bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of Gut Microbiome Modulation on Gut Inflammation
Authors: Ballou Anne , Ali Rizwana , Croom Jim , Koci Matt
Published: June 2021

Direct fed microbial products have been the focus of considerable scientific and public interest as we learn how the gut microbiome impacts host health and immune function. Previous research in our lab has demonstrated supplementation with a lactic acid bacteria (LAB) product results in an anti‐inflammatory host phenotype. It is also associated with an anti‐inflammatory cytokine profile in the ileum (↑ IL‐10, ↓ IL‐12RB2). However, the mechanisms behind this effect remain unknown. We hypothesized that the LAB product would stimulate the presence of bacteria with an anti‐inflammatory effect on the gut mucosa and/or suppress inflammatory species. Following supplementation with 3x105 CFU LAB (Lactobacillus acidophilus, Lactobacillus casei, Enterococcus faecium, and Bifidobacterium bifidium)/g feed from day of hatch, we evaluated the gut microbiome of 28‐day old chickens at 5 gastrointestinal locations. Microbial DNA was isolated from 5 samples/treatment x location and sequenced using Illumina MiSeq paired end sequencing. The sequence reads were processed and analyzed with Qiime and Primer‐E. Analysis of intestinal samples revealed upward shifts in Lactobacillus following supplementation, as well as a sharp decline in segmented filamentous bacteria (SFB) in the ileum. Though they have been observed in the ileum of many species for decades, the taxonomic identification of these bacteria is relatively recent, as is understanding of their role in stimulating gut inflammation through TH17 cells. These data suggest this LAB consortium works to decrease levels of SFM in the ileum, which may result in less ileal inflammation. This could reduce gut inflammation, reducing irritation and improving digestive efficiency.
Metabolomic and Clinical Disturbances in Canine Inflammatory Bowel Disease
Authors: Jackson Matthew , Jewell Dennis
Published: June 2021

Canine inflammatory bowel disease (IBD) is a progressive disease with poorly characterized etiology. We sought to gain insight into the metabolic disturbances preceding and co‐incident with canine IBD by comparing the plasma metabolome and clinical indices of dogs (n =30) prior and subsequent to diagnosis of IBD by histopathological assessment of intestinal tissue collected by endoscopy. Archived samples were generated from annual health screens and adventitious health‐related collections curated according to the IBD status of their donors. Metabolomic and clinical analysis was also performed on bioarchived time‐matched samples from disease‐free, healthy control dogs (n = 30). Blood collections were carried out under IACUC approved protocols. Results showed several circulating metabolites of colon microbiota were decreased in IBD dogs, including phenol and indole derivatives. Further disruptions were noted in antioxidant status and lipid metabolism. A stepwise discriminant model selected a subset of clinical markers including liver enzymes, plasma and urine protein, platelets, basophils and monocytes as capable of correctly classifying >90% of samples as derived from post‐IBD versus healthy controls. The clinical findings imply that disruption of the gut and liver barriers, protein‐losing enteropathy and immune perturbation was co‐incident with IBD. In summary, assessment of dogs pre‐, peri‐ and post‐diagnosis implies hindgut dysbiosis, altered redox status and disrupted lipid metabolism accompany clinical disturbances in canine IBD.
Cognitive Development in Rural Gambia: Relating Behavior and Growth from 6 – 24 Months of Age
Authors: Lloyd‐Fox Sarah , Halliday Drew , Begus Katarina , Vellekoop Perijne , Maris Helen , Sanyang Lamin , Drammeh Saikou , Darboe Momodue , Prentice Andrew , Moore Sophie , Elwell Clare
Published: June 2021

The goal of our project is to establish assessments to evaluate the efficacy of nutritional interventions during the first 1000 days of life. Here we examine behavioral performance on a standardised developmental assessment scale (Mullen Scales of Early Learning; MSEL) and anthropometric growth indices in infants at a research station in West Africa.

The MSEL is a developmental assessment battery for use from birth to five years of age. We adapted the MSEL for use in The Gambia. A committee of native, dual‐language speakers and trained MSEL administrators reviewed and adapted the measure where appropriate (i.e. linguistic translation of the item‐by‐item verbal prompts, as well as the substitution of stimuli that were deemed culturally inappropriate). 225 infants ranging from 6 – 24 months of age were assessed and their MSEL scores compared with an age‐matched group of infants tested in the UK.

Relative to the U.K infants, scores of cognitive development declined over the first two years of life, particularly for the language subscales of the MSEL. An analysis of the relationship between growth and cognitive development revealed that height for age z‐scores correlated with scores on the MSEL. In particular for infants in their second year of life lower scores of cognitive development (MSEL) were related to lower height for age scores.
Neutrophil‐Derived Microvesicles Migrate into Cell‐Impenetrable Cartilage to Protect the Joint During Inflammatory Arthritis
Authors: Headland Sarah , Norling Lucy , Kim Andrew , Jones Hefin , Jan Lily , Perretti Mauro
Published: June 2021

