bluuebunny/crossref_metadata_2025 huggingface dataset:
Bite Force and Feeding Traces of Tyrannosaurusthroughout Ontogeny: A Review
Authors: Peterson Joseph
Published: May 2022

Bite marks attributed to adult Tyrannosaurus rexare common in the fossil record and have been subject to numerous studies, though relatively few bite marks attributed to T. rexhave been traced to juveniles. However, a series of recent studies have contributed new information on bite force, behavior, and ecological interactions of juvenile Tyrannosaurus rex, providing insight into ontogenetic changes in lifestyle and ecological roles of juvenile tyrannosaurs in the late Cretaceous of North America. Recent bite force estimates for a juvenile Tyrannosaurus rexwere based on mechanical tests designed to replicate bite marks previously attributed to a T. rex of approximately 13 years old in the centrum of a caudal vertebra of Edmontosaurus(BMR P2007.4.1) and the left maxilla and nasal of a juvenile Tyrannosaurus (BMR P2002.4.1). Results indicate an estimated bite force of 5,641.19 N, and suggest that bite forces were increasing parallel to growth and were utilizing the same prey as adults. Additionally, evidence of intraspecific aggression in the form of lesions resulting from face biting are present in over 50% of juvenile and adult tyrannosaurs. The high frequency of facial scars positively correlates with the inferred onset of sexual maturity. Furthermore, lesions attributed to a Trichomonas gallinae‐like parasite are present on the dentaries and surangulars of adult tyrannosaurs. Presented here is the first evidence of similar pathologies on a juvenile Tyrannosaurus rex that also possesses facial scars from intraspecific aggression, supporting a cause‐and‐effect relationship between the two groups of facial pathologies. Collectively, these findings suggest tyrannosaurs increased interactions, aggressive bouts, and potential transmission of parasites at the onset of sexual maturity as they were moving into different ecological niches. Furthermore, the frequency and category of pathologies in late Cretaceous tyrannosaurs and late Jurassic theropods of North America are compared with the unique guild structures of tyrannosaurs in the late Cretaceous of North America, suggesting a possible relationship between theropod diversity, behavior, and parasitism.
Hammock, Diaphragm, or Floor: Pelvic Anatomy for Healthcare Students
Authors: Carroll Melissa A.
Published: May 2022

The pelvic region is a complex three‐dimensional conglomeration of muscles, fascias, nerves, vessels, and viscera in an osteoligamentous ring. Unfortunately, the use of dissection limits the comprehension of the pelvic region because of the donor‐cadaver’s thin, possibly atrophied muscles. Virtual reality is also limited in accurately rendering the dimensions, interactions, and relationships with essential organs, nerves, and vessels – not to mention the inconsistency of branching patterns. However, knowledge of pelvic anatomy is essential to pelvic health. The presence of incontinence, pelvic pain, sexual discomfort or dysfunction, and the use of episiotomies are just a smattering of clinical reasons for improving competence in pelvic anatomy. This portion of the Master Class will present the current evidence regarding pelvic anatomy, specifically regarding the recent imaging, histological, and structural publications.
Mapping Brain‐wide Pathologies in Mouse Models of Neurodegeneration
Authors: Harris Julie
Published: May 2022

Neurodegenerative diseases are characterized by deposition of abnormal protein aggregates in the brain in predictable spatial and temporal patterns. This selective vulnerability to pathology, which encompasses vulnerabilities of cell types, regions, and brain networks, is a fundamental feature of disease that is not yet understood. Mouse models of human disease are required for preclinical investigations into molecular and cellular mechanisms underlying vulnerability and resilience. Yet, little data exists on how well aligned commonly used models are with the brain‐wide human spatiotemporal vulnerability maps. To address this gap and more deeply characterize existing models, we built a high‐throughput whole brain imaging and informatics pipeline to systematically quantify spatiotemporal patterns of disease‐relevant pathologies in mice. Using whole brain clearing, antibody labeling, and lightsheet imaging, we have quantified tyrosine hydroxylase levels in a mouse model of Parkinson’s Disease with nigrostriatal degeneration and show the utility of 3D imaging to identify disease‐relevant regional vulnerabilities.
Neuronal and Non‐neuronal Cell Type Mapping in the Mouse Brain
Authors: Kim Yongsoo
Published: May 2022

The brain contains multiple neuronal and non‐neuronal cell types with distinct spatial relationship that serve as a basic building block in the brain. I previously developed high resolution mapping methods to visualize and to quantify these individual brain cell types across the entire mouse brain. In this talk, I will present my lab’s effort to understand spatial arrangement of several major cell types in the mouse brain across lifetime. I previously applied my mapping methods to examine GABAergic neuronal cell types, c‐Fos‐labeled cells representing functional network activation, and long‐range anatomical connectivity of oxytocin neurons. Moreover, I used the method to trace the complete network of cerebrovasculature and pericytes that showed striking correlation with cortical parvalbumin and neuronal nitric oxide synthase (nNOS) positive neurons. Recently, my lab established a new online anatomical atlas for the adult mouse brain that combines the segmentation of the Allen Mouse Brian Atlas and Paxinos Mouse Brain Atlas, and developed new atlas framework for early postnatal brains. Using these resources, I have mapped developmental trajectories of oxytocin receptor‐expressing neurons during early postnatal development, unveiling spatial temporal patterns of the brain cell type. In summary, high resolution anatomical mapping of cell types helps to understand overall anatomical and functional organization of the brain.
Mechanistic Insights into Deregulated TGF‐β Family Signalling in Human Disease
Authors: Hill Caroline S. , Gori Ilaria , Kjaer Svend , George Roger , Ramachandran Anassuya , Mehic Merima , Tolar Pavel , Hyvonen Marko , Jones Chris
Published: May 2022

Deregulation of TGF‐β family signalling underlies some serious human diseases, including cancer, the Marfan syndromes, and other connective tissue disorders. Recent work in my lab has focused on working out how mutations in key components of TGF‐β family signalling pathways cause these diseases.

