human microscopic anatomy. Although such a course exists in the Biological Sciences course catalog, it had not been offered for two years, and was scheduled to be eliminated from the catalog, In response to the students’ petition, I designed a laboratory‐only course focusing on close observation and critical thinking. Students were challenged to seek out sources for their learning about cells, tissue types, and organ anatomy, to supplement class presentations and discussions. Tests consisted of clinical case studies which the students interpreted and related to their laboratory studies. The final project was a poster, incorporating information from a clinical case study, which contrasted the appearance of normal human tissues with pathological specimens. The posters were submitted for presentation in Wellesley's undergraduate research conference. Students have now petitioned for an advanced microscopic anatomy course in systems anatomy and pathology.
Watching development: an easy ex ovo culturing method to directly observe late‐staged chicken embryos
Authors: Franz-Odendaal Tamara Anne , Duench Kellie
Published: June 2021

The ability to view an embryo in vivo during development is important for the teaching of developmental biology. Viewing vertebrate embryos that are protected by a shell presents a challenge. Researchers have developed techniques to grow chicken embryos outside of their shell yet this method has typically focused on the study of relatively young embryos. We have optimized the ex ovo culturing method so that embryos can be maintained for the majority of embryonic development (until at least stage HH41), enabling students to directly observe many of the aspects of development over time (e.g. limb development, eye development, etc). This technique is suitable for introductory and advanced developmental biology, embryology and anatomy classes. For advanced classes, this culturing technique enables access to late stage embryos in order to perform manipulations. An example of a manipulation that can be performed on shell‐less embryos is the implantation of beads with inhibitors in order to gain further understanding of the molecular pathways involved in the development of anatomical structures. After any manipulation, the chamber can be sealed and the embryo can be cultured further. This method enables the direct observation of normal development and manipulated development.
The novel ‘syncretion’ approach to learning gross anatomy with clay models: Is it a plausible alternative for learning the muscles in the anterior forearm?
Authors: Kim Hanna , Johnson Marjorie , Wilson Timothy
Published: June 2021

Traditionally, dissection has been the method of choice for learning gross anatomy at the undergraduate level. However, rising problems in medical curriculums, such as reduced hours dedicated to anatomy education, has yielded a plethora of research in alternatives. A novel approach to learning gross anatomy, called ‘syncretion,’ has yet to be proven efficacious. The purpose of the current study is to evaluate student performance on a “bell‐ringer” examination following exposure to a syncretic or dissection model of the anterior forearm. Students will be randomly assigned into three test groups: the syncretion group will reconstruct, or build up, the anterior forearm using pre‐formed clay representations of the 8 muscles; the dissection group will “dissect” through a fully‐assembled clay model of the anterior forearm with a scalpel to visualize deeper muscles; and, the control group will learn the 8 muscles without a model. All groups will complete pre‐ and post‐tests to evaluate spatial identification, spatial reasoning and comprehension of function of musculature and osteology. It is hypothesized that the change in scores from pre‐test to post‐test in the syncretion group will mirror those exhibited by the dissection group. Following this, it is also thought that the change in scores in the two treatment groups will be greater than that of the control group, due to the added benefit of a tactile model.

Grant Funding Source: Western Graduate Scholarship
A digital atlas for ultrasound guided nerve blocks of the upper limb
Authors: Rawski Ryan James , Brookes Jon , Johnson Marjorie , Ganapathy Sugantha
Published: June 2021

Ultrasound guidance (UG) ameliorates the dependency on surface landmarks to perform nerve blocks by providing real‐time imaging of subcutaneous tissues, allowing for purposeful needle movements to accurately deliver anesthetic. UG has been associated with improvements in the quality of sensory blocks, the onset time, and the success rate. The objective of this project is to create a digital atlas (DA) to aid trainees with their interpretation of ultrasound (U) anatomy, so that benefits of UG are actualized. Surface anatomy, serial cadaver dissections, U, and Visible Human Project images have been integrated into the DA to illustrate relationships between surface landmarks, physical anatomical relationships, and the planar U image. Each block and its indications are described in text and subsequently demonstrated in narrated videos. The DA includes an introductory module on mechanics and operation of the U device. Feedback solicited at monthly anesthesiology resident training will guide the design of the DA as content for trunk and lower limb blocks is developed. We anticipate that having online access to the DA will allow residents to acquire the requisite foundation of knowledge to interpret U anatomy. This would allow training programs to efficaciously use limited lab time to reinforce key concepts and develop students procedural skills (needle/ultrasound probe manipulation) through kinesthetic learning.