Microvesicles (MVs) mediate inter‐cellular communication by transferring cellular components to target cells, endowing them with an enhanced lipid and protein repertoire. We aimed to determine the function of neutrophil‐derived MVs in arthritis, as their concentration was augmented in synovial fluid of rheumatoid arthritis patients compared to paired plasma. Neutrophils adoptively‐transferred into arthritic mice migrated to inflamed joints and released MVs, observed abundantly within cartilage; the first evidence that MVs can penetrate cartilage matrix. Recent studies implicate TMEM16F as crucial for the exposure of phosphatidylserine on the cell surface, a key step in MV formation. Congruently, TMEM16F knockout mice released fewer neutrophil MVs and exhibited exacerbated cartilage damage in a model of inflammatory arthritis. Exogenous neutrophil MVs activated anabolic gene expression leading to extracellular matrix accumulation and cartilage protection. Mechanistic studies support a model whereby MV‐associated Annexin A1 interacts with its receptor FPR2/ALX, increasing TGF‐β production by chondrocytes, ultimately leading to cartilage protection. We envisage that MVs can be harnessed as a novel unique therapeutic strategy for the protection of cartilage and treatment of cartilage‐erosive diseases, where current therapy is completely lacking.
Simultaneous Quantitation of Lipid and Apo‐Protein Synthesis: Diet‐Mediated Reduction in De Novo Lipogenesis is Associated with a Stimulation of ApoB and A1 Production In Vivo
Authors: Previs Stephen , Zhou Haihong , Herath Kithsiri , Xie Dan , Castro‐Perez Jose , Wagner David , Kelley David
Published: June 2021

We recently demonstrated the ability to simultaneously quantify lipid and protein flux in vivo using oral D2O dosing. The uniqueness of using D2O is the rapid equilibration with total body water, use of oral and not intravenous dosing, and ease to measure the true precursor enrichment (either plasma, urine, or saliva). To examine whether dietary modulation of de novo fatty acid and cholesterol synthesis would lead to parallel changes in apoB and/or A1 production, non‐human primates were either fed a standard carbohydrate‐based diet or a high‐fat, high‐cholesterol diet for several months. Lipid and protein kinetics were studied by giving a single oral bolus of D2O and sampling blood for ~ 96 hours thereafter. The dietary perturbation altered the plasma lipid profiles. The analyses of lipid labeling (measured via GC‐IRMS) demonstrated a marked suppression of fatty acid and cholesterol synthesis with high‐fat feeding. Despite these changes we observed a substantial increase in both apoB and A1 synthesis (measured via LC‐MS). In total, we demonstrate that diet‐induced modulation of pathways involving de novo lipid synthesis do not necessarily translate into a comparable protein‐flux phenotype. Our data suggest that the dietary lipid, as compared to de novo synthesized lipid, has a pronounced effect on circulating apo‐protein flux. The methodology applied here can be readily adapted for studies in other pre‐clinical models (e.g. rodents) as well as applied in humans. This protocol design provides a translational biomarker approach for interrogating modulations that affect dyslipidemia.
The Cardiovascular System for Fourth Graders: Heart Rate and Physical Activity during PhUn Week
Authors: Brown Jaylyn , Jarrett Steven , Miller Breena , Hayslip Rachel , Gurovich Alvaro
Published: June 2021

Physiology Understanding (PhUn) week is the APS K‐12 outreach program that helps increasing awareness about STEM education. Here, we combined PhUn week and health education to show the cardiovascular system (CV) and how the heart rate (HR) increases through physical activity, integrated into the 4th grade science curriculum. The activity was divided in 5 different sessions, one per room, in two different days, reaching 116 elementary school students. Each room was divided in small 4‐student groups with the following general activity set‐up was: 1) brief presentation about the CV system, 2) measuring HR during 3 different conditions, 3) analyzing collected data, and 4) discussing obtained results. Two children per group were connected to an EKG via Biopac MP40 system and data was analyzed with the Biopac Science Lab software. Electrodes were placed on the right wrist, right ankle, and left ankle. Resting, hula hooping, and running EKGs were recorded. After collecting EKGs, other two students analyze the data, using the computer, to determine HRs during each activity. The students recorded these values in a data table that they would later use in their science class to plot and compare HR values. Finally, a discussion was performed to integrate findings with CV physiology. By the end of PhUn week, 4th graders collected data from 40 class mates and each teacher had enough HR data to enhance their science curriculum. Overall, we expect that the goal of enhancing the elementary students' knowledge of the cardiovascular system was achieved; however, we are sure that at least they have ‘PhUn’.

Supported by IU AHEC 14/15 #PIU081 and APS PhUn week program
Open Inquiry‐Based Learning Elicits Deeper Understanding of Complex Physiological Concepts Compared to Traditional Lecture‐Style or Guided‐Inquiry Learning Methods
Authors: Franklin Brandon , Xiang Lin , Collett Jason , Rhoads Megan , Osborn Jeffrey
Published: June 2021

Inquiry‐based teaching (IBT) pedagogy has been theorized to be a more effective method of conveying deeper understanding of scientific concepts compared to traditional lecture‐style instruction. This study compared 2 levels of IBT (guided‐problem based inquiry, GPBL; and open ended‐problem based learning, OPBL) with traditional lecture style instruction (LI) in an upper level, undergraduate animal physiology (AP) course. Students enrolled in AP were randomly assigned to one of three experimental groups: LI, GPBL or OPBL. Student grade point averages were not different between groups at the onset of instruction (p>0.05). Student assessments included multiple choice (MC) and short answer (SA) questions at 3 time points across the semester. Students in the OPBL group scored higher on both MC (56.48±6.92pts) and SA (55.38±8.78pts) compared to the LI group (MC: 53.52±8.64pts; SA: 50.95±12.7pts; p<0.05). Conversely, the GPBL group showed no difference in either MC scores (53.54±7.27pts) or SA scores (50.28±11.75pts). These results indicate that students with OPBL instruction, who have been challenged to think about conceptual problems and work with their peers to find a solution, gain a better understanding of concepts compared to LI students. Future studies will assess the writing sample performances of each test group in the same experimental design.
Construction and Implementation of Multiplatform Virtual Microscopy for Histology
Authors: Ettarh Raj , Frasch Tripp , DeVore John , Rhodes Jim
Published: June 2021

