bluuebunny/crossref_metadata_2025 huggingface dataset:
Aerospace medical factor gene expression patterns in live subjects persist in aviation accident victims within muscle, blood, and brain (981.6)
Authors: Burian Dennis , Williams Daniel , Budd Austin , White Vicky , Hutchings David
Published: June 2021

In support of our efforts to validate gene expression markers for aerospace medical factors such as mild hypoxia, sleep deprivation, and alcohol use, we compared gene expression patterns between aviation accident victim samples and live subject samples. RTqPCR was performed against a panel of markers and 15 potential normalizers. Microarray data was taken from a publically available dataset of fresh post‐mortem tissue samples or our own marker discovery efforts in blood.

RNA integrity from tissues was poor, ranging from RINs of 0 in most brain samples to 5 in some muscle samples. RINs in blood ranged from 2 to 6. In both tissues and blood, purity was high: A260/A280 ratios between 1.9 and 2.1. Post‐mortem interval did not correlate with quality measures or yield.

Expression patterns of the gene panel within tissues between the microarray (live subjects) and RTqPCR (accident victims) data were compared by Spearman correlation coefficient. Because in qPCR data, higher Ct values reflect lower gene expression, negative coefficients were expected. Skeletal muscle (‐0.50), blood (‐0.48), brain (‐0.48), cardiac muscle (‐0.47), and lung (‐0.44) had the best correlations. Expression patterns within accident victim blood samples were highly correlated (r>0.80) suggesting that degradation was irrespective of accident conditions or post‐mortem interval.

In the future, it is expected that accident investigators will utilize gene expression data to determine aviation accident causality. However, to translate tests developed in live subject studies to the field, it must be demonstrated that assays are robust to RNA degradation. These results demonstrate that in some tissues, relative gene expression patterns remain stable post‐mortem and assay development can remain focused on discovering the most accurate markers without regard for potential differential stability between mRNAs.
Protein quantification in dried blood spots by MRM mass spectrometry (981.8)
Authors: Chambers Andrew , Percy Andrew , Yang Juncong , Borchers Christoph
Published: June 2021

Dried blood spot (DBS) sampling offers proven advantages over intravenous blood collection for clinical diagnostics targeting a wide array of biomarkers. The simplicity of this approach enables minimally‐trained staff to collect less than 100 microliters of blood from patients. Furthermore, many analytes are stable in the DBS format at room temperature reducing the challenges of sample storage and transportation. The most common clinical application of DBS sampling is the screening newborns for metabolism disorders by targeting small molecules by multiple reaction monitoring mass spectrometry (MRM‐MS). In addition, DBS‐MRM is increasingly employed for pre‐clinical toxicology and pharmacokinetics studies supporting small molecule drug development. The goal of our work is to integrate DBS methodology with multiplexed MRM assays for the quantification of endogenous proteins in human blood. Highly reproducible methods were developed for extracting dried proteins from collection cards (coefficient of variation <15% for full process technical replicates). These samples were then digested with trypsin and spiked with stable isotope‐labeled standard peptides to improve the precision of the assay. Finally, peptides were separated by reversed‐phase liquid chromatography and detected by an Agilent 6490 triple quadrupole mass spectrometer. Robust MRM assays were generated for over 30 proteins and most were stable in DBS samples over a wide range of storage temperatures. This work demonstrates considerable promise for clinical MRM assays targeting endogenous proteins in DBS samples.

Grant Funding Source: Supported by Genome Canada, Genome BC, and the Western Economic Diversification of Canada
Development of peptidomic assays for discovery of endogenously processed peptides from the healthy and juvenile idiopathic arthritis human synovial fluid (981.9)
Authors: Clement Cristina , Santambrogio Laura
Published: June 2021

Juvenile idiopathic arthritis (JIA) is the most common chronic condition seen by pediatric rheumatologists. We and others hypothesized that joint destruction could be partly due to an adaptive immune response mediated by antibodies, and that the characterization of the peptidome (together with the degradome) found in arthritic synovial fluid (SF) (from children with JIA) will help to advance the present studies and shed new molecular mechanisms on the pathogenesis of JIA. Herein, we present the development of peptidomics assays coupled with high resolution nanoLC‐MS/MS on LTQ/Orbitrap Velos mass spectrometer aimed to characterize the endogenously processed peptides from the SF of children with JIA and control patients. The SF peptidome was fractionated by ultrafiltration and peptides with MW<5 kDa were sequenced by HCD/ETD nanoLC Orbitrap‐ESI‐MS/MS or CID nanoLC LTQ‐ESI‐MS/MS. The MS/MS spectra were analyzed with two searching engines, Mascot and PEAKS, against SwissProt database. The peptidome analysis highlighted the breakdown products of the fibrinogen alpha and beta chains and cartilage, mainly proteoglycan 4, fibronectins and multiple collagen subtypes (I, II, III, IV, V, among others). Ingenuity Pathway Analysis (IPA) revealed that the SF from JIA patients is enriched with acute phase reactants, as well as structural components having more than 80% of the proteome connected into the pathways characterizing the inflammatory phenotype: activation of the complement, coagulation systems, intrinsic snd extrinsic prothrombin and the amplification of the acute phase signaling. We proposed that the peptidome biomarkers found in the JIA‐SF could help to elucidate the mechanism for the amplification of the autoimmune process during inflammation. Moreover, the peptidomic assays described herein could be applied to other biological samples of biomedical interest and further used to investigate the proteases (the degradome) responsible for regulating the bioactive peptides.
Integrative analysis of microbiome and metabolome in mouse model simulating features of post‐traumatic stress disorder (982.1)
Authors: Gautam Aarti , Sowe Bintu , Donohue Duncan , Kumar Raina , Muhie Seid , Chakraborty Nabarun , Hoke Allison , Hammamieh Rasha , Jett Marti
Published: June 2021