Shprintzen‐Goldberg syndrome (SGS) is a Marfan‐related syndrome caused by heterozygous point mutations in the co‐repressor SKI, which is a negative regulator of TGF‐β signalling that is rapidly degraded upon TGF‐β or Activin stimulation. The molecular consequences of these mutations, however, have not been well understood. Using a combination of structural biology, genome editing and biochemistry we have shown that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3, which are the downstream signal transducers of the TGF‐β and Activin signalling pathways. This results in stabilization of SKI and consequently attenuation of TGF‐β responses, in both knockin cells expressing an SGS mutation, and in fibroblasts from SGS patients. Thus, we conclude that SGS is associated with an attenuation of TGF‐β‐induced transcriptional responses, which has important implications for other Marfan‐related syndromes.

Fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG) are debilitating diseases that share causal mutations in ACVR1, a TGF‐β family type I receptor. ACVR1R206H is a frequent mutation found in patients with these diseases. The relevant ligand required to activate this mutant receptor is thought to be Activin A. To obtain insights into the underlying molecular mechanism we have used a combination of biochemistry, optogenetics and live imaging in both knockin cells and in cells from DIPG patients. Our results demonstrate that in contrast to wild type TGF‐β family receptors, ACVR1R206H activation does not require any upstream kinases. Instead we show that it is predominantly activated via Activin A‐dependent receptor clustering, which induces its auto‐activation, leading to downstream SMAD1/5 phosphorylation. The relevance of this for the pathogenesis of FOP and DIPG will be discussed.
BMP Signaling and Iron Homeostasis
Authors: Babitt Jodie L.
Published: May 2022

Iron is an essential nutrient that is critical for fundamental cellular and organismal processes including oxygen transport and energy generation. However, excess iron can catalyze the formation of toxic free oxygen radicals. Cellular and systemic iron homeostasis are therefore tightly regulated to titrate the iron supply to match iron needs. The master regulator of systemic iron homeostasis is the liver hormone hepcidin, which binds and degrades the iron exporter ferroportin to limit iron release from dietary sources and body stores. To maintain normal systemic iron homeostasis, liver hepcidin production is coordinated by a number of signals that communicate body iron needs. Iron deficiency and anemia suppress hepcidin production to increase iron availability for red blood cell production and other essential functions. Conversely, increases in serum or tissue iron stimulate hepcidin production to prevent iron overload. Inflammation also stimulates hepcidin production to restrict iron availability to infectious microorganisms. Abnormal regulation of the hepcidin‐ferroportin axis has a pathogenic role in most disorders of systemic iron homeostasis including the anemia of inflammation, the iron overload disorder hereditary hemochromatosis, and iron‐loading anemias such as beta‐thalassemia. We have discovered a key role for the bone morphogenetic protein (BMP)‐SMAD signaling pathway in controlling liver hepcidin transcription. This talk will present recent insights into how BMP‐SMAD signaling coordinates liver hepcidin transcription in response to body iron levels and the iron demand of red blood cell production to maintain systemic iron homeostasis.
Disparities in Cancer Incidence and Outcomes
Authors: Adebamowo Clement , Adebamowo Sally
Published: May 2022

Considerable progress has been made in reducing the incidence, morbidity, and mortality of several cancers across the United States in recent decades. Nevertheless, the progress has been uneven with many areas of persistent disparities based on racial/ethnic, rural/urban, and geographical factors. In many cases, these determinants of disparities intersect to increase the overall burden of cancer on affected populations. The intersectionality of these determinants ‐ race, class, and gender – interact and overlap with genomic factors. Thus dedicated resources and funding are required to dissect the roles of these factors and the genomic inheritance.

In this panel, speakers will discuss recent research findings, novel methods, and emerging technologies – polygenic genomic methods, epigenomics, gene‐environment interaction research, artificial intelligence, etc. ‐ that would enable appropriate interventions to reduce disparities in cancer incidence and outcomes. Clinical and research ethics, community engagement and community participatory and co‐produced research that is responsive to the highest priorities of populations most affected by health disparities would also be discussed as ways to earn and secure community participation in research production, dissemination and implementation of research findings.
Evidence for Continuity of Interstitial Spaces across Tissue and Organ Boundaries in Humans
Authors: Theise Neil
Published: May 2022