For students studying tissue and organ microstructure, histology is an image‐based approach to understanding the relationship between form and function. Borne out of a collaborative effort between members of the Digital Histology Interest Group of the American Association of Anatomists, this project examined the conversion of specialized digitized images to a newer digitized open‐source format that would allow for compilation and delivery in a browser‐based environment in a cost‐efficient way. 214 images (76GB) were converted from the Bacus format to a format that is compatible with the Google Maps application programming interface (API) using high speed image processing and conversion facility at Pacific Northwest University of Health Sciences (courtesy of J. Rhodes). Converted images were then compiled and assembled as a virtual microscopy application using the open‐source Google API code API, and deployed on servers at Tulane University. Implementation has involved the construction of image collections for lab as well as embedding into web‐driven html guides for use during and outside of interactive laboratory sessions in Histology. The outcomes following this implementation have included the introduction of interactive microscopy‐based sessions, the creation of single window viewing arising from integration of the virtual microscopy application with html‐driven lab guides, and enhanced accessibility on a range of desktop and mobile smart‐devices.
Perceptions of a Flipped Histology Class: as Polarized as Politics?
Authors: Klein Barbie
Published: June 2021

Incorporating flipped classrooms in the medical curriculum is gaining popularity, yet the perceived benefits by students are less well known. At the request of the course director, the author of this study developed a flipped class for the immune and lymphatic systems in a traditional semester‐based medical‐level histology course. Students were to prepare prior to class by watching a podcast, reviewing a presentation of laboratory terms, and reading a chapter. Class began with an online quiz over the material followed by a discussion about the questions. Groups then completed three computer‐based clinical cases. To emulate the process of diagnosis, cases were constructed as hyperlinked text documents permitting students to decide between various outcomes at each step before proceeding.

Students were invited to complete a survey regarding their experience. Most responded positively, however, were divided about adding more flipped classes. Several students expressed dislike for the amount of preparation required to get the most benefit and some felt that flipped classes are a poor substitute for traditional lectures. The interactive clinical cases were well received by most students and several commented that the cases lead to effective collaboration with classmates. This study indicates that some learners prefer the design of this particular flipped class while others are strongly opposed to deviating from traditional lecturing. The majority of students suggested incorporating a few flipped classes throughout the semester to supplement lecturing.
Assessment of Brazilian Students' Views about the Use of Human Bodies in Anatomy Education
Authors: Rocha Andrea , Pedron Julia , Thomaz Leonardo , Girotto Marina , Simoneti Lucas
Published: June 2021

The new technologies available for teaching anatomy and the shortage of human bodies for anatomy classes in Brazil make it necessary to discuss the use of bodies in anatomy education nowadays. Thus, this study aims to assess the views of health sciences university students about the use of human bodies in practical anatomy classes. Students were asked to fill out an online questionnaire about the use of bodies in anatomy teaching and its impact on their academic training. Two different questionnaires were applied to two groups of students based on their experience with dissection in the course of gross human anatomy. The questionnaires were available from July, 2013 to August, 2014. A total of 723 students from 83 universities around Brazil participated in the study. Of those, 53% had never performed human dissection, among which 96% think that dissecting a body might improve their anatomical knowledge. Among those who had performed dissection, 0,3% agree that dissection could be replaced by virtual training in 3‐dimension models, and 89% believe that dissecting made it easier to learn anatomy. Of all the students who participated in the study, 94% believe that the use of human bodies is essential in anatomy education and 89% disagree that the learning of anatomy using only anatomical models is enough. Finally, 93% believe that anatomical models complement but do not replace the use of human bodies in anatomy classes. Data analysis showed that students believe that the use of bodies has a positive impact on their learning of anatomy and on the quality of their academic training. Moreover, the students' opinions might reflect their will to learn anatomy using human bodies in spite of the technologies available these days.
Acquiring Skills Beyond the Bench Through the Great Diseases ‐ a Teacher‐Scientist Partnership
Authors: Tammen Stephanie , Malanson Katherine , Jacque Berri , Meiri Karina
Published: June 2021

Young scientists know that being competitive in the job market requires skills and experience beyond the bench. Given the overproduction of biomedical scientists with PhDs relative to the number of openings for secure academic positions, it is critical that young scientists acquire broad skill sets. Participating in curriculum design partnerships with teachers offers scientists a novel career opportunity. Our collaborative curriculum development team of biomedical scientists has partnered with teachers to build inquiry‐based biomedical curricula focused on “Great Diseases” that impact global health (Infectious Disease, Neurological Disorders, Metabolic Disease and Cancer), and now the scientists are also leading dissemination of the curricula in classrooms nationwide. Teachers often feel unprepared to lead students in formulating questions, designing experiments and interpreting data, a major obstacle to implementation of curricula with novel scientific content or authentic science practice. Interacting directly with scientists helps solve this problem. Scientists work with teachers via one‐on‐one virtual meetings, during which the biomedical scientist offers the teacher structured real‐time mentoring in content and helps devise strategies to implement the material in the classroom, at the same time augmenting their knowledge of effective teaching methods. We have found that when teachers participate in structured mentoring their ability to teach about cutting‐edge biomedical science improves, which is passed on to the high school students, the ultimate beneficiaries.

