bluuebunny/crossref_metadata_2025 huggingface dataset:
The Value of Autopsy in Medical Education
Authors: Yagoda William D , Severson Elizabeth
Published: June 2021

Background

By allowing medical students the opportunity to observe and assist in autopsies, the Kansas City University of Medicine and Biosciences Pathology Club has created a beneficial learning experience. The students' outlook on the experience was collected which reflected the positive influence that this learning tool has in medical education.

Discussion

Autopsy, as an experimental learning tool, has been underutilized in the medical education curriculum.1 In light of this fact, the Pathology Club at Kansas City University of Medicine and Biosciences has implemented a program that provides students with a different perspective on clinical medicine that is currently not being employed. By allowing students the opportunity to engage in spatial learning, we have introduced the art of medicine from a different viewpoint. The advantages of spatial or experimental learning include a hands‐on approach in real time by working in an atmosphere emphasizing clinical pathological approach by increasing understanding and retention.

Conclusion

Students who participated in our study found the experience to be instrumental in relating clinical medicine to death and disease. Based on our findings, observing or participating in an autopsy is a valuable learning experience that enhances the quality of students' education.
A mechanical model of the cardiovascular system – a pedagogical tool
Authors: Sweeney Terrence E , Miller John W
Published: June 2021

We have developed a mechanical model of the cardiovascular system for instructional use. The water‐circulating system includes a cardiac chamber for which the strength, duration and frequency of contraction may be varied; arterial and venous elements that each have variable compliance; and an arteriolar element with variable resistance. A PowerLab system (ADInstruments) is used to control the system and to display measured parameters. The cardiac element consists of a balloon enclosed in a plexiglass chamber that is cyclically pressurized via a FairchildTM electronic pressure regulator. Windkessels are used to mimic the compliant arterial and venous compartments. Two windkessels arranged in series mimic the peripheral and central venous compartments. Each is outfitted with a FairchildTM regulator to allow mimicry of the skeletal and thoracic pumps. Small bore silastic tubing arrayed in a parallel configuration comprise the variable peripheral resistance. An in‐line flow transducer and multiple pressure transducers enable measurement and evaluation of critical cardiovascular parameters in real time as system elements are manipulated. The highly visual and hands‐on nature of the system maximizes its use as a pedagogical tool at many different levels of instruction. Supported by The University of Scranton.
TIMP‐1 protein but not secretion is increased by shear stress in the skeletal muscle microvasculature
Authors: Uchida Cassandra , Milkiewicz Malgorzata , Fudalewski Tomasz , Gee Eric , Haas Tara L.
Published: June 2021

Luminal splitting is a form of angiogenesis caused by increased shear stress. A feature of luminal splitting is a lack of extracellular matrix proteolysis correlated with decreased production of matrix metalloproteinase (MMP)‐2. We hypothesized that an MMP inhibitor, tissue inhibitor of matrix metalloproteinase (TIMP)‐1, is upregulated by shear stress. TIMP‐1 mRNA increased in the EDL of prasozin‐treated rats (p<0.05, n=4) as measured by RT‐PCR. Rat skeletal muscle microvascular endothelial cells were exposed to shear stress (12 dyne/cm2). TIMP‐1 mRNA increased in cells after 2 and 24 hours of shear (p<0.05, n=3), and TIMP‐1 protein increased at 2 and 24 but not 4 hours (p<0.05, n=3) as assessed by Western. TIMP‐1 secretion decreased at 2 hours of shear and returned to control levels at 24 hours (p<0.01, n=3) as measured by reverse zymography of concentrated media. Similarly, MMP‐2 activity increased in media at 2 but not 24 hours of shear (p<0.05, n=7) as assessed by an in vitro MMP‐2 activity assay (Anaspec). 2 hours of shear caused no change in TIMP‐1 surface staining (ns, n=3) as assessed by confocal microscopy, but did cause an increase in intracellular TIMP‐1 staining (p<0.05, n=3). Our results show that intracellular TIMP‐1 protein is increased by shear stress, due to increased production and decreased secretion. The function of intracellular TIMP‐1 is unknown.