The gastrointestinal tract is colonized by an enormous array of microbial symbiotes and pathogens. The symbiote influence of gut microbiota on the nervous system and behavior has been receiving profound interest. Rodent studies have provided suggestive evidence that probiotics (e.g. Lactobacillus and Bifidobacterium) can influence behavior. Using “cage‐within‐cage resident‐intruder” model, subject C57BL/6J mice were exposed to aggressors for 6 h daily for 5 or 10 days. After one day of recovery inflammatory cardiac histopathologies were prevalent. Here we report metagenomics and metabolomics approaches to understanding the microbiome activity in our social stress model of mice. Plasma samples were compared between aggressor‐exposed and control mice using various Mass Spec‐based techniques. Large effects of microbiome composition on plasma biochemistry were observed, and several microbiome‐affected molecules such as hippurate, phenylpropionylglycine, and 3‐phenylpropionic were identified in plasma. We also sequenced the 16S rRNA gene V4 variable region on DNA from duodenum tissue using an ion torrent Personal Genome Machine platform. A significant increase of Lactobacillus in the socially stressed mice compared to the control group was observed. Together, these data demonstrate that prolonged stress can significantly change the composition of the intestinal microbiome. We are in the process of analyzing possible correlations between host microbiome population structure and accumulation of metabolites in plasma.

DISCLAIMER “Research was conducted in compliance with the Animal Welfare Act and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.”
Dephosphorylation of acyl‐CoAs in the rat ischemic heart (982.4)
Authors: Zhang Guofang , Li Qingling , Berthiaume Jessica , Kemerer Mary
Published: June 2021

Ischemia and reperfusion cause injury to the heart. Metabolomics has been attempted to study the pathological mechanism of ischemia and reperfusion. Coenzyme A and its esters play essential roles in many physiological processes, such as fatty acid oxidation, lipid synthesis/remodeling, ketone body synthesis, xenobiotic metabolism, signaling pathways, and regulation via acylation and deacylation. We investigated how acyl‐CoAs in the rat hearts correspond to the ischemic and reperfusion injuries. Isolated rat (Sprague Dawley rats, 300‐350g) hearts were used for Langendorff perfusion with Krebs‐Ringer bicarbonate buffer. Heart was subjected to various ischemic durations from 5 to 30 minutes after initial 15 minutes equilibrating perfusion. Different reperfusion durations (5 and 45 minutes) after a 30‐minutes ischemia were also conducted. The data showed that: (i) acyl‐CoAs were found to lose 3’‐phosphate to form acyl‐dephospho‐CoAs in the ischemic heart; (ii) acyl‐dephospho‐CoAs constantly went up with the increasing ischemic duration, and returned to baseline after reperfusion; (iii) heart perfusion and in vitro incubation experiment indicated the link between ATP level and acyl‐CoAs dephosphorylation. The dephosphorylation of acyl‐CoA may be due to the energy demanding during the ischemia. Supported by grants from AHA award 12GRNT12050453 and Cleveland Mt. Sinai Health Care Foundation.
Genesis of antibiotic resistance VII: implementation of unused prescription antibiotics/antimicrobial recycling and incidence of antibiotic resistant bacterial infections in Texas (986.7)
Authors: Kannan Subburaj , Amezcua Judy , Harbison Tiffany , Hernandez Lizeth , Subburaj Prabavathy
Published: June 2021

An effort has been made to explore the implementation of a prescription medicine (Antibiotics) recycling program in Texas. According to The Office of the Regional Medical Director, Texas Department of State Health Services (TDSHS) Region 8 at present there are no programs for recycling the unused prescription medications, Antibiotics in particular in existence in the State of Texas. http://www.dshs.state.tx.us/contact.shtm. Pertaining to the infectious diseases and antibiotic resistance as per the information available and Region 8 Medical director, VISA/VRSA Vancomycin‐Intermediate/Resistant Stapylococcus aureus are the only diseases that are reportable to the TDSHS. Of the 609 infections related to Staphylococcus aureus isolated reports through TxHSN in 2012, 55% tested sensitive to oxacillin/methicillin. NDM‐1, which stands for New Delhi metallo‐beta‐lactamase 1 has been considered to become a global public health problem, but is not detected in Texas. Though 4% of US hospitals had at least one patient with a CRE (carbapenem‐resistant Enterobacteriaceae) infection during the first half of 2012, none were reported in Texas. In 2011, 1,325 cases of tuberculosis (TB) were reported in Texas however, the total number of the antibiotic resistant form of TB among this population is not known. The implications of the discrepancy in the Annual Federal Prescription Drug retake program and lack of recycling of the prescription medicines (Antibiotics) in the evolution of antibiotic resistance shall be presented in the EB2014.