Bodies have continuous reticular networks, comprising collagens, elastin, glycosaminoglycans, and other extracellular matrix components, within and around all organs. Fibrous tissue coverings of nerves and blood vessels create structural continuity beyond organ boundaries. We recently validated fluid flow through human fibrous tissues, though whether these interstitial spaces are continuous through the body or discontinuous, confined within individual organs, remains unlcear. We investigated continuity of interstitial spaces using two approaches. Non‐biological particles (tattoo pigment, colloidal silver, anthracotic pigment) were tracked within colon, skin, and lung interstitial spaces and into adjacent fascia. Hyaluronic acid, a macromolecular component of interstitial spaces, was also visualized, analyzing skin and subcutaneous fascia, colon and mesenteric fascia, lung and pleura, gynecologic organs and their fascial sheathes, breast, and heart and great vessels. Both techniques demonstrate interstitial space continuity within and between organs including within perineurium and vascular adventitia traversing organs and the spaces between them. We suggest that there is a body‐wide network of fluid‐filled interstitial spaces that has significant implications for molecular signaling, cell trafficking, and the spread of malignant and infectious disease.
The Microbiome and Bone
Authors: Hernandez Christopher J.
Published: May 2022

Eukaryotic organisms evolved in environments containing bacteria and archea. Findings over the past decade have demonstrated that the physiology of vertebrates is greatly influenced by the microbes that reside in the body– a population of organisms known as the microbiome. The composition of the gut microbiome has co‐evolved with vertebrates leading, to species‐specific populations linked to vertebrate phylogeny. Bones are among the most visually compelling parts of vertebrate anatomy and are integral to the interpretation of phylogeny in the fossil record. A connection between the microbiome and bone morphology therefore has the potential to have substantial influence on the interpretation of the fossil record. Here I provide an introduction to the microbiome, including the state of the art in the field and current concepts regarding how the gut microbiome may influence distant organs such as bone. The effects of the gut microbiome on bone morphology were established in the first animals studies using antibiotics in the 1930s but only recently have studies attempted to explain the connection between the gut microbiome and bone. Laboratory studies over the past few years have described profound effects of changes in the composition of the gut microbiome on cortical and cancellous bone morphology. These studies suggest that the gut microbiome can regulate whole bone length and appositional bone growth leading to changes in cortical bone morphology. Additionally, recent studies in mice suggest that, in some cases, a bone phenotype can be transferred from one mouse strain to another by transfer of the gut microbiota alone. Together with recent studies suggesting co‐adaptation of commensal microbes and their hosts, these studies suggest regulation of bone morphology by the microbiome.
The pathophysiology of cobalamin (vitamin B12) deficiency syndromes impacting development
Authors: Poche Ross A.
Published: May 2022

CblC is the most common inborn error of intracellular cobalamin metabolism and due to mutations in MMACHC. Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC‐like disease. However, additional de‐regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated the first mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously observed in cblC, we uncovered reduced expression of target genes that encode ribosome protein subunits. We also identified specific phenotypes that we ascribe to deregulation of ribosome biogenesis impacting normal translation during development. These findings identify HCFC1/RONIN as transcriptional regulators of ribosome biogenesis during development and their mutation results in complex syndromes exhibiting aspects of both cblC and ribosomopathies.
Glyphosate increases anxiety‐like behavior and threat response to novel neutral stimuli
Authors: Cáceres‐Chacón Mauricio , Figueroa‐Pérez Alexdiel , Rodriguez‐Rosado Sian , Hernández‐Busot Gabriela N. , Haddock‐Martínez Hector A. , Rivera‐López Melissa , Martínez‐Guzmán Osmarie , Sierra‐Mercado Demetrio
Published: May 2022

Glyphosate is the active ingredient in the most used herbicides worldwide. Glyphosate was initially considered safe for mammals because it acts by inhibiting a metabolic route that is not present in mammals. Recent studies have correlated an increase in the diagnosis of anxiety with the increased use of glyphosate. Studies in rodents have shown that high doses of glyphosate increase anxiety‐like behaviors. Glyphosate exposure is regulated by the Environmental Protection Agency, which has established a chronic reference dose for glyphosate of 2.0mg/kg daily. The effect of this dose on anxiety has not been explored. Therefore, we aimed to assess the effect of prolonged oral consumption of glyphosate‐contaminated drinking water on anxiety‐like behaviors. Furthermore, we also aim to assess the effect of glyphosate on threat behaviors in response to novel, neutral stimuli. To achieve this, rats were given access to glyphosate‐contaminated drinking water ad libitum. Water was prepared for a target dose of 2.0mg/kg daily. Control rats received filtered drinking water. No differences were observed in the volume of water consumed. After 4 weeks of exposure, we assessed for anxiety‐like behavior using the open field test. No difference was observed in the amount of time spent in the center (glyphosate: 40.42.68, control: 49.01; p = 0.2601). After an additional 10 weeks of exposure, anxiety‐like behavior was reassessed in the elevated plus maze. Here, glyphosate decreased the time spent in the open arms (glyphosate: 48.95, control: 103.7; p=0.0104). Next, after 4 more weeks of exposure, animals were assessed for threat response in an open field containing a novel object in the center. Glyphosate decreased time spent exploring the novel object (glyphosate: 44.35, control: 89.69; p=0.0365). Lastly, after a total of 16 weeks of exposure, animals were exposed to 5 repetitions of a novel auditory tone (30s, 4kHz, 77dB; 3 min intertrial interval) within a familiar context to further explorer threat response. Threat response was measured as time spent freezing during the tone. We observed that glyphosate increased overall freezing to a novel tone (glyphosate: 14.05, control: 4.949; p=0.0201). For these reasons, we conclude that glyphosate exposure increases anxiety‐like behaviors as well as negative valence interpretation of different types of neutral, novel stimuli. Future directions include performing immunohistochemistry on brain regions involved in anxiety‐like behaviors such as the basolateral amygdala and bed nucleus of the stria terminalis.
RTI‐263, a biased NPS receptor agonist that retains anxiolytic effect, attenuates cocaine‐seeking behavior in rats
Authors: Huang Yuanli , Feldman Kyle , Runyon Scott , Clark Stewart
Published: May 2022