Funded by the Heart and Stroke Foundation of Canada.

Grant Funding Source

Heart and Stroke Foundation of Canada
Cooperation between Vezf1 and RhoB during vascular development
Authors: Stuhlmann Heidi , Benjamin Laura , Victor Andrea , Ocaya Pauline
Published: June 2021

Vascular endothelial zinc finger 1 (Vezf1) encodes a zinc finger transcription factor that is essential for developmental angiogenesis and lymphangiogenesis. Vezf1−/− embryos show vascular remodeling defects, loss of vascular integrity, internal bleeding and embryonic death. Haploinsufficiency is observed in 20% of Vezf1+/− embryos, resulting in lymphatic hypervascularity, edema and hemorrhaging (Kuhnert et al Dev.Biol.2005).

Recent studies suggested that Vezf1 might functionally interact with Rho GTPases. We studied cooperation between Vezf1 and RhoB during vascular development by generating embryos with compound Vezf1;RhoB null alleles. Strikingly, loss of RhoB exacerbates the phenotypes associated with loss of one Vezf1 allele. The phenotypes include: 1) enlarged head vessels 2) vascular anomalies at multiple sites in the head, hypervascularity, and hemorrhaging of internal organs, and 3) hypervascular lymphatic phenotype with blood‐filled vessels in the neck. These structures display similarities with human vascular and lymphatic anomalies. Thus, the results suggest that Vezf1 and RhoB cooperate in vivo, and indicate their potential role in human vascular pathologies.
Hypoxia Response and Stroke Susceptibility with Sickle Cell Anemia (SCA)
Authors: Milbauer Liming C , Enenstein Judy A , Kiley Jim , Johnson Darrell , Roney Mark , Solovey Anna , Hebbel Robert P
Published: June 2021

Individuals with SCA are susceptible to stroke at an early age. Since hypoxia is a hallmark of stroke, we hypothesize that hypoxic responses of some genes may differ constitutively in individuals at risk for sickle‐stroke. Blood outgrowth endothelial cells (BOEC) were isolated from African American donors without SCA (NS) as well as SCA donors at risk (AR) or not at risk (NAR) of early stroke. Risk was assessed as occlusive disease at the Circle of Willis. BOEC were incubated under hypoxic (0.5% O2) conditions and induction of 9 known hypoxia response genes were assessed by qRT‐PCR. Combined data from 30 min and 1h hypoxia, indicated significant differences (p<.05) between groups in fold induction of several genes: AR>NAR (Elk‐1, tPa, HIF1α, HIF1β and Endothelin‐1), AR>NS (ELK‐1, tPa), and NS>NAR (Elk‐1, HIF1α). Slightly significant differences (p<.1) included AR>NAR (Egr‐1, Flk‐1) and AR>NS (Flk‐1, Egr‐1, HIF1α, Endothelin‐1). Under normoxic conditions, most genes did not differ at p<.05 between groups. The 9 genes are known for complex impacts on inflammation, angiogenesis, apoptosis and cerebral ischemia. However, the high hypoxia response of AR compared to NAR BOEC suggests that AR individuals may lack protective mechanisms that NAR have. In conclusion, individuals with SCA at risk for stroke respond to hypoxia with exaggerated expression of several genes.

Funding: NIH (HL076540 & HL55552)
TB Meningitis, Reactive Oxygen Species (ROS) and the Pathological Ossification of the Petroccipital Fissure and Cochlear Cannaliculus
Authors: Balboni Armand L , Bergemann Andrew D , Laitman Jeffrey T
Published: June 2021