Grant Funding Source: Professional Development Funds from SWTJC, Eagle Pass, Texas
Genesis of antibiotic resistance VIII: monitoring of non‐therapeutic use of antibiotics in biomedical, agricultural ‐ livestock and pharmaceutical research and development process (986.8)
Authors: Kannan Subburaj , Lehler Raul , Miller Monica , Subburaj Prabavathy
Published: June 2021

A systematic approach has been taken to demonstrate that the unregulated antibiotic use in the R&D is a potential cause for concern in regards to the genesis of Antibiotic Resistance in bacterial pathogens leading to a causing difficulty in treating infectious diseases. Research and development activities are funded either by private foundations or through federal agencies such as National Science Foundation, National Institutes of Health (NIH), Centers for Disease Controls and various other organizations. Federal funding from the NIH require strict has guidelines be met for use and disposal of the reagents originating from recombinant DNA technology including antibiotic resistant gene encompassing plasmids and transgenic plant and animal models. Procurement of antibiotics from commercial vendors such as Sigma‐Aldrich for R&D activities is easy to acquire. Use of antibiotics in lab experiments as well as storage and disposal of unused antibiotics is at the discretion of the lab personal such as lab technicians, undergraduate, and graduate students as well as research fellows who are shuffled around with limited supervision including abrupt termination. The quantity of antibiotics used in the R&D to date is unknown by any standards of Governmental or Non‐Governmental regulation. With a menacing uncertainty of the quantity of antibiotics used in R&D, the disposal of unused medications and the wastes produced which are considered as Non‐Biohazard is poignant in a regular waste disposal.

Grant Funding Source: Prefessional Developement Funds from SWTJC, Eagle Pass, texas
Secretory kinases in parasites block innate immunity (99.2)
Authors: Sibley David
Published: June 2021

Toxoplasma gondii is a widespread parasite of animals that is transmitted between cats and rodents, although it also infects a wide range of vertebrates including humans. Genetic mapping studies have identified a family of polymorphic serine threonine (S/T) kinases that control acute virulence in the mouse. These kinases are released from organelles called the rhoptries (ROP) and they target different host pathways. One of the key virulence determinants is ROP18, which phosphorylates switch region I in the GTPase domain of a family of immunity related GTPases, which are important in pathogen control. Phosphorylation in this key region prevents oligomerization and blocks GTPase function, thus protecting the parasite within the host cell. Recent studies have revealed that ROP18, and other active ROP kinases, are control by the pseudokinase ROP5, which lacks catalytic activity, but is required for acute virulence. ROP5 regulates the activity of ROP18, as well as binding to its substrates to facilitate their phosphorylation. Collectively, these studies reveal a complex network of S/T kinases in the parasite that control acute virulence of T. gondii by blocking innate immunity in the mouse.
Probing herpesvirus membrane glycoprotein interactions using proximity biotinylation (993.2)
Authors: Connolly Sarah , Lajko Michelle , Haddad Alexander
Published: June 2021

Herpesviruses cause significant disease within the human population, ranging from simple cold sores to life‐threatening lymphomas. The critical first step of any viral infection is entry into a host cell. For herpesviruses, the conserved mechanism of entry is initiated when glycoproteins embedded in the viral membrane bind to specific cellular membrane receptors. This receptor binding elicits interactions among viral membrane glycoproteins that trigger the viral fusion protein to execute virus‐cell membrane fusion, by inserting into the cell membrane and refolding from a prefusion to a postfusion conformation. Details of the viral protein interaction cascade are poorly understood because the interactions are likely transient and/or low affinity. To examine how the proteins interact, we adapted a proximity biotinylation assay (ProB). In this assay, one protein is linked to a bacterial biotin ligase and a second candidate protein is linked to a target acceptor peptide. If the proteins interact during membrane fusion, the ligase on the first protein catalyzes site‐specific biotinylation of the acceptor peptide on the second candidate. Labeling is covalent and occurs within live cells, thus weak and transient interactions can be detected. Positive controls show strong biotinylation, indicating that proximity of the viral membrane proteins can be detected. Unexpectedly, proteins that do not functionally interact also show a lower level of biotinylation. The results demonstrate the special circumstances that must be considered when examining interactions among proteins that are constrained within the two‐dimensional boundaries of a membrane.
Understanding mechanisms of multidrug resistance with guidance from evolution (994.2)
Authors: Gokirmak Tufan , Campanale Joseph , Shipp Lauren , Moy Gary , Hamdoun Amro
Published: June 2021

Cancer accounts annually for over half a million deaths in the U.S. and 7.6 million deaths worldwide. Although effective chemotherapeutics are available for different types of cancers, multidrug resistance (MDR), mediated by ATP‐binding cassette (ABC) transporters, is responsible for the failure of chemotherapy in most metastatic cancers. Active transport of drugs out of tumor cells by efflux transporters: ABCB1, ABCC1, ABCC2, and ABCG2 (a.k.a., P‐gp, MRP1, and BCRP), is the major mechanism of acquired MDR in cancer. MDR transporters have promiscuous substrate recognition, which makes the design of chemotherapeutic drugs a challenging task. Because high‐resolution crystal structures are not available for every clinically important MDR transporter, comparative mutagenesis is a useful method to understand drug‐transporter interactions. In this study, we adopt a comparative transporter efflux assay approach to define the structural features that mediate the conserved promiscuous substrate recognition using MDR transporter homologs from a basal deuterostome, the purple sea urchin (Strongylocentrotus purpuratus). Despite the vast evolutionary distance, we found that sea urchin MDR transporters have conserved efflux activities for canonical substrates of their closest human homologs. We also found that relatively a few changes in the drug binding pocket could fine‐tune substrate specificity through evolution without destroying the broader pattern of substrate selectivity. Our current efforts focus on determining the conserved residues in the drug‐binding pockets between sea urchin and human MDR transporters and testing their role in canonical substrate efflux by site‐directed mutagenesis. Identification of the critical residues involved in the promiscuous efflux function will broaden our knowledge of how substrate‐transporter interactions occur, and help in the design of better drugs that can traverse the plasma membrane without being recognized by MDR transporters.