Neuropeptide S (NPS) is a modulatory peptide, activating a G protein‐coupled receptor (NPSR) that has been shown to have memory‐enhancing, anxiolytic‐like, and hyper‐locomotive, and drug reinforcement effects in rodents. The NPS system has emerged as a candidate therapeutic target as NPSR antagonists were shown to attenuate reinstatement responding of drug seeking behavior (cocaine). A recently disclosed NPSR biased agonist, RTI‐263, blocks NPS induced hyperlocomotor but produces anxiolytic‐like and learning effects similar to NPS in vivo. Therefore, as triggers of drug taking behavior are often related to stressful life circumstances, RTI‐263 may be an ideal pharmacotherapeutic for substance use disorders due to its anxiolytic effects. Our current research demonstrates that the NPSR biased agonist RTI‐263 (1 and 10nmole, intracerebroventricular) attenuates cue‐induced cocaine reinstatement under a concurrent alternating FR4 schedule in self‐administration experiment. Future research will focus on whether the effect of RTI‐263 is specific to drug reward or is more generalizable (e.g. food reward). This research is supported by NIH grant R21DA045825 (Dr. Stewart Clark).
Identification and Stratification of Cultured Microorganisms from Wind Cave
Authors: Nuehring Caitlyn , Peters Justin
Published: May 2022

Wind Cave National Park in the Black Hills of South Dakota contains many fungal, bacterial, and archaeal organisms. These biotic communities tend to be low in diversity and sensitive to changes in external inputs of organic matter from seasonal snow melts and tourist traffic. Tourist traffic in particular can affect the natural state of the cave environment and alter species composition and population densities of the resident biota. The goal of this project was the molecular identification of the various fungal and bacterial samples between remote and public sites of Wind Cave to better understand the impact of tourist traffic on cave biota. DNA obtained from cultured samples was used to perform PCR‐based sequencing analysis using primers targeting either a specific region of the 16S small ribosomal subunit for identifying bacteria or an internal transcribed spacer (ITS) for identifying fungal lineages. Phylogenetic trees were created of both the fungal and bacterial genra stratified by cave location.
Mechanism of Inosine‐Uridine Nucleoside Hydrolase (IU‐NH) from Arabidopsis thaliana
Authors: Griner Jason G. , Kline Paul C.
Published: May 2022

IU‐NH catalyzes the hydrolysis of selected nucleosides to the corresponding base and pentose sugar. This enzyme plays a central role in the salvage pathway of nucleosides. Kinetic studies with inosine and uridine along with other analogs of these nucleosides were used to characterize the substrate specificity of the enzyme. Uridine was found to be the preferred substrate over inosine with a KM of 2,099 μM compared to 509 μM. The enzyme was found to have broad specificity. Substitution at sites in the nucleoside substrate were used to determine the role these sites played in the catalytic mechanism. Site‐directed mutagenesis studies on selected amino acid residues were used to determine the role these residues play in the binding of the nucleoside to the enzyme and the catalytic mechanism. Molecular dynamic studies in conjunction with the experimental determination of substrate specificity were used to build a visual picture of the catalytic mechanism. A flexible loop around the active site acts in a pincer motion to maintain and stabilize the bound nucleoside conformation and cleave the glycosidic bond. Calcium is suspected to play a central role in how the flexible loop interacts with residues of the active site by using calcium to complete a hydrolytic union between water, C1’ of the nucleoside, and N9 of a purine base or N1 of a pyrimidine base. The mechanism of the enzyme isolated from Arabidopsis was compared to the well‐characterized enzyme isolated from parasitic protozoans.
Monitoring of hemoglobin and erythropoiesis‐related mRNA in athletes and patients Dried Blood Spots
Authors: Loria Francesco
Published: May 2022

Assess the feasibility of monitoring hematological parameters, such as hemoglobin (HGB) and Reticulocyte mRNA in Dried Blood Spots (DBSs), to improve the Athlete Biological Passport (ABP) and patient care. Here, we measured HGB and erythropoiesis‐related mRNA from ALAS2and CA1 in venous blood (VB) and DBS from healthy athletes and hemodialysis patients on MIRCERA® treatment. Finally, EPO was directly measured in DBSs using ELISA. Changes in HGB over time were well captured with both VB and DBS. When combining HGB and mRNA analysis in DBSs, the DBSs OFF‐Score was more sensitive than the classical ABP OFF‐Score to detect EPO use. Moreover, DBSs are more efficient also for direct EPO detection. To conclude, DBS represents a practical new tool for the analysis of HGB and OFF‐score calculation, and could help to enhance blood doping detection and predict the response to EPO in hemodialysis patients.
Unique Biochemical Properties of Eukaryotic RNA polymerases I, II, and III
Authors: Jacobs Ruth Q. , Ingram Zachariah M. , Lucius Aaron L. , Schneider David A.
Published: May 2022