Funded through: NIH Grant: R25OD010953‐04, gift from Boston Scientific and Cubist Pharmaceuticals Grant
Biochemistry Summer Academy ‐Assistance in the Development of Curricula for the Next Generation Science Standards
Authors: Blaha Gregor , Niks Dimitri , Halford Joshua , Baldwin Thomas
Published: June 2021

The Biochemistry Summer Academy is a 2‐week intensive workshop for in‐service science teachers with emphasis on biochemistry laboratory techniques and molecular modeling. By focusing on the underlying science behind these methods, we enhance the understanding of protein and nucleic acid structures, the nature of the forces maintaining these structures, and the fundamentals of mechanistic enzymology. Through discussions with departmental faculty, participants gain familiarity with topics of current interest. With the pilot program described here, we have established communication between the Department of Biochemistry and local school districts to assist local high school science teachers in their transition into the Next Generation Science Standard (NGSS). Our aim is to develop a program that empowers chemistry and biology science teachers without cross‐disciplinary training to teach biochemistry with authority and enthusiasm while showcasing the interdisciplinary nature of science. We hope that the academy will be a model for other molecular life science programs.
Biochemical Mechanism for Recruitment of the Checkpoint Activator TOPBP1 to Stalled Replication Forks
Authors: Michael Matthew
Published: June 2021

During a replication stress response, the ATR protein kinase is activated on stalled replication forks to allow a delay in cell cycle progression, a block to further origin firing, and stabilization of the stalled fork. ATR is recruited to stalled forks via an interaction between its binding partner ATRIP and RPA‐coated single‐stranded DNA (ssDNA). However, binding of ATR‐ATRIP to RPA‐coated ssDNA is not sufficient for ATR activation as both the 9‐1‐1 clamp protein and the checkpoint activator TOPBP1 must also be recruited so that TOPBP1 may activate ATR. The biochemical mechanism for assembly of the checkpoint‐signaling complex has been an important unanswered question in the cell cycle field for many years.

Previous work from our laboratory, and other groups, has shown that ATR/ATRIP and TOPBP1/9‐1‐1 are recruited independently to stalled forks, and then come together on the DNA to form an active checkpoint‐signaling complex. Furthermore, we have previously shown that TOPBP1 is required for loading of the 9‐1‐1 clamp, however the means by which TOPBP1 senses the stalled fork to allow checkpoint complex assembly was not previously known. We will present data, obtained using Xenopus egg extracts, showing that direct interaction between TOPBP1 BRCT repeat 2 and RPA‐coated ssDNA is the mechanism for TOPBP1 recruitment. In addition, we present evidence that direct interaction with RPA‐coated ssDNA is a general feature of a subclass of BRCT domains from other proteins, including PTIP and REV1.
Directed Evolution of DinB in Escherichia coli by Hydroxylamine Mutagenesis and UV Selection
Authors: Hollander Judith , Beuning Penny
Published: June 2021

DNA polymerase III (pol III) replicates undamaged DNA in E. coli under normal conditions. However, pol III is unable to replicate past DNA damage and when this happens, the SOS response is induced. The SOS response induces the expression of at least 57 genes that help repair or tolerate the damage. Some of the genes expressed during the SOS response include those encoding the Y‐family polymerases, which are capable of translesion synthesis (TLS). Translesion synthesis is the process by which DNA polymerases replicate damaged DNA without correcting the damage.

The dinB gene product (pol IV) is capable of replicating DNA containing minor groove deoxyguanine adducts, such as N2‐furfuryl‐dG and N2‐benzo[a]pyryl‐dG adducts, whereas pol V, a product of the umuC and umuD gene products, is capable of replicating DNA containing lesions caused by ultraviolet (UV) light. Our goal was to induce TLS across UV lesions with variants of pol IV. We used DinB and chimeras of DinB containing fragments of UmuC, with additional random mutations via treatment with hydroxylamine, to identify variants of DinB that have obtained the ability to replicate past UV lesions. This work is supported by the American Cancer Society.
ATR Promotes the Replication Traverse of DNA Interstrand Crosslinks
Authors: Huang Jing , Bellani Marina , Xue Yutong , Wang Weidong , Meetei Amom Ruhikanta , Seidman Michael
Published: June 2021

DNA instability disorders are associated with cancer, neurodegeneration, and premature aging. The genome integrity is most vulnerable during S phase during which the replication apparatus encounters many impediments. Among the most severe are interstrand crosslinks (ICLs) which are absolute blocks to helicases, and have always been regarded as absolute blocks to replication. Cells from patients with the genome instability disorder Fanconi Anemia (FA) are highly sensitive to crosslinking agents. We have developed a novel single molecule strategy, based on DNA fibers, to visualize collisions of replication forks with ICLs. Remarkably, we found that DNA synthesis can resume past an ICL, leaving behind still crosslinked parental strands, a process that requires only a few minutes. Traverse of ICLs requires the activity of the Fanconi Anemia translocase FANCM. Furthermore, while the FA “core” complex proteins, which monoubiquitinate the FANCD2 protein, were not required, FANCD2 was. These results indicate that non ubiquitinated FANCD2 is involved in replication traverse of the ICLs. The most profound suppression of replication restart patterns was observed in cells deficient in the replication stress kinase ATR, among whose substrates are FANCM and FANCD2. ATR is required for the recruitment of FANCM to ICLs. Cells expressing a mutant form of FANCM that cannot be phosphorylated by ATR show a marked decline in the frequency of replication traverse events at ICLs. This research was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging.
Expression and purification of putative Y‐family polymerase DinB from Sinorhizobium meliloti
Authors: Kramer Caitlin , DeLauteur Nicholas , Brewer Tess , Jones Kathryn , Beuning Penny
Published: June 2021