Grant Funding Source: Supported by NIH and University of California, Cancer Research Coordinating Committee
Ferroportin deficiency impairs manganese metabolism in flatiron mice (995.1)
Authors: Seo Young , Wessling‐Resnick Marianne
Published: June 2021

Mn homeostasis must guard against neurotoxic effects of excess metal while providing a sufficient source for growth and development. Mn is absorbed from the diet by the intestine, while excess metal is excreted into bile by the liver. In vitro evidence suggests that ferroportin (Fpn), a known Fe exporter, can also export Mn. Here we examine the physiological role for Fpn in Mn metabolism using flatiron mice, a genetic model of Fpn deficiency. Flatiron mice harbor a missense mutation (H32R) affecting Fpn localization and iron export activity. These mice fully recapitulate the human iron loading disorder “ferroportin disease”. Compared to littermate wild‐type controls, Mn levels in flatiron mice are reduced in liver by 10% and in blood by 13% (P < 0.05). Using MnSOD activity as a biomarker, Mn deficiency appears as early as 6 weeks of age in flatiron mice. Pharmacokinetic studies show that intestinal 54Mn absorption is reduced in flatiron mice by 60% (P < 0.05). The sum of evidence indicates that Mn deficiency develops in flatiron mice due to reduced intestinal absorption implicating a role for Fpn in this process. Notably, flatiron mice also have 51% less Mn in bile compared to controls (P < 0.05). These data suggest that Fpn plays a key role in Mn homeostasis.

Grant Funding Source: Research supported by NIH R01ES014638.
Reprogramming of hepatic lipoprotein metabolism by small molecule inducers of TRIB1 expression (998.1)
Authors: Nagiec Marek , Skepner Adam , Negri Joseph , Eichhorn Michelle , Comer Eamon , Muncipinto Giovanni , Musunuru Kiran , Subramanian Aravind , Clish Clary , Duvall Jeremy , Foley Michael , Perez Jose , Palmer Michelle
Published: June 2021

Recent genome wide association studies have identified TRIB1 as a novel locus associated with the risk of CAD and myocardial infarction. Since increased expression of the TRIB1 gene is associated with lower plasma levels of LDL cholesterol and triglycerides and higher levels of HDL cholesterol, we set up a qRT‐PCR‐based phenotypic assay for inducers of TRIB1 expression in human HepG2 cells. In the screen of the diversity‐oriented synthesis (DOS) compound collection we identified a class of benzofuran‐based compounds that upregulate TRIB1 expression and phenocopy the effects of TRIB1 overexpression as they inhibit the triglyceride synthesis and ApoB secretion in cells. In addition, the compounds downregulate expression of MTTP and APOC3, encoding key proteins involved in lipoprotein assembly. Unexpectedly, active benzofurans ‐ as well as natural product TRIB1 inducers ‐ also modulate multiple components of hepatic cell cholesterol metabolism including elevating the expression of LDLR transcript and the level of LDL receptor while reducing the levels of PCSK9 transcript and secreted PCSK9 and stimulating the LDL uptake. The effects of the TRIB1 inducers are not masked by cholesterol depletion and are MAPKerk dependent. These chemically tractable compounds represent a novel type of modulators that reprogram cellular lipoprotein metabolism in HepG2 cells from lipogenesis to scavenging.
Specific nonlipid agonists of the lysophosphatidic acid type 2 GPCR with subnanomolar potency mitigate radiation injury (998.2)
Authors: Szabo Erzsebet , Patil Renukadevi , Fells James , Norman Derek , Balogh Andrea , Miller Duane , Tigyi Gabor
Published: June 2021

Previous studies have identified ligands of the LPA2 GPCR as mitigators of high‐dose radiation injury in vitro and in vivo. These early hit compounds originating from the hit scaffold GRI977143 had EC50 values in the hundred nanomolar range. Lead optimization guided by a validated computational model of the LPA2 GPCR led us to explore the structure‐activity relationship of a group of aromatic sulfonamides. The apparent EC50 value for oleoyl lysophosphatidic acid (LPA 18:1) at LPA2 is 1.4 nM. Synthetic modifications of the aromatic sulfonamide scaffold led to the synthesis of compounds RP 10‐73 and 10‐83, which have subnanomolar potency at LPA2. The EC50 value of ligand activated Ca2+ transient in LPA2 reconstituted mouse embryonic fibroblasts derived from LPA1&2 double knockout mice was 0.15 ± 0.02 nM and 6 ± 3.9 pM for RP 10‐73 and 10‐83, respectively. These two nonlipid compounds lack agonist or antagonist activity at other LPA GPCR up to 10 µM when tested for eliciting or inhibiting ligand‐activated Ca2+ responses. RP 10‐73 and 10‐83 rescued cells from apoptosis induced by ionizing radiation or by the radiomimetic chemotherapeutic agent Adriamycin. The protective effect was not detectable in cells lacking the LPA2 receptor subtype indicating that the antiapoptotic effect requires functional LPA2 receptors in the target cell. The pharmacological properties of RP 10‐73 and 10‐83 make them ideal drug candidates for the specific activation of LPA2 in various disease conditions.