Fibrocartilage ossification is a well‐controlled process in which chondrocytes within the tissue stop proliferating and differentiate into hypertrophic chondrocytes before undergoing apoptosis and the induction of tissue mineralization. We previously reported a pathological ossification of the fibrocartilagenous petroccipital fissure (POF) and cochlear cannaliculus (CC) in individuals with TB meningitis (Balboni et al., 2008). However, a mechanism by which such ossification could occur has not been shown. Recent work by Yang et al. (2007) demonstrates that TB meningitis causes a marked increase in the production of ROS in the central nervous system. To test whether an increase in ROS could cause POF/CC ossification we cultured ATDC5 chondrocytes in the presence of control medium(low ROS activity), differentiation medium(high ROS activity), or differentiation medium plus the antioxidant Deferiprone®, and observed them for evidence of hypertrophy. ATDC5 cells were also tested for the expression of the hypertrophic markers mmp13, typeX collagen, and runx2 via RT‐PCR. We show that increased ROS levels induce chondrocyte hypertrophy in vitro. Markers of elevated ROS levels are detected in hypertrophic ATDC5 chondrocytes while Deferiprone® protects cells from oxidative stress and inhibits these markers. These data demonstrate that POF/CC ossification could occur via ROS formed during TB meningitis.

Grant Funding Source

Faculty Research Award
We can't teach everything: Less can be more
Authors: Drake Richard L
Published: June 2021

There is just not enough time! Current course surveys show that the amount of class time available for Gross Anatomy, Embryology, Microscopic Anatomy and Neuroanatomy continues to decrease. This has a big impact on courses that spend a significant amount of time in the laboratory, like Gross Anatomy. What am I going to do? I have to look at different approaches regarding how material is presented. Is the lecture format still the best approach? Can we modify the approach in the laboratory making it more time efficient? Is it possible to get time in the 2nd and 3rd years to reinforce anatomical knowledge? What level of understanding of anatomy does a medical student need to be adequately prepared for 3rd year clinical rotations? Approaches, options and possibilities related to these questions will be discussed.
Effect of Autologous Platelet‐Rich Plasma on Distraction Osteogenesis of the Mandible in Rabbits.
Authors: Jurjus Abdo R , Rahme Mark , Saghieh Said
Published: June 2021

Distraction osteogenesis of the jaws has been used in the treatment of pediatric craniofacial deformities. Successful clinical outcome depends on a coordinated multidisciplinary treatment.

Platelet rich plasma (PRP) containing growth factors extracted from autologous blood and used to increase the healing potential of bone and soft tissue has been, in addition to bone marrow cells (BMC), recently tested during distraction osteogenesis of long bones in humans. The specific aim of this research is to study the effect of PRP on bone healing after mandibular distraction in rabbits and explore future possible application in humans. Right mandibular distraction was performed in 12 rabbits divided into 2 groups of 6 each. PRP and physiological saline were injected in the callus following distraction in the tested and control group respectively. The rabbits were sacrificed after consolidation period of 45 days and the mandibles were surgically removed. Two rabbits, 1 from each group, were excluded from the study the first due to lack of adequate distraction, the second died on the 30th day of consolidation period. Bone mineral density, radiographic analysis, mechanical properties and histological features of the lengthened bones were assessed on 10 rabbits 5 in each group.

The data analysis showed a faster bone remodeling with an increase in the amount of trabeculation but no significant increase in bone mineral density and content in the experimental group in comparison to the control group.

The value of this research lies in the potential acceleration of healing after mandibular distraction using PRP which is greatly needed since the mandible is usually subjected to complex functional demands.
Regulation of meiotic recombination by a multifunctional ATF/CREB protein
Authors: Gao Jun , Davidson Mari K , Wahls Wayne P
Published: June 2021