Grant Funding Source: Ontario Graduate Scholarship
Restoration and Preservation of Pathological Surgical Specimens
Authors: Zekhtser Mitchell , Tugbenyoh Mawuli , Nusse Gloria
Published: June 2021

San Francisco State University received a historically significant pathology specimen collection from Stanford University in the 1970s. Originally, the specimens had been preserved in formaldehyde and water, but as the years went by the formaldehyde degenerated, causing decomposition of the tissues. As a result, the anatomy lab had stopped using them as teaching tools because they had become cloudy and dark.

Ethanol has become the widely accepted professional method of liquid preservation partly because it is a less toxic and more environmentally friendly solution for long‐term storage and display. However, not much has been written about its use for human tissue revitalization in collections such as this.

We found that the tissues dried out significantly when we changed solvents immediately from formaldehyde and water to an ethanol solution. After researching the problem, we decided to use sequential positive step‐gradient incubation in alcohol. First, the specimens were drained of the formaldehyde and water. Then, the ethanol was introduced in 20% increments. The slow steps of the ethanol incubation prevented the tissue from drying. Four months after this revitalization, the results show that the tissues have not shrunk and have sustained their integrity.

By restoring these rare specimens to prime condition, we are once again able to use them as invaluable learning tools in the anatomy lab.

Grant Funding Source : San Francisco State University Biology Department
Foundations for a lifetime: A qualitative inquiry into the salient aspects of cadaver dissection over time and their impact on medical professionals
Authors: Skinner Miki D , Morrow Sue , Morton David A
Published: June 2021

Cadaver dissection has been a central part of the education of medical professionals for centuries. Throughout that time, anatomists have claimed that dissection is a learning experience rich with life lessons encompassing more than simply gross anatomy. Yet, no published empirical data exists of the long‐term impact that dissection has on medical professionals. The objective of the current study was to explore the aspects of cadaver dissection that remain salient following the transition from medical student to medical professional. This qualitative research utilized grounded theory methodology exploring the autobiographical narratives of physicians (n=36) 5 to 57 years post‐graduation. Data were drawn from multiple sources: interviews, written reflections, focus group, and archival data. Results indicate that physicians reflect on their dissection experience as a foundation for skills in teamwork, deportment with patients, and diagnostic visualization among others. Implications of these results add to the pedagogical value of dissection, providing empirical evidence for the multi‐faceted learning within the anatomy laboratory. The study was funded in part by the Frank L. Christensen Fellowship.

Grant Funding Source : Frank L. Christensen Fellowship
The Anatomy Training Program (ATP) by Distance Learning Co‐sponsored by AAA and the Anatomical Societies: Experiences of First Cohort of AAA Trainees and Mentor
Authors: Vasan Cheryl , Smoot Betty , Vasan Nagaswami
Published: June 2021

The ATP, co‐sponsored by AAA & the Anatomical Society, aims to develop trainee competence in topographical, systematic, functional and developmental anatomy. The ATP promotes development of knowledge, pedagogical skills, and attitudes to become a competent teacher. Individualized training allows trainees to work within the ebb and flow of their research and teaching; online resources and course syllabi allow trainees to work with a mentor at their home institution. This includes self‐directed learning, guided by an anatomy faculty mentor, with opportunities for mentored teaching, dissection, and self‐reflection. Radiological anatomy, clinical correlates and case studies are included throughout. The ATP can be completed in 2 years and includes 2 learning modules per year: Limbs and Head & Neck, and Trunk and Neuroanatomy, offered in alternate years. Training may include all modules or only those that fit trainee career plans. In July 2011, AAA sent two trainees and one mentor to the annual intensive Residential School in England, run by a team of anatomists with support from clinicians. Trainee admission to the Residential School required satisfactory completion of the yearly program, as determined by the Steering Committee and the trainee's mentor. AAA provided tuition for each participant, plus travel/room/board expenses and expenses of one selected mentor to the Residential School. Supported by the AAA.
Increased stress during gross anatomy course negatively affects students’ performance
Authors: Ramos Amanda , Bringe Kariline
Published: June 2021