Eukaryotes express at least three nuclear, DNA‐dependent RNA polymerases (Pols I, II, and III) responsible for synthesizing all genome‐encoded RNA. Based on the Pols’ cellular locations, target loci, and transcriptional demands, it is reasonable to hypothesize they have diverged enzymatically over the course of evolution. Since the discovery of the Pols over 50 years ago, the Pols have been largely investigated separately and under disparate experimental conditions, making direct comparisons of the Pols unjustifiable. To directly compare the Pols under identical experimental conditions, we utilized our promoter‐independent in vitro transcription assays. We use a rapid mixing instrument, the chemical quenched‐flow, to analyze single‐ and multi‐nucleotide addition catalyzed by the Pols. We find that Pol I incorporates a single AMP faster than Pols II and III. Interestingly, Pol III incorporates multiple nucleotides faster than Pol I on average. Pol II is consistently the slowest Pol over single‐ and multi‐nucleotide incorporation events. Outside of elongation kinetics, we find additional distinct biochemical properties. Pol II forms the most stable elongation complex of the Pols and is the least error prone Pol. Taken together, our results reveal unique enzymatic characteristics of the Pols that provide new insights into their evolutionary divergence.
Beyond the Gastrointestinal Tract: Sex‐Specific Skin Microbiota Are Associated With Hypertension in Rats With Genetic Disparities
Authors: Mei Xue , Mell Blair , Cheng Xi , Yeo Ji‐Youn , Yang Tao , Chiu Nathaline , Joe Bina
Published: May 2022

Current knowledge of microbiota in hypertension is restricted to the gut and mostly to males. Other than the kidney, the skin is the largest organ playing a role in sodium homeostasis and the mouth is the first site exposed to dietary salt, whereby, we hypothesized that microbiota in these sites associate with salt‐sensitive blood pressure (BP). To test this hypothesis, we compared microbiota from the skin, mouth and feces of both male and female young Dahl salt‐sensitive (S) rats and 4 genetically disparate congenic strains (RNO1, RNO5, RNO9 and RNO10). Skin microbiota was from 4‐6 days old rats before they developed their coats. Oral and fecal samples were collected at 4 weeks old. Microbiota were profiled by 16S rRNA sequencing. Compared to the gut, which is an anerobic site, microbiota from the aerobic skin and oral sites were consistently different in composition across all the strains. Unlike in the fecal samples where Firmicutes and Bacteroidetes were enriched, Cyanobacteria and Actinobacteria were enriched in the skin and oral samples, respectively. Female S rats had greater abundance of skin Cyanobacteria than male S rats. Strain effects were not observed in any of the strains or sites except for RNO5, where a strain × sex × site interactive effect was specifically noted for skin Cyanobacteria. Cyanobacteria contain sodium transporters and are salt‐tolerant microbiota, whereby they could potentially regulate sodium excretion and contribute to BP regulation. In summary, our study is the first to discover microbiota beyond the gut as being associated with BP.
Genomic Study of GABA Receptor Ligand Binding Sites of the Bivalve Mollusc Crassostrea virginica
Authors: Phoenix Tamia , Foster Tia M. , Catapane Edward J. , Hinkley Craig , Carroll Margaret A.
Published: May 2022

GABA (γ‐aminobutyric acid) an inhibitory neurotransmitter in molluscs and other animals has not been well studied in bivalves. In humans, impairment of GABA neurotransmission can cause epilepsy. In the bivalve mollusc Crassostrea virginica, as well as other bivalves, serotonin is an excitatory neurotransmitter that increases beating rates of gill lateral cell cilia. Previously our lab demonstrated in C. virginica serotonin’s increase of cilia beating rates is blocked by applying GABA to the visceral or cerebral ganglia. Additionally, bicuculline methchloride, a GABAa receptor antagonist, blocked the effects of GABA. By using HPLC we previously detected GABA in low ng amounts in cerebral and visceral ganglia of C. virginica. Our immunofluorescence studies showed the presence of GABA neurons in cerebral and visceral ganglia; and that some serotonin neurons had GABA receptors on their soma. Recently the genome of C. virginica and other bivalves has begun to be mapped. By conducting BLAST searches of the NCBI (National Center for Biotechnology Information) database using DNA and protein sequences of C. virginica and other invertebrate and mammalian species we found matches for GABAa and GABAb receptor genes on C. virginicachromosomes 3 and 5, respectively. Various invertebrates had Percent Identity above 60%, while humans and mice had Percent Identity of about 40% to 50% for GABAa and GABAb. We hypothesize that the ligand binding sites (LBS) for GABAa and GABAb receptors in C. virginica are evolutionarily conserved and will closely match those of other animals. To study this, we conducted searches of the NCBI database for GABAa and GABAb LBS of C. virginica and compared them to other animals. We found GABAb LBS contained 4 amino acids (N, L, A, Y) in positions 34, 122, 123, 267 that were highly conserved in LBS of other bivalves, gastropods, insects, mice, rats and humans. GABAa LBS for C. virginica has not yet been identified. We did find LBS of other animals contained 2 amino acids (L, Y) that were highly conserved among the animals in which it has been identified. The C. virginica GABAa receptor does contain L and Y amino acids. The study complements our earlier physiology and cell biology studies demonstrating the presence and a function for GABA in C. virginica and shows the genome of C. virginica contains genes to produce GABA receptor LBS that are similar to those of other animals. This new information is valuable as it shows the simple nervous system of C. virginica can be used to conduct studies on GABA neurotransmission.
Housing Temperature Alters Energy Expenditure and Adipose Tissue Bioenergetics in Mice
Authors: Sadler Daniel , Treas Lillie , Porter Craig
Published: May 2022