Grant Funding Source: Supported by NIAID AI‐107331
Second Verse, Same as the First? Structures of Thioredoxin Proteins TrxA and TrxC from Mycobacterium tuberculosis (LB103)
Authors: Junior Amari , Rios Llamosa Brigitte , Richmond Anthony , Rios Llamosa Jhordy , Osademe Isioma , Cobb Michaun , Camacho Alexis , Gonzalez Cruz Jose , Johnson Carol , Neumann Terrence
Published: June 2021

Mycobacterium tuberculosis, the causative agent for tuberculosis (TB), infected 8.6 million and killed 1.3 million people in 2012 (WHO). TB is most prevalent in countries with a high incidence of infectious diseases, such as HIV, due to weakened immune systems. TB mainly affects the lungs, but can also affect other systems. When a host organism is infected wtih TB, the Mycobacteria in the lungs multiply, often resulting in pneumonia, chest pain, and prolonged coughing. In response, host macrophages, a part of the natural immune system, engulf the Mycobacteria and oxidize bacterial cell proteins in an attempt to destroy it. To protect itself against this attack, the bacterial thioreductase system, consisting of the redox protein thioredoxin reductase (TrxR) and the thioredoxin proteins TrxA, TrxB, and TrxC, gives electrons back to the oxidized proteins. As this system works to maintain cellular redox homeostasis, finding ways to stop it might provide a new method for treating people with TB. TrxA and TrxC have similar structures, thus it can be hypothesized that their functions are similar. Comparing binding sites between the proteins can provide insight if TrxA can react with TrxR similarly to TrxC. By modeling TrxA and TrxC with 3D printing technology, the Messmer SMART Team (Students Modeling A Research Topic) can compare the structures of the thioredoxins which may lead to new strategies for curing or preventing TB.

Grant Funding Source: Supported by a grant from NIH‐CTSA.
Incidence and Structural Variety of the Anterior Lateral Ligament of the Knee Joint (LB12)
Authors: Alimohammadi Majid
Published: June 2021

The overall objective of this study is to show the incidence of presence of this ligament and its structural variations in both genders. I began to search for this ligament using 34 embalmed cadavers (14 females and 20 males) in December 2013.

Each specimen was carefully dissected and photographed. Based on the findings in this study, the “ALL ligament” was present in 64% (9) of female and 85% (17) of male specimens.

From structural point of view, in 46% (12) of cases with the “ALL ligament”, this ligament was in the form of a flat band arising from the lateral collateral ligament of the knee and gradually blending with the capsule of the knee joint above the Gerdy’s tubercle. In the remaining cases (14 = 54%) the “ALL ligament” could be followed down to the tibia close to the Gerdy’s tubercle. In this second group, the “ALL ligament” always had some attachments to the anterior end of the body of the lateral meniscus.
The B. subtilis accessory helicase PcrA facilitates replication through transcription units genome‐wide (LB126)
Authors: Merrikh Christopher , Merrikh Houra
Published: June 2021

In bacteria, transcription and DNA replication occur concurrently, inevitably causing replication‐transcription conflicts. Conflicts can occur both co‐directionally (leading strand genes) and head‐on (lagging strand genes), leading to replisome stalling and restart. Because the majority of genes are co‐oriented with replication in most bacteria, co‐directional conflicts are expected to be highly prevalent, impacting replication speed and genomic stability. Consequently, mechanisms that deal with these conflicts should be critical for the timely and accurate duplication of genomes. Here we report that the essential Bacillus subtilis accessory helicase, PcrA, mitigates both co‐directional and head‐on conflicts between the replication and transcription machineries. Using ChIP‐seq, we have identified genomic regions where PcrA associates and facilitates replication progression. We find that PcrA predominantly associates with co‐directional rDNA and tRNA genes, in a transcription‐dependent manner. When PcrA is depleted, and only when transcription is active, replisome stalling and restart increases in the same areas. PcrA also reduces transcription‐induced replication stalling and restart at other regions, including some head‐on genes. Differential levels of PcrA at engineered head‐on and co‐directional conflicts indicate that its association and activity correlate with conflict severity, rather than transcription levels. These results show that PcrA reduces replisomes stalling, and restart, caused by head‐on and co‐directional replication‐transcription conflicts. Furthermore, these data indicate that during vegetative growth, and in the absence of exogenous stress, the most difficult regions to replicate are co‐directionally transcribed genes. Both the genome co‐orientation bias and these accessory helicases are widely conserved, suggesting that our results are relevant for many other bacterial species.

Grant Funding Source: NIH
Mitochondrial quality control and autophagosomal process in Parkinson’s disease model (LB170)
Authors: Sung Hyun , Hollenbeck Peter
Published: June 2021

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, and can be caused by genetic mutations of PTEN‐induced putative kinase 1 (PINK1) and the E3 ubiquitin ligase, Parkin. It has been proposed that Parkin contributes to policing mitochondrial fidelity by binding to dysfunctional mitochondria via PINK1 and targeting them for autophagic degradation. However, the actual role of PINK1/Parkin in controlling autophagy is unknown. To define more clearly the roles of Parkin in neurons, we quantified the behavior of mitochondria and autophagosomes in motor neurons of live Drosophila melanogaster larvae by time lapse microscopy. We analyzed the life cycle of mitochondria in Parkin mutant animals expressing mitochondrially‐targeted GFP by three distinct criteria: mitochondrial axonal transport, mitochondrial fission‐fusion and mitophagy. Autophagosomal processes were analyzed by studying autophagosomal movement and density via expressed RFP‐atg8. In motor axons, parkin mutants showed reduced flux of both anterograde and retrograde mitochondria and an increase in pause time compared to controls. However, these mutants did not show significant differences in mitochondrial velocities. Morphological measurements from parkin mutants showed that normal fission‐fusion balance was preserved in the axons of segmental nerves, while tubular and hyper‐fused mitochondria were found in the cell bodies. In addition, we did not observe a correlation between mitochondrial length and velocity or direction of axonal movement in parkin mutants. To understand the organization of mitophagy, we quantified mitochondrial density and found that parkin mutants displayed a significantly reduced mitochondrial density in motor axons. Analysis of autophagic vacuoles in parkin mutants showed reduced retrograde axonal flux and reduced density in the motor cell bodies. These results suggest that the differences in axonal mitochondrial flux in disease mutants result from the combined effects of altered mitochondrial motility and density in motor axons. Additionally, Parkin perturbation may disrupt autophagosomal retrograde transport in axons and affect mitochondrial morphology in motor cell bodies.