A modular design of gross anatomy curriculum encompasses learning a large amount of content in a short time frame. Studies have identified academic responsibilities as a stressor which may negatively impact students’ comprehension of material. At Mayo Medical School, the academic demands of the 7 week anatomy course provide an environment that may predispose students to increased stress. Our research explores the relationship between perceived stress and academic performance. A validated survey instrument identified a baseline stress level prior to the course and was used to reassess at timed intervals during the course. Stress levels were compared to course performance using quiz and laboratory practical grades as performance indicators. Preliminary results demonstrated a 46% increase in perceived stress levels (10.8 to 15.8) by day 21 with an increasing correlation between perceived stress and decreased academic performance. From day 7 to day 21, the correlation between perceived stress and quiz scores increased from R2=0.0002 to 0.29. The correlation between stress and laboratory practical grades increased from R2=0.09 to 0.29. Understanding the impact of stress on academic performance may aid in helping students with high perceived stress develop coping strategies which may increase performance as well as retention of anatomical knowledge.

Grant Funding Source : Department of Anatomy, Mayo Clinic College of Medicine
Structural Insights into the Role of PHD Fingers in the MOZ/MORF and HBO1 HATs
Authors: Glass Karen C , Saksouk Nehmé , Ullah Mukta , Yang Xiang-Jiao , Côté Jacques , Kutateladze Tatiana G
Published: June 2021

The MOZ/MORF and HBO1 histone acetyltransferases (HATs) are important for regulation of many cellular processes including transcription activation, DNA repair and development. They form multi‐subunit complexes that contain several distinct plant‐homeodomains (PHD). PHD fingers were originally discovered to bind post‐transcriptional modifications (PTMs) on the histone H3 tail, particularly tri‐methylation at lysine 4 (H3K4me3). Current research indicates PHD fingers are divided into functional classes that bind other PTMs as well. The ING4, ING5, BRPF1 and Jade1 subunits of MOZ/MORF and HBO1 all harbor PHD fingers, however, the functional role of these PHD modules has not been determined. We aimed to elucidate the different substrate specificity and function of PHD fingers within MOZ/MORF and HBO1 towards histone tails using X‐ray crystallography, NMR, fluorescence spectroscopy and mutagenesis. Here we report the crystal structures of ING4 and ING5 in complex with the H3K4me3 histone peptide, the binding of Jade1 and BRPF1 to the unmodified H3 tail, and affinities of the PHD finger:histone interaction. Functional analysis indicates both the ING4/5 subunits and methylated H3K4 regulate enzymatic activity. Together these studies provide an essential link between HAT activity and chromatin remodeling, and impart a greater understanding on how the histone code is read.
Structural basis for excision of deaminated and oxidized 5‐methylcytosine by thymine DNA glycosylase
Authors: Maiti Atanu , Pozharski Edwin , Drohat Alexander C
Published: June 2021

The DNA base excision repair (BER) pathway is essential for maintaining genomic integrity and is implicated in active DNA demethylation, a key element of epigenetic transcriptional regulation. Thymine DNA glycosylase (TDG) excises thymine from mutagenic G·T mispairs, initiating repair of deaminated 5‐methylcytosine (mC). TDG also excises 5‐formylcytosine (fC) and 5‐carboxylcytosine (caC), oxidation products of mC produced by Tet enzymes. These seemingly disparate activities are consistent with TDG specificity for acting at CpG sites and its essential role in active DNA demethylation and embryonic development. Understanding how glycosylases excise lesions and avoid acting on undamaged DNA is an important problem in DNA repair. Structural and biochemical results here reveal how TDG attains broad specificity for G·T and G·fC lesions while avoiding A·T pairs. A crystal structure of TDG (catalytic domain) bound to substrate analogue suggests G·T glycosylase activity is suboptimal owing to unfavorable interactions between flipped dT substrate and two active‐site residues. Remarkably, mutating these residues greatly increases G·T activity and confers substantial activity for normal A·T base pairs. The results suggest TDG evolved with suboptimal G·T repair capability in order to minimize aberrant activity on undamaged DNA, an unprecedented finding for a repair enzyme. Supported by NIH (R01‐GM072711).
Termination of exonuclease 1‐catalyzed mismatch excision requires physical interaction between exonuclease 1 and MutLα
Authors: Lee Sanghee , Fan Zhanmin , Zhang Yanbin , Tian Lei , Yuan Fenghua , Gu Liya , Li Guo-Min
Published: June 2021