Background

Sub‐thermoneutral housing temperatures increase facultative thermogenesis in mice, a process which has been attributed in part to uncoupling protein 1 (UCP1). Typical vivarium temperatures (i.e., ~22‐26°C) likely represent mild cold stress for mice, yet the metabolic impact of modest changes in housing temperature in mice remains unclear. Here, we determined the impact of transitioning mice from 24°C to 30°C on total energy expenditure (TEE) and adipose tissue bioenergetics.

Methods

Male and female C57BL/6J mice were studied for 6‐weeks. All mice were housed at 24°C (room temperature, RT) for two weeks, and then either remained at RT (n=16 per group, 8M/8F) or were transitioned to 30°C (thermoneutrality, TN) (n=16 per group, 8M/8F) for 4 weeks. Mice were housed in metabolic cages to determine TEE and its components via respiratory gas exchange. Interscapular brown adipose tissue (iBAT) and inguinal white adipose tissue (iWAT) were harvested at the end of the 6‐week study to assess mitochondrial respiratory function by high resolution respirometry.

Results

Compared to RT, total daily energy expenditure (TEE) was 25% and 16% lower in males and females at TN (M: TN, 8.4±0.6 vs. RT, 11.1±1.0 kcal/day; F: TN, 8.1±0.6 vs. RT, 9.6±1.6 kcal/day; P<0.05), which was partly due to a 36% and 40% decrease in basal metabolic rate (BEE) at TN (M: TN, 3.6±0.2 vs. RT, 5.6±0.8 kcal/day; F: TN, 3.2±0.2 vs. RT, 5.4±0.9 kcal/day; P<0.01). Wheel running distance completed by males was 50% lower at TN versus RT (P<0.05), and female mice ran 101% further than males at TN (P<0.05). iBAT depot mass was 73% and 76% greater at TN versus RT (P<0.01) in males and females, respectively, whereas iWAT mass was similar between conditions. At RT, total iBAT depot UCP1 protein content was 2‐fold greater than that at TN (P<0.01). Further, iBAT depot UCP1 protein content was respectively 20‐ and 40‐fold greater than the iWAT depot at TN and RT (P<0.01). However, iBAT UCP1‐dependent respiration per depot was unaltered by housing temperature, whereas iWAT UCP1‐dependent respiration per depot was 70% and 62% lower in males and females, respectively, at TN versus RT (P<0.01). Still, the absolute thermogenic capacity of UCP1 in the iBAT depot was 45‐ and 16‐fold greater than the iWAT depot at TN and RT, respectively.

Conclusion

A modest change in housing temperature markedly alters BEE and thus TEE in mice. Cooler housing temperature (even those within the National Research Council’s guide for housing laboratory rodents) results in marked hypermetabolism and recruitment of UCP1 in both iWAT and iBAT. Despite UCP1 recruitment in iWAT, iBAT is the principal site of UCP1‐dependent thermogenesis in mice housed at 24°C. These data may have important implications regarding the optimization of preclinical models of human disease.
Effects of aging and type 2 diabetes on cardiac structure and function: Underlying mechanisms
Authors: Nguyen Justina , Garate‐Carrillo Alejandra , Navarrete‐Yanez Viridiana , Moreno‐Ulloa Aldo , Ramirez‐Sanchez Israel , Villarreal Francisco
Published: May 2022

This study characterized long‐term changes in cardiac structure and function in a high‐fat diet/streptozotocin mouse model of aging and type 2 diabetes mellitus (T2D) and how the intersection of both conditions alters plasma metabolomics. Furthermore, we wanted to assess the possible mechanistic roles played by oxidative stress, increases in arginase activity and pro‐inflammatory cytokines levels. For this purpose, 38, 13‐month‐old C57BL/6 male mice were utilized. Control animals (n=13) were fed a regular chow for a period of 10 months. T2D (n=25) animals were provided a single injection of streptozotocin (75 mg/kg/IP) and fed a 60% high fat diet (HFD) for the same period of time. To track changes in cardiac structure and function serial echocardiography was performed. At the end of the study, blood samples were collected via a cardiac puncture and plasma samples frozen. Hearts were sectioned and allocated for either rapid freezing (biochemical studies) or fixed in formalin for histological analysis. Biochemical assays using blood and/or tissue samples included determinations for arginase activity, pro‐inflammatory cytokines, protein carbonylation, glucose tolerance tests, Western blots and plasma metabolomics. Glucose tolerance tests confirmed the development of T2D in the HFD animal group. Serial echo yielded moderate increases in cardiac mass with aging that were greater in T2D animals. Fractional shortening (figure) significantly decreased by 16 months of age in T2D animals yielding a value of 28% at 23 months vs. 40% in aged controls. Ejection fraction also significantly decreased by 16 months of age in T2D animals yielding 55% at 23 months vs. aged animals (70%). Western blots of myocardium samples documented increases in fibronectin, type III collagen and smooth muscle actin with T2D which were confirmed by immunohistochemistry. Significant increases in plasma protein carbonyls, TGF‐β1, TNF‐α, IL1β and IFN‐γ occurred with aging and further increased with T2D. Untargeted metabolomics and cheminformatics revealed clear differences in the plasma metabolome of the HFD mice vs. controls. Multiple lipid metabolites linked to insulin resistance were dysregulated, including C16:0 and C18:0 acyl carnitines (up‐modulated) and saturated and unsaturated phosphocholine‐containing lipids (up and down‐modulated). In summary, we document the development of cardiac hypertrophy with aging that is aggravated by T2D. Systolic dysfunction occurred as a late age associated event with T2D. Increases in OS markers and pro‐inflammatory cytokines occurred with aging and were aggravated with T2D. These changes were accompanied by increases in myocardial and blood arginase activity and TGF‐β1 levels which may account for the development of fibrosis and may be associated with altered plasma metabolomics.
Detecting RNA dynamics in live mammalian cells with fluorescence lifetime‐based sensors
Authors: Braselmann Esther , Sarfraz Nadia , Oertel Therese
Published: May 2022