The conserved Y‐family polymerase DinB is involved in translesion synthesis (TLS) of damaged DNA. The beneficial nitrogen‐fixing bacterium Sinorhizobium meliloti is a symbiont of leguminous plants. Because TLS polymerases enhance tolerance to stress, it is of interest to characterize these genes from S. meliloti, which has to survive an oxidative burst in order to establish the symbiosis. Genome annotation of S. meliloti indicates the presence of dinB genes on the endosymbiotic plasmid pSymA, which contains genes largely implicated in stress response, colonization, and nodulation, as well as on the chromosome. We cloned both putative dinB genes for protein expression and characterization. The pSymA DinB protein was overexpressed in Escherichia coli Rosetta pLysS cells with an N‐terminal 6His tag using the pET system, and purified by affinity chromatography. Characterization of the protein is ongoing. This work is supported by a Matz Scholarship and the American Cancer Society.
Nutritional Status Modulates Box C/D snoRNP Biogenesis by Regulated Subcellular Relocalization of the R2TP Complex
Authors: Houry Walid
Published: June 2021

Box C/D snoRNPs, which are typically composed of box C/D snoRNA and four core protein components (Nop1, Nop56, Nop58 and Snu13), play an essential role in the modification and processing of pre‐ribosomal RNA. The highly conserved R2TP (Rvb1‐Rvb2‐Tah1‐Pih) complex has been found to be required for box C/D snoRNP biogenesis and assembly. However, the molecular basis of R2TP chaperone‐like activity is not yet known. Here, we describe an unexpected finding in which the activity of the R2TP complex is required for Nop58 protein stability and is controlled by the dynamic subcellular redistribution of the complex in response to growth conditions and nutrient availability. In growing cells, the complex localizes to the nucleus and interacts with box C/D snoRNPs. The R2TP‐snoRNP interaction is mediated by Pih1. This interaction was found to be significantly reduced in poorly growing cells as R2TP predominantly relocalizes to the cytoplasm. Hence, the R2TP complex exerts a novel regulation on box C/D snoRNP biogenesis that affects their assembly and consequently pre‐rRNA maturation in response to different growth conditions.
Role of Pore Lining Residues in Drug Modulation of Ligand‐Gated Ion Channels
Authors: Schneider Thomas , Facey Jody‐Ann , Joyce Glenna , Voigt Thomas , Stater Evan , Hyde Michael , Venner Laura , Howard Rebecca
Published: June 2021

Pentameric ligand‐gated ion channels (pLGICs) such as GABAA, glycine, and nicotinic acetylcholine receptors mediate synaptic transmission by converting chemical signals into electrical impulses. Alcohol, anesthetics, and other drugs have been shown to directly modulate the function of pLGICs, however the precise mechanism through which they act remains to be determined. Although high‐resolution structural details of functionally relevant pLGICs are limited, the structure of the prokaryotic homolog GLIC has been determined in multiple conformations and in the presence of drugs, making it a potentially valuable model system. In order to elucidate the influence of alcohol and other drugs on pLGIC function, site‐directed mutations were introduced to an ethanol‐sensitized GLIC variant at pore‐facing residues that have been shown to play a critical role in channel gating. Mutant ion channels were expressed in Xenopus laevis oocytes and characterized using two‐electrode voltage clamp electrophysiology. Polar substitutions to the pore altered channel sensitivity to proton activation, alcohol potentiation, and anesthetic inhibition, consistent with a role for pore‐lining residues in bridging direct effects of alcohol binding on the ion pathway.

This work was support by startup funds from Skidmore College.
Rescuing Nod2, an innate immune receptor of bacterial cell wall fragments, in Crohn Disease
Authors: Grimes Catherine , Mohanan Vishnu
Published: June 2021

Nucleotide binding oligomerization domain‐containing protein 2 (NOD2, CARD15) is an intracellular host pattern recognition receptor that recognizes fragments of bacterial cell wall. Nod2 is important to human biology, as when it is mutated, it loses the ability to respond properly to bacterial cell wall fragments. In order to determine the mechanisms of misactivation in the Nod2 Crohn's mutants, we developed a cell based system to screen for protein‐protein interactors of Nod2. We identified Hsp70 (heat shock protein 70) as a protein interactor of both wild‐type and mutant Nod2. The objective of this study is to determine the mechanism by which Hsp70 stabilizes Nod2. Induced Hsp70 expression in cells increased Nod2 mediated NF‐kB activation in response to bacterial cell wall stimulation. We found Hsp70 to regulate Nod2′s half‐life, as increasing the Hsp70 level in cells increased Nod2′s half‐life, and down‐regulating Hsp70 decreased Nod2′s half‐life. The expression level of the Crohn's associated Nod2 variants were less compared to wild‐type. The over‐expression of Hsp70 significantly increased Nod2 levels as well as the signaling capacity of the mutants. Thus our study shows that restoring the stability of the Nod2 Crohn's mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand. Further, we have identified the minimal region of Hsp70 that facilitates the interaction with Nod2. Studies are currently under way to identify pharmacoperones that can potentially stabilize the Crohn's associated Nod2 variants.