The human mismatch repair (MMR) reaction was previously reconstituted in vitro using purified recombinant proteins that include MutSα or MutSβ, MutLα, RPA, EXO1, HMGB1, PCNA, RFC, polymerase δ, and DNA ligase I. Although MutLα is not essential for 5′ nick‐directed excision, it promotes termination of mismatch‐provoked EXO1‐ catalyzed DNA excision upon mismatch removal. However, the mechanism of the excision termination reaction is unknown. This study examines whether the physical interaction between the MLH1 subunit of MutLα and EXOI, which is mediated by a conserved motif in EXO1 known as an MLH1 interacting protein (MIP)‐box, is responsible for the excision termination. For this purpose, alanine substitution mutations were engineered at residues F506 and F507 in the MIP‐box of EXO1, and the resulting FF‐EXO‐AA mutant protein was tested for its role in MMR in vitro. As expected, FF‐EXO1‐AA behaves similarly as wild type EXO1 in catalyzing 5′‐directed excision and interacting with MutSα, but the EXO1 mutant protein fails to interact with MutLα. Our in vitro MMR assay show that mismatch‐provoked excision is efficiently terminated in reactions containing wild type EXO1, but not in reactions containing FF‐EXO1‐ AA. These observations strongly suggest that the termination of EXO1‐catalyzed mismatch excision is executed via physical interaction between MutLα and EXO1.
Hydrogen sulfide reduces GFP‐tagged autophagosomes in vitro
Authors: Duarte Maria J , Julian David , Akin Debra , Dunn William A , Wohlgemuth Stephanie E
Published: June 2021

Autophagy, a process by which intracellular components are degraded within lysosomes, has been implicated in protection from ischemia/reperfusion (I/R) injury. The gasotransmitter H2S has also been associated with I/R protection. H2S activates the PI3‐Kinase/AKT pathway. However, activation of this pathway has been shown to inhibit autophagy.

Objective

To test whether H2S exposure attenuates autophagy.

Methods

Saos‐2 human osteosarcoma cells, stably expressing the autophagosome (AP) marker GFP‐LC3, were placed in Krebs‐Henseleit Buffer (“starved” cells), which induces autophagy, or in growth medium (“fed” cells) for 4 h. H2S gas was then added to a final concentrations of 1, 10, or 100 ppm for 1 h. The number of GFP‐tagged APs per cell was quantified using epifluorescence microscopy.

Results

Addition of H2S significantly reduced the number of GFP‐tagged APs per cell in starved cells from 4.1 ± 0.9 (SE) to 2.0 ± 0.5, and in fed cells from 1.8 ± 0.3 to 1.1 ± 0.1 (H2S‐effect: p = 0.016). The effect of H2S on autophagy was similar at 1, 10 and 100 ppm, with the lowest H2S concentration producing sub‐micromolar dissolved H2S. Addition of H2S did not reduce fluorescence intensity per AP or the fluorescence of cytosolic GFP, suggesting that H2S did not have a direct effect on GFP fluorescence. These data suggest that the role of H2S as a protective agent in I/R injury is likely independent of autophagy. Supported by UF funds to SEW.
HS1 is required for neutrophil polarization, LFA‐1 activation and leukocyte recruitment
Authors: Schnoor Michael , Logermann Silke Maria , Jing Ding , Li Hang , Butz Stefan , Vestweber Dietmar
Published: June 2021

Neutrophil extravasation is a critical step in innate immunity in response to tissue injury or invading pathogens. The interaction of neutrophils with the endothelium requires activation of adhesion molecules such as the β2‐integrin LFA‐1 leading to firm adhesion, polarization and intraluminal crawling to the actual site of diapedesis. These steps require coordinated cytoskeletal remodeling. The cortactin homologue in leukocytes, hematopoietic cell‐specific lyn substrate (HS1) is an actin‐binding protein that regulates actin dynamics and may thus be relevant in the regulation of leukocyte extravasation. Indeed, we found that leukocyte adhesion and extravasation in the TNF‐α‐stimulated cremaster were strongly inhibited. This was accompanied by an increased rolling velocity pointing to the fact that the transition from rolling to firm adhesion is disturbed in the absence of HS1. This could be explained by impaired LFA‐1 activation and disturbed pseudopod formation in response to TNF‐α as revealed by electron microscopy. Importantly, we unraveled a novel signaling complex containing HS1 that is involved in the regulation of LFA‐1 activation upon inflammatory stimuli. Our results are the first physiological evidence that HS1 is crucial for orchestrating molecular events in neutrophils to mediate firm adhesion and leukocyte recruitment in vivo.