Grant Funding Source: Supported by grant, 5 R01 NS027073 from National Institutes of Health/NINDS
Histological changes in the structure of lateral and medial menisci following the destabilization of the knee in a rat model (LB18)
Authors: Riccardi Margaret , Fraticelli Nicole , Jordan Rebecca , Boehm Karl , Ginsberg Zachary , Marberry Kevin , Kondrashov Peter
Published: June 2021

Three month‐old rats underwent destabilization of the medial meniscus (DMM) surgery, to replicate meniscal damage, or sham surgery as a control, and were sacrificed 8 weeks later. Knees were fixed in formalin, decalcified, embedded in paraffin, and sectioned coronally at 12 µm. Sections were stained with hematoxylin and eosin and scored by 2 investigators using point counting with a grid probe. Based on existing data, each square was scored as one of the 4 tissue types normally present in the meniscus: dense connective tissue, fibrocartilage, hyaline cartilage, or bone. Percentages of these tissue types were used to quantify the histological composition of the menisci.

Preliminary data on the histological composition of the lateral and medial menisci showed a significant difference between the two. In the control group, the lateral meniscus contained significantly more hyaline cartilage than the medial. In the DMM group, the lateral meniscus contained more bone and hyaline cartilage than the medial. The comparison of the histological composition of the menisci between the DMM and control groups revealed a statistically significant increase in the percentage of bone in the lateral meniscus in the DMM group.

The changes in the architecture of the knee following the destabilization of the medial meniscus in the rat model resulted in the increased ossification of the lateral meniscus compared to the control.
Comparative analysis of cancer driver genes between human and six non‐human primates (LB182)
Authors: Lopez Darryl , Montague Michael , Warren Wesley
Published: June 2021

Cancer incidences among non‐human primates compared to humans are very low, however the causative factors are unknown. The difference could be derived from a combination of immunity, environmental factors, longer life expectancy in humans, and genetic variation. Detailed genome sequences for human and six other non‐human primates (Pan troglodytes, Gorilla gorilla, Pongo abelii, Callithrix jacchus, Macaca mulatta, and Otolemur garnettii) make it possible to assess whether genetic differences contribute to cancer susceptibility. We obtained a list of high‐confidence driver genes common to tumors from twelve different cancer types and examined a subset of these across non‐human primate orthologs. Empirical Bayes methods were used to test for adaptive evolution by examining the rate of non‐synonymous substitutions, and a number of genes contained amino acid sites with evidence for positive selection along the human lineage. We suggest that some of the driver genes in human common to various cancer types have a higher variability compared to non‐human primates, and that further examination of these genes is warranted.

Grant Funding Source: HG00668702
Creating a comparative map of the facial nerve: A cadaveric study (LB19)
Authors: Jenkinson Ashley , Nusse Gloria
Published: June 2021

This study will explore the facial nerve through detailed dissection of the left and right sides of a male and female cadaver with respect to major facial landmarks. A map will be created using qualitative analysis in order to compare the position and variability of the nerve branches. A photo expose will demonstrate the dissections and the comparative map in order to gain a better appreciation for the location, functionality, and importance of the nerve.

The facial nerve (CNVII), controls the muscles involved in facial expression, aids in the perception of taste and is involved in the production of tears. The facial nerve is composed of 10,000 neurons, 7,000 of which are directly involved in the expression of emotion. CNVII exits the skull through the stylomastoid foramen and from there, branches into five distinct rami under the parotid gland. The frontal branch controls the movement and expression of the forehead. The zygomatic branch is involved in ear movement. The buccal branch controls the movement of the upper lip, nostril, and corner of the mouth. The marginal mandibular branch is involved in depression of the lower lip and chin. And finally, the cervical branch controls the chin muscles and superficial neck muscles.

Grant Funding Source: none
Novel variations in the anatomical relationship between the piriformis muscle and sciatic nerve: a cadaveric study (LB2)
Authors: Harrell Kelly
Published: June 2021

The goal of this study is to assess variations in the anatomical relationship between the piriformis muscle and sciatic nerve in a cadaveric population. Currently, Beaton and Anson’s 1937 classification of piriformis‐sciatic arrangements is widely accepted and used for clinical evaluation and diagnosis of Piriformis Syndrome—a dynamic sciatic nerve impingement by the piriformis muscle. This study (n=164 limbs) revealed two clinically important differences to Beaton and Anson’s original findings. First, the Type “B” classification was observed in 18.3% of the cases, representing a 63.9% increase from Beaton and Anson’s original report. Additionally, two Type “B” subsets with potential clinical consequences were observed. Type “B2” included a vascular component, wherein the inferior gluteal artery and vein coursed between the common fibular and tibial divisions of the sciatic nerve (4.3% of total cases). Type “B3” included an additional nerve component, wherein the inferior gluteal nerve coursed between the piriformis muscle and adjacent to the common fibular division, to reach the gluteus maximus muscle (1.0% of total cases). Collectively, these observations expand the current classification of piriformis‐sciatic arrangement, and carry clinical significance for the evaluation and treatment of Piriformis Syndrome.