Funding: This project was supported by the Delaware COBRE program, with a grant from the NIH: NIGMS (1 P30 GM110758‐01) from the National Institutes of Health.
Visualization of Pronation States in PLP Dependent Enzymes
Authors: Dajnowicz Steven , Mueser Timothy , Hanson Leif , Schall Constance
Published: June 2021

Pyridoxal phosphate (PLP) dependent enzymes are essential proteins that catalyze a diverse number of reactions: transamination, racemization, phosphorylation, decarboxylation, aldol cleavage, elimination, and replacement. Despite completing a myriad of chemical reactions, these proteins have a unifying Schiff base catalytic intermediate but the overall mechanisms remain ambiguous. Based upon previous X‐ray structures and abundant literature of the different families of PLP dependent enzymes, one question must be answered: How do the different families of PLP dependent enzymes utilize different protonation profiles of pyridoxal phosphate that enable them to promote catalysis of a specific reaction? The objective of this research will be development of detailed model mechanisms for the different classes of PLP dependent enzymes, based on the protonation state of the coenzyme. Aspartate aminotransferase (AAT) and tryptophan synthase (TS) are two model PLP dependent enzymes that have shown to grow neutron diffraction quality crystals (1mm3). Neutron crystallography is an unparalleled tool to determine the atomic coordinates of the hydrogen/deuterium in macromolecules undetectable by other means. Initial neutron data collection and refinement of these proteins gives insight to the protonation state of PLP during catalysis.
Mechanistic Investigation of the Radical SAM Enzyme DesII Using Fluorinated Analogs
Authors: Lin Geng‐Min , Ruszczycky Mark , Choi Sei‐Hyun , Liu Hung‐wen
Published: June 2021

DesII is a radical S‐adenosyl‐L‐methionine (SAM) enzyme that catalyzes the deamination of TDP‐4‐amino‐4,6‐dideoxy‐D‐glucose to form TDP‐3‐keto‐4,6‐dideoxy‐D‐glucose during the biosynthesis of TDP‐D‐desosamine. Generation of a C3‐centered substrate radical has been demonstrated as the first key step, but the subsequent steps remain unclear. Two plausible radical‐mediated mechanisms include 1,2‐migration of the amino group followed by elimination of ammonium versus direct elimination facilitated by deprotonation of the C3‐hydroxyl group. DesII can also catalyze the C3‐dehydrogenation rather than C4‐dehydration of TDP‐D‐quinovose, which is the C4‐hydroxy analog of the natural substrate. To get more insight into the reaction mechanism of DesII, C3‐fluorinated and deuterated analogs of these two substrates were synthesized as mechanistic probes. Substitution of the C3‐hydroxyl group with fluorine is expected to prevent direct elimination of the C4‐amino group but not its migration. Careful investigation showed that no product except 5′‐deoxyadenosine‐5′‐sulfinate and a small amount of 5′‐deoxyadenosine (5′‐dAdo) was produced from the reaction of either analog with DesII. However, deuterium was found to be transferred to both SAM and 5′‐dAdo. This indicates that the fluorinated analogues do undergo C3‐hydrogen atom abstraction by 5′‐dAdo radical. Taken together, these results suggest a mechanism that does not involve migration of the amino functionality and demonstrate the free reversibility of H‐atom transfer when forward partitioning is inhibited.
Effect of High Light Intensity on Polyketide Synthase Gene Expression in the Toxic Dinoflagellate, Karenia brevis
Authors: Chauhan Unnati , Lupo Daniel , Monroe Emily
Published: June 2021

Karenia brevis is the toxic dinoflagellate endemic to the Gulf of Mexico that causes detrimental human health, environmental, and economic impacts through the production of brevetoxins. Brevetoxins are polyketide compounds produced by polyketide synthase enzymes (PKS), but no gene or protein has been linked to their biosynthesis. Previous work on two PKS proteins, KB2006 and KB5299, suggests a link between chloroplast physiology and toxicity. To test the effect of high light (HL) intensity on PKS gene expression in NTB, 1‐liter cultures were exposed to HL treatment (~ 100 μmol photons m‐2s‐1 ) after reaching mid‐log phase in control light intensity (~ 60 μmol photons m‐2s‐1 ). Samples for cell counts and gene expression were taken at T0 (prior to HL treatment) and T6 (6 days post HL treatment) for control and HL cultures. Cultures in the HL treatment entered stationary phase four days post‐treatment and rapidly declined while control cultures entered and maintained stationary phase two days later. There were no differences in expression of KB2006 or KB5299 between the control and HL cultures at the transcript level analyzed by qPCR. At the protein level analyzed by western blotting, KB2006 was approximately four times more abundant in HL cultures than control cultures, and KB5299 was half as abundant in HL conditions compared to control at T6. High light intensity affects growth and protein abundance of PKS proteins in the non‐toxic sub‐strain of K. brevis providing additional evidence linking chloroplast physiology and toxicity. Additional analyses are underway to examine effects of HL on brevetoxin production. Developing a better understanding of biosynthetic pathways involved in brevetoxin production will play a pivotal role in management of future algal blooms.
Gene expression profiles suggest iron transport pathway in human lactating epithelialcell
Authors: Cai Chenxi , Eck Peter , Castillo San Juan Sandra , Friel James
Published: June 2021