RNA function is closely linked with subcellular localization and interaction with other biological molecules such as other RNAs and proteins. Acute cellular perturbations and disease mutations may alter RNA dynamics and hence function. Therefore, visualizing RNAs in live cells is critically important for a mechanistic understanding. However, robust fluorescence labels for specific RNA molecules to achieve quantitative detection in the complex cellular environment for diverse RNA species are under‐developed. Typically, fluorescent RNA tags are engineered where a genetically encoded short RNA binds a small molecule that conveys fluorescence properties. These efforts aim to generate large fluorescence intensity changes of the fluorophore upon binding the RNA. Here, we use fluorescence lifetime as an orthogonal approach for a fluorescence RNA sensor. We used the Riboglow platform, an RNA tagging platform that relies on binding of a small fluorescent molecule to a short RNA sequence. First, we compared probe fluorescence signal in live cells in the presence and absence of the RNA tag. We found that fluorescence lifetime provides substantially improved contrast compared with fluorescence intensity. Second, we assessed sensitivity of monitoring subcellular RNA localization dynamics live via fluorescence lifetime. Third, we are exploring modularity of the system by varying the RNA‐ and probe component to enable multi‐parameter RNA detection. Together, we find that fluorescence lifetime offers advantages to track RNAs and quantify localization versus fluorescence intensity. We demonstrated the versatility of this system in diverse cellular examples. Our system is the first platform that exploits fluorescence lifetime for live RNA tracking in cells. We plan to expand this imaging modality further to quantitatively assess RNA dynamics.
An Enterprise in 3‐D Printed Smilodon fatalis Mandible Reconstruction: Experimentation Where No Fossil or Cast Has Gone Before
Authors: Haji‐Sheikh Misty , Naples Virginia L. , Haji‐Sheikh Michael
Published: May 2022

Introduction and objective

Smilodon fatalis went extinct approximately 10,000 years ago. During the fossilization process, changes occurred, including shrinkage, deformation, and misalignment. With no modern analog, studying how S. fatalis killed its prey, used its canines, and what bite force may have been exerted requires an accurate skull. 3‐D printing is a new way to manipulate, explore, and study fossils, in this case, the mandible of S. fatalis.

Materials and methods

Our work uses modern techniques, specifically a 3‐D scanner, a 3‐D printer, and thermal plastic polyester, to accurately rebuild the mandible. These tools allowed the mandible to be manipulated from the original cast size, and width to the more correct alignment by widening the mandible so that the markers we took matched all along the maxilla.

Results

The mandible casts we studied had narrowed over time and the upper teeth and the lower teeth did not occlude. Our novel 3‐D approach eliminates taphonomic problems including deformations that are often encountered when studying S. fatalis. This experiment created a mandible in which homologous points on the maxilla and mandible aligned correctly, and permitted the carnassial teeth to occlude properly.

Conclusions

Our method allows repeatable experimental manipulation. The 3‐D printed mandible we modified became wide enough to be occluded more accurately with the glenoid fossa. The important aspects include differences between the cast mandible and the 3‐D printed one, specifically the size difference; the alignment of the incisor teeth, and the alignment of the carnassial teeth.

Significance/importance

These manipulations would not be possible with real fossils due to the rarity, texture, and fragile nature of the fossil. This is also not possible with a cast as the cast is usually a preformed plasticized unit. With this 3‐D method, researchers can make multiple copies for a variety of experiments. Until now there was no way to manipulate a fossil for better placement or alignment. Our method increases the opportunity for anatomical and functional details that can be obtained by no other means. Researchers can now use this method in studying the fossils they have on hand and the result will be a better understanding of extinct animals. These 3‐D methods are a new set of tools that can revolutionize the study of not only Smilodon but also of all other fossils.
Repurposing SARS‐CoV‐2 Main Protease Inhibitors for HIV‐1 Protease Inhibition
Authors: Minkkinen Jacob P. , Gentile Lisa
Published: May 2022