Grant Funding Source: n/a
MicroRNA‐130b suppresses metastasis of colorectal cancer through downregulation of its target genes (LB200)
Authors: Zhao Yanyang , Li Yao , Miao Gang
Published: June 2021

MicroRNA 130b (miR‐130b) is significantly dysregulated in various human tumor types. In this study, using a microarray assay, we characterized the upregulation of miR‐130b expression in colorectal cancer (CRC) specimens. However, there is limited knowledge about the roles of aberrant miR‐130b expression in CRC. Our studies in CRC cells demonstrated that miR‐130b significantly decreases cell migration and invasion in vitro and in vivo. In the overexpression miR‐130b CRC cells and the CRC specimens, we observed a decreased level of integrin β1 protein, which is considered as a key molecule involved in cell motility. The targeting of the 3’‐UTR region of integrin β1 gene by miR‐130b was revealed using a luciferase reporter assay. The impaired motility of the miR‐130b overexpression cells is recovered partly by the expression of integrin β1 lacking the 3’‐UTR. Furthermore, the inverse expressions of miR‐130b and integrin β1 were observed in CRC specimens. In summary, these data demonstrate that miR‐130b downregulates its target‐integrin β1, leading to the impaired migration and invasion of CRC cells. It also reveals the potential therapeutic function of miR‐130b for preventing metastatic colorectal cancer.
Changing Plasmid Copy Number by Mutation of the pMB1 Origin of Replication (LB209)
Authors: Grieshaber Brandon , Baker Erich , Campbell Malcolm , Eckdahl Todd , Poet Jeffrey , Heyer Laurie
Published: June 2021

We are developing Programmed Evolution as a system to optimize metabolic pathways in E. coli. The system allows introduction of variation in the regulatory elements of a genetic circuit encoding enzymes that control the desired orthogonal metabolism. Bacteria with variations that contribute best to metabolic output are selected through a fitness module. We reasoned that variation in plasmid copy number (PCN) of vectors containing the genetic circuit would affect metabolic output. We designed and cloned mutations in the pMB1 origin of replication to produce a change in PCN. We mutated the promoter regions of the RNA I and RNA II genes. PCN was measured by the use of Real Time Quantitative Polymerase Chain Reaction (RT‐qPCR) by comparison of five serial dilutions of chromosomal and plasmid DNA. Analysis of Ct values of plasmid and chromosomal DNA suggested altered PCN in some of the mutants. PCN measurements from RT‐qPCR were corroborated with measurements of DNA yield from plasmid preps of bacteria carrying the mutated origins. The results provide a collection of origins with several PCNs that can be used to introduce variation that is expected to affect metabolic output during Programmed Evolution.
Structural biology of heteromeric class B G protein‐coupled receptors (LB216)
Authors: Moad Heather , Pioszak Augen
Published: June 2021

The calcitonin family peptides calcitonin (CT), amylin (AMY), calcitonin gene‐related peptide (CGRP), and adrenomedullin (AM) regulate diverse processes including calcium homeostasis, blood glucose levels, vasodilation, and vascular development. Their receptors are heteromeric cell surface protein complexes comprised of a class B GPCR subunit, either the calcitonin receptor (CTR) or the calcitonin‐like receptor (CLR), in association with a peptide selectivity‐determining receptor activity modifying protein (RAMP) subunit. CTR alone is the CT receptor and CTR complexes with any of three RAMPs are AMY receptors. The CLR‐RAMP1 complex is a CGRP receptor, whereas CLR complexes with either RAMP2 or ‐3 are AM receptors. How RAMPs determine calcitonin family peptide selectivity is unclear and the receptor stoichiometries are a topic of debate. Here, we report unique methods for bacterial production of recombinant CLR‐RAMP extracellular domain (ECD) complexes engineered for structure/function studies and use of the novel constructs for peptide binding studies and crystallization. In addition, we describe recombinant CTR ECD production in HEK293T cells, and production of the integral membrane portions of the receptors in the E. coli inner membrane for structure/function studies. Our results indicate that the CTR and CLR ECDs are sufficient to determine peptide binding selectivity, identify peptide residues critical for receptor binding, shed light on the receptor stoichiometries, and provide methods that will facilitate structural studies of these therapeutically important GPCRs.

Grant Funding Source: Supported by NIH grant R01GM104251
TLR4 in monocytes plays a pivotal role on monocyte adhesion to vascular endothelium (LB226)
Authors: Lee Seung‐Jin , Seo Kyo‐Won , Bae Jin‐Ung , Kim Chi Dae
Published: June 2021

Toll‐like receptor 4 (TLR4) is known to mediate monocyte adhesion to endothelial cells via an increased expression of adhesion molecules on endothelial cells (EC), however, its role on the expression of monocyte adhesion molecules is unclear. In the present study, we investigated the role of TLR4 on the expression of monocyte adhesion molecules, and determined the functional role of TLR4‐induced adhesion molecules on monocyte adhesion to vascular endothelium. When THP‐1 monocytes were stimulated with Kdo2‐Lipid A (KLA), a specific TLR4 agonist, Mac‐1 expression was markedly increased in association with an increased adhesion of monocytes to EC. These were attenuated by anti‐Mac‐1 antibody, suggesting a functional role of TLR4‐induced Mac‐1 on monocyte adhesion on EC. In monocytes treated with MK886, a 5‐lipoxygenase (LO) inhibitor, both Mac‐1 expression and monocyte adhesion to EC induced by KLA were markedly attenuated. Moreover, KLA increased the expression of mRNA and protein of 5‐LO, suggesting a pivotal role of 5‐LO on these processes. In in vivo studies, KLA increased monocyte adhesion to aortic endothelium of wild‐type (WT) mice, which was attenuated in WT mice treated with anti‐Mac‐1 antibody as well as in TLR4‐deficient mice. Taken together, TLR4‐mediated expression of Mac‐1 in monocytes plays a pivotal role on monocyte adhesion to vascular endothelium, leading to increased foam cell formation in the development of atherosclerosis.