Early determination of the factors predisposing an infant to iron deficiency during breastfeeding may enable timely intervention via dietary supplementation. However, the molecular background of iron excretion into breast milk has not been determined in humans. We determined the expression of known iron transporters in the lactating and non‐lactating human and mouse mammary epithelial cell by RT‐PCR to deduce which transporters are responsible for iron excretion into breast milk. The mRNA extracted from breast milk was mainly from epithelial and macrophage cells. In addition, there were only two iron transporters (TFRC and DMT‐1) present in both the lactating human and mouse epithelial cell. SLC40A1, the gene of the only identified cellular iron exporter (FPN), was not expressed in lactating human cells but was previously reported to be expressed in the rat epithelial cell. Our findings indicate that, rat, the only reported model for the iron secreting pathway may not be the best corresponding model for the iron transport system in the human mammary gland. We propose a potential iron transport pathway in lactating human mammary epithelial cell based on the gene expression profiles of the mRNA extracted from breast milk. Supported by CIHR.
Household Food Insecurity Assessed by the Food Access Survey Tool: A Comparison between the Item‐wise Internal Validity in Rural Bangladesh and Rural Zambia
Authors: Na Muzi , Palmer Amanda , Talegawkar Sameera , Lewis Bess , Wu Lee , West Keith
Published: June 2021

Food insecurity can be accompanied by stressful experiences, which may be common across different cultures. Researchers have developed perception‐based scales that summarize stressful experiences in order to assess severity of household food insecurity (HFI). Stressful experiences evolve with progressive HFI, therefore different items can reflect different HFI severity. However, the performance of individual items on perception‐based scales in different cultural settings is unknown. We have adapted the Food Access Survey Tool, a 9‐item Likert scale, to assess HFI in the past 6 months in rural Bangladesh (n=11,992) and Zambia (n=840). We used the Rasch partial credit model (PCM) to study item‐wise internal validity and severity. Other statistical methods examined unidimensionality, monotonicity and measurement invariance assumptions. In Bangladesh, all 9 items fit well in the PCM and all assumptions were met. In Zambia, the item “frequency of square meals” displayed an out‐of‐range fit statistic on the PCM and violated model assumptions. We noted differences in the distribution of item severity: e.g., “frequency of buying staple foods” versus “worrying about food” scored lowest in item severity in Bangladesh and Zambia, respectively. “Borrowing food” and “taking food for credit” both ranked low in item severity in Bangladesh but high in Zambia. In both settings, “switching from a preferred staple to other grains” had the highest item severity. Though core experiences of HFI may be common, caution is necessary when adapting, interpreting, and comparing individual items commonly used in HFI assessment tools across cultures.
Iodine supplementation reduces elevated total‐ and LDL‐cholesterol in iodine‐deficient, overweight women: a randomized, placebo‐controlled trial in Morocco
Authors: Herter‐Aeberli Isabelle , El Ansari Nawal , Chabaa Laila , Cherkaoui Mohamed , Zimmermann Michael
Published: June 2021

Objective

Whether higher serum TSH caused by iodine deficiency adversely affects serum lipids is unknown. Our objective was to determine if correction of iodine deficiency in adults decreases serum TSH and thereby improves the lipid profile. We also measured the effect of iodine repletion on parameters of glucose metabolism and subclinical inflammation.

Methods

In an area of moderate iodine deficiency in Morocco, we randomized overweight women (n=163; mean±SD age 38.6±6.7 y and mean BMI 32.4±4.1 kg/m2) to receive 200 µg iodine daily for 6 months or placebo tablets. At baseline, 3 and 6 months, we measured serum TSH, free T3 and T4, Tg, total, LDL‐ and HDL‐cholestrol, triglycerides, leptin, glucose, insulin, C‐peptide, C‐reactive protein, and urinary iodine concentrations (UIC).

Results

At baseline, median (95%CI) UIC was 41.6 (37.3, 47.1) µg/L and prevalences of subclinical hypothyroidism, hypothyroxinemia, and hypercholesterolemia were 4, 14.5, and 40.5%, respectively. There were significant time by treatment effects on: UIC and free T4 (higher with treatment; p<0.001), serum TSH and Tg (lower with treatment; p<0.024, p<0.001). Treatment decreased the prevalence of hypercholesterolemia and elevated LDL cholesterol (at 6 months, comparing treatment vs control: 21.5% vs. 34.8% and 35.4% vs. 44.9%; p=0.005, p=0.068)

Conclusions

Correction of iodine deficiency in overweight women decreases serum TSH, reduces hypercholesterolemia and improves the lipid profile. Thus, iodine prophylaxis may help reduce cardiovascular disease risk in populations with high rates of obesity.
Beneficial Effects of a Nutritional Composition on Diabetic Risk Factors and Complications in LDLr‐/‐. Leiden Mice
Authors: Schoemaker Marieke , van Tol Eric , Kooistra Teake , Kleemann Robert , Wielinga Peter
Published: June 2021

Obesity frequently associates with chronic inflammatory diseases, including type 2 diabetes. In this study, a combination of a protein hydrolysate, LCPUFAs and a probiotic strain was investigated on the development of high fat diet ‐induced diabetic risk factors and complications in LDLr‐/‐.Leiden mice.

Male LDLr‐/‐.Leiden mice at 12 wks of age received a high fat diet (HFD) for 21 wks with or without a combination of an extensive casein hydrolysate, docosahexaenoic acid (DHA), arachidonic acid (ARA), and Lactobacillus Rhamnosus GG (LGG). Both HFD and intervention diet were isocaloric and casein from HFD was replaced with casein hydrolysate in the test diets. The addition of DHA/ARA in the test diets was controlled for in the HFD. Moreover, a PBS gavage control group was included to control for potential effects of LGG gavage.