Homologous recombination occurs at a high frequency in meiosis, is clustered at hotspots, and aids chromosome segregation. In the fission yeast S. pombe, Atf1‐Pcr1 heterodimer (ATF/CREB proteins) binds to and activates a meiosis‐specific recombination hotspot, ade6‐M26. The stress‐activated protein kinase Spc1 (a homolog of human p38) phosphorylates Atf1 and is also essential for hotspot activity. We determined the roles of Atf1, Pcr1, and Spc1 in hotspot recombination. Deletion analyses revealed that only the bZIP (DNA binding/protein dimerization) domain of Pcr1 was required. In contrast, bZIP and non‐bZIP regions of Atf1 affected hotspot recombination. Remarkably, a mutant form of Atf1 that can not be phosphorylated by Spc1 kinase still activated the hotspot. Furthermore, artificial tethering of Atf1 to ade6 promoted recombination in the absence of its own bZIP domain, or Pcr1, or Spc1 kinase. Finally, we successfully developed methods to purify and identify proteins associated with Atf1. Therefore: 1) An amino‐terminal "activation domain" of Atf1 is necessary and sufficient to promote hotspot recombination. 2) Pcr1 confers DNA binding site specificity to Atf1‐Pcr1 heterodimer in vivo, thus targeting the Atf1 activation domain to specific chromosomal sites. 3) Phosphorylation of Atf1 by Spc1 kinase is, unexpectedly, not required for hotspot activity. 4) The Spc1 kinase regulates hotspot recombination only at or prior to binding of Atf1‐Pcr1 to ade6‐M26 DNA. 5) Protein complexes associated with Atf1.
DNA polymorphism of the European Percids
Authors: Islam Asiful , Gorshkov Oleg , Chernov Vladislav , Kuznetsov Vjatscheslav
Published: June 2021

The DNA polymorphism of European Percids of Russian Kuibyshev reservoir revealed a close phylo‐evolutionary relationship among their populations. Pikeperches (Stizostedion lucioperca and S. volgense), river perch (Perca fluviatilis) and ruffe (Gymnocephalus cernuus) are the most precious species which are sharply declining from this reservoir. This situation urges us to investigate their genetics relationship. Two microsatellites' DNA of American walleye (S. vitreum) were used as primers for this study. Both of the DNA markers (Svi‐4 and Svi‐18) have 4‐6 alleles with a size range of 102‐124 bp. The DNA analysis revealed complete polymorphisms in Percid populations. S. volgense have randomly amplified DNA polymorphism of 100‐1200 bp sizes. Most of the dominant DNA bands of Percids existed in 100 bp. S. lucioperca have diversified DNA amplifications with a size range of 100‐400 bp. The 2‐3 dominant DNA bands of S. lucioperca were located at 100‐250 bp regions. River perch only had one band at 100 bp and ruffe had two DNA amplification signals at 100 and 180 bp. The DNA polymorphisms of S. lucioperca and S. volgense showed close and specific size ranges of DNA amplifications. Thus, these two species emerge as closely phylo‐genetics relatives. These analyses could also help us further to understand the human phylo‐genetics variations.
A disease‐linked DNA repeat array rich in potential guanine quadruplexes
Authors: Chen Desheng , Xu Xueqing , Tsumagari Koji , Ehrlich Melanie
Published: June 2021

Potential G‐quadruplex sequences (PQS) can form non‐B structures containing four equal‐length runs of Hoogsteen‐paired G residues. They have extremely high thermodynamic stability and form hairpin structures from four clustered G runs, bimolecular‐type structures from two clustered G runs, or tetramolecular‐type structures (e.g., at telomeres) from four G runs on different strands. D4Z4 arrays at 4q35.2, which are linked to facioscapulohumeral muscular dystrophy (FSHD), have a high density of PQS. In D4Z4's 3.3‐kb repeat unit we found seven hairpin (G3‐5N1‐7G3‐5N1‐7G3‐5 N1‐7G3‐5) PQS. Our evidence for G‐quadruplex formation from these consensus sequences and other D4Z4 oligonucleotide sequences that contain runs of only 2 G's or only two runs of 4 or 5 G's came from characteristic circular dichroism (CD), gel electrophoresis, partial resistance to PCR, and highly targeted deletion and mutation products arising spontaneously upon DNA replication in vitro. D4Z4 arrays of 3.3 ‐ 33 kb at 4q are usually pathogenic, and those of >36 kb are almost always phenotypically neutral. Given the overrepresentation of PQS in promoters of myogenic genes, it is attractive to propose that G‐quadruplexes help organize D4Z4 chromatin structure as part of special changes in D4Z4's conformation accounting for the near‐threshold