Grant Funding Source: None
PLC‐gamma1 signaling plays a subtype‐specific role in postbinding cell entry of influenza A virus (LB258)
Authors: Liang Yuying , Zhu Liqian , Ly Hinh
Published: June 2021

Host signaling pathways and cellular proteins play important roles in the influenza viral life cycle and can serve as antiviral targets. In this study (Zhu, et al., 2014, J. Virol.), we report the engagement of host phosphoinositide‐specific phospholipase gamma 1 (PLC‐gamma1) in mediating cell entry of influenza virus H1N1 but not H3N2 subtype. Both PLC‐gamma1‐specific inhibitor and short hairpin RNA (shRNA) strongly suppress the replication of H1N1 but not H3N2 viruses in cell culture, suggesting that PLC‐gamma1 plays an important subtype‐specific role in the influenza viral life cycle. Further analyses demonstrate that PLC‐gamma1 activation is required for viral postbinding cell entry. In addition, H1N1, but not H3N2, infection leads to the phosphorylation of PLC‐gamma1 at Ser 1248 immediately after infection and independent of viral replication. We have further shown that H1N1‐induced PLC‐gamma1 activation is downstream of epidermal growth factor receptor (EGFR) signaling. Interestingly, both H1N1 and H3N2 infections activate EGFR, but only H1N1 infection leads to PLC‐γ1 activation. In summary, we have identified for the first time the subtype‐specific interplay of host PLC‐gamma1 signaling and H1N1 virus that is critical for viral uptake early in the infection. This is the latest study in our series of recent publications that reveal novel insights into how influenza virus interacts with the cellular signaling network (Zhu, et al., 2014 J. Virol., Kumar, et al., 2011 J. Virol., 2011 Antimicrobial Agents Chemother., 2008 J. Virol.).

Grant Funding Source: NIH R21 grant AI094133 to Y.L.
Seed germination and recovery responses of suaeda heterophylla to abiotic stresses (LB284)
Authors: HAMEED ABDUL , AHMED MUHAMMAD , GULZAR SALMAN , GUL BILQUEES , ALAM JAN , HEGAZY AHMAD , Alatar Abdulrahman , KHAN M.
Published: June 2021

Seed germination and recovery from salt stress of an annual halophyte Suaeda heterophylla (Kar. & Kir.) Bunge to different iso‐osmotic concentrations (0, ‐0.46, ‐0.92, ‐1.38, ‐1.84, and ‐2.30 MPa) of NaCl and PEG‐6000 at 15/25, 20/30 and 25/35oC in both 12‐h temperature and light regimes and in complete darkness were studied. Maximum number of seeds germinated in distilled water and increase in concentrations of both NaCl and PEG‐6000 decreased germination at all temperature regimes, light and dark conditions, with higher inhibition in NaCl than PEG‐6000. Recovery of germination and viability of seeds were lower in NaCl than PEG‐6000 both in the light and dark. Moderate alternate temperatures (20/30oC) and 12‐h photoperiod were found to be the optimal for seed germination and recovery. Better seed germination of S. heterophylla when osmotic potential caused both by NaCl and PEG 6000 is lower, temperature regime of 20/30oC and light regime is for 12 h.

Grant Funding Source: ABDEL REHMAN A. ALATAR
Assessment of factors related to carbohydrate intolerance I: OGTT glucose AUC (LB299)
Authors: Levers K , Galvan E , Coletta A , Dalton R , Jung Y , O'Connor A , Goodenough C , Simbo S , Seesselberg C , Bonin B , Koozehchian M , Sanchez B , Barringer N , Rasmussen C , Greenwood M , Kreider R
Published: June 2021

The OGTT is a gold standard in assessing carbohydrate intolerance (CI) and insulin resistance. However, the test is costly and time consuming. This study examined whether a CI survey could predict response to an OGTT. 108 women (31.6±13 yr, 34.7±7% body fat, 25.3±4 kg/m2) donated fasting blood samples, completed a CI inventory, had body composition and health measures determined, and underwent a 75 g OGTT in which glucose samples and perceptions of CI symptoms were obtained at 0, 30, 60, 90, and 120 minutes. Pearson product correlations were performed to determine which factors correlated with OGTT glucose AUC. Results revealed significant correlations (p<0.05) in GAUC (264±46 mg/hr/dL) to G120 AUC (r=0.43), glucose AUMC (r=0.97), Cmax (r=0.91), fasting insulin (r=0.26), HOMA (r=0.30), height (r=‐0.28), resting HR (r=0.19), BMC (r=‐0.36), BMD (r=‐0.32), FFM (r=‐0.28), being overweight since very young (r=0.27), and slowly gaining weight after age 30. These findings indicate that OGTT GAUC is positively correlated with G120, insulin, and HOMA. Further, shorter women who gained weight as they got older with a higher resting HR and lower FFM, BMC, and BMD were more related to GAUC during a OGTT.

Grant Funding Source: Curves International, Inc. (Waco, TX)