There were significant beneficial effects of the hydrolysate/ARA/DHA/LGG composition versus the HFD control group including reduced body weight gain, lower plasma levels of insulin, cholesterol and triglycerides, lower systemic inflammation, improved adipose tissue quality and mass, and improved kidney and liver function. In a follow up study, evaluating the individual components of the test formulation, some of the outcomes were attributable to the hydrolysate or LGG.

A combination of an extensive casein hydrolysate, ARA, DHA and LGG reduces the detrimental effects of HFD on the development of obesity and its metabolic complications. Main risk factors for the metabolic syndrome such as adipose tissue and chronic inflammation were markedly reduced which could provide a rationale for the beneficial effects observed.
Unbiased measurements of postnatal cerebellar cortical growth interpreted quantitatively through topological constraints suggest prolonging of cell cycle time in Superficial EGL progenitors
Authors: Chang Joshua , Leung Mark , Gokozan Hamza , Gygli Patrick , Catacutan Fay , Czeisler Catherine , Otero Jose
Published: June 2021

Postnatal cerebellar folial surface area expansion promotes cerebellar cortical cytoarchitectural lamination. We developed a stream‐lined sampling scheme/work‐flow to generate objective/unbiased estimates of cerebellar surface area and volume using stereological principles. Our data demonstrate that during the proliferative phase of EGL and folial surface area expansion, EGL thickness does not change and thus is a topological proxy for progenitor self‐renewal. These topological constraints indicate that during proliferative phases, migration out of the EGL is balanced by self‐renewal. Progenitor self‐renewal must therefore include mitotic events yielding either two cells in the same layer to increase surface area (b‐events) and mitotic events yielding two cells, with one cell in a superficial layer and one cell in a deeper layer (a‐events). Using a mathematical model constrained by these macroscopic observations, we were able to relate easily‐obtained global measurements of volume and surface area to the underlying cellular physiology. We used this overall method to quantify the lengthening of inter‐mitotic times for b‐events on a per‐cell basis. Similar methodology may prove useful by providing another layer of depth to the interpretation of results from stereological studies.
Behavioral Analysis of Nicotine Self‐Administration
Authors: Kohut Stephen , Bergman Jack
Published: June 2021

Despite the high prevalence of nicotine use in humans, robust nicotine self‐administration has been difficult to demonstrate in laboratory animals. We conducted a parametric analysis of nicotine self‐administration in non‐human primates to better understand the conditions that support nicotine intake. Adult rhesus monkeys (N=6) were trained to self‐administer intravenous nicotine (0.01 mg/kg) under a fixed ratio (FR) 1 schedule of reinforcement during daily 90 min sessions. After self‐administration of vehicle and a range of nicotine doses (0.001‐0.1 mg/kg) was evaluated, the dose was returned to 0.01 mg/kg nicotine and the FR was increased across multiple sessions in an ascending order (i.e. 1, 3, 6, 10, 18, 30, 60, 100, etc). To compare nicotine self‐administration with that of another stimulant, monkeys were subsequently given access to 0.01 mg/kg cocaine using identical testing procedures. Finally, the effects of FR change on other doses of nicotine (0.0032 and 0.032 mg/kg) were assessed. Results indicate that nicotine self‐administration followed an inverted‐U pattern with the peak injections per session at 0.0032 mg/kg. Self‐administration of nicotine (at each dose) and cocaine gradually decreased as the fixed ratio size was increased. Application of the exponential model of demand to the FR data found that essential value for cocaine was significantly higher than that for nicotine. Interestingly, elasticity of demand differed according to nicotine dose with the order: 0.0032 < 0.01 < 0.032 mg/kg/inj. These data show that high levels of nicotine self‐administration can be achieved in non‐human primates but that it's reinforcing strength is limited. Supported by NIH DA026892.
PKC Activation Leads to Increased RGS2 Protein Levels
Authors: Aschermann Lauren , Sjogren Benita
Published: June 2021

Regulator of G protein Signaling 2 (RGS2) is a member of a family of proteins that regulate G protein‐coupled receptor (GPCR) mediated signaling by accelerating GTP hydrolysis on active Gα subunits. RGS2 has been indicated to play an important role in regulating cardiovascular function. RGS2‐/‐ mice are hypertensive, are prone to heart failure and show increased responses to vasoconstrictors, such as Angiotensin II. Strategies to increase RGS2 protein levels could thus be a beneficial therapeutic strategy. More detailed information on how RGS2 protein levels are regulated would facilitate these drug discovery efforts.

A previous high‐throughput cell‐based screen of natural product extracts identified Indolactam V (ILV), a known PKC activator, to selectively increase protein levels of RGS2. Utilizing the PathHunter ProLabel assay; a β‐galatosidase complementation assay that measures relative protein levels, we found that ILV, as well as phorbol 12‐myristate 13‐acetate (PMA), selectively increase RGS2 protein levels in a time and concentration dependent manner. This effect was blocked by PKC inhibitors indicating a mechanism mediated by PKC. Stimulation of a Gq coupled receptor also results in an increase in RGS2 protein, indicating that this could be a novel feedback mechanism for Gq signaling. This study is continuing to dissect the mechanism by which PKC activation leads to increased RGS2 protein levels. Overall, these studies will lead to increased understanding of how RGS2 protein levels are regulated.