Severe acute respiratory syndrome (SARS‐CoV‐2) led to the COVID‐19 global pandemic, with over 178 million people infected since the start of the pandemic. SARS‐CoV‐2 is a retrovirus that utilizes a main protease (Mpro). Mpro is a catalytic cys/his protease. Several treatments were proposed to stop the pandemic including repurposing drugs to inhibit the Mpro. Another retrovirus that uses a protease is human immunodeficiency virus (HIV‐1); which has been a global epidemic for 40 years and is a devastating disease that attacks the immune system. HIV‐1 has infected 77.5 million people since the start of the epidemic in 1981 and is still prevalent today. HIV‐1 retrovirus utilizes the host cell to transcribe viral RNA and translate gag‐pol protein. HIV‐1 protease is a dual aspartyl protease used to cleave this gag‐pol protein, thereby activating the protein, allowing for viral replication and infection of other cells. The focus of this research is to investigate whether these two proteases have a similar enough mechanism that SARS‐CoV‐2 Mpro inhibitors will also inhibit HIV‐1 protease. Four repurposed SARS‐CoV‐2 inhibitors: carmofur, leupeptin, rosuvastatin, and withanone were chosen to be repurposed for HIV‐1 protease due to their affordability and affinity for SARS‐CoV‐2 Mpro. Initially, computational analysis was utilized to acquire binding information of the repurposed SARS‐CoV‐2 inhibitors on HIV‐1 protease. POCASA (Pocket Cavity Search Algorithm) was used to identify the main binding pockets of HIV‐1 protease. Swissdock identified the center of the HIV‐1 protease homodimer being the most prevalent binding pocket. Analysis of this pocket showed thermodynamically favorable binding to leupeptin, carmofur, rosuvastatin, and withanone. Since computational analysis showed promising prevalence for the inhibitors, the next step was to test these experimentally; therefore HIV‐1 protease was purified from E. Coli cells. The pure HIV‐1 protease was used in a fluorescent binding assay to derive binding affinity of the inhibitors. Preliminary experimental binding results show carmofur binding (Kd= 1 ± 0.63 mM), and rosuvastatin binding (Kd= 0.025 ± 0.007 mM); leupeptin did not show binding. The next steps are to evaluate binding for withanone as well as activity inhibition of HIV‐1 protease using these repurposed SARS‐CoV‐2 Mpro inhibitors to further investigate whether these two proteases have a similar enough mechanism that SARS‐CoV‐2 Mpro inhibitors will also inhibit HIV‐1 protease.
Microprobe‐based technology for antigen‐specific purification of exosomes for OMICS analysis
Authors: Nestorova Gergana G. , Nwokwu Chukwumaobim D. , Ishraq Bari Saif Mohammad
Published: May 2022

Exosomes encapsulate genomic and proteomic biomarkers for non‐invasive diagnosis and disease monitoring. However, exosome surface‐markers heterogeneity is a major drawback of current isolation methods. Here, we report a direct, one‐step exosome sampling technology, ExoPRIME, for selective capture of CD63+ exosome subpopulations using an immune‐affinity protocol. Microneedles (300μm × 30 mm), functionalized with anti‐ CD63 antibodies, were incubated under various experimental conditions in conditioned astrocyte medium and astrocyte‐derived exosome suspension. The probe’s capture efficiency and specificity were validated using Flu‐oroCet assay, immunofluorescent imaging, and OMICS analyses. Significantly higher exosomes were captured by probes incubated for 16 h at 4 0C in enriched exosomal suspension (23 × 10 6 exosomes per probe) vis‐'a‐vis 2 h at 4 0 C (12 × 10 6) and 16 h at 22 0C (3 × 10 6) in conditioned cell media. Our results demonstrate the application of ExoPRIME over a broad dynamic range of temperature and incubation parameters, offering flexibility for any desired application. ExoPRIME permits the use and re‐use of minimal sample volumes (≤200 μL), can be multiplexed in arrays, and integrated into a lab‐on‐a‐chip platform to achieve parallel, high‐throughput isolation of different exosome classes in a semi‐automated workstation. This platform could provide direct exosomal analysis of biological fluids since it can elegantly interface with existing room‐temperature, picomolar‐range nucleic acid assays to provide a clinical diagnostic tool at the point of care.
Troubleshooting Real‐time PCR Data Online and on Ground to Develop Critical Inquiry and Problem Solving Skills
Authors: Bistulfi Gaia
Published: May 2022

One of the most important skills for molecular biology students upon entering the workforce is their ability to problem solve. From answering research questions to diagnosing and troubleshooting, learning to interpret data and draw pertinent conclusions is essential for scientists.

In the last decade, we devised a series of laboratory experiences during which students extract RNA from their own cells, retro‐transcribe it, design expression specific primers for a house‐keeping transcript using tools freely available online, perform polymerase chain reaction (PCR), amplicon purification and quantification, serial dilutions of the amplicon to prepare real‐time‐PCR standards, run real‐time PCR experiments, and finally analyze their data. To maximize student engagement and discussion during the labs, each module is weaved though with a gaming experience awarding students points for answering questions by themselves or as part of a team.

During the pandemic, the curriculum was adapted to run online as a simulation.

The gamification approach significantly increased student participation, both online and on ground, and it also increased camaraderie on ground. The discussion‐focused report made a significant difference in student understanding; 85% of the students, as compared to 40% with a regular lab report, where able to draw pertinent conclusions from their dataset pinpointing errors to one or more of the following: standard preparation, addition of standards to the plate, inconsistent pipetting, or contamination.

The robust laboratory modules described here have been improved for the past decade and can be adapted to study any gene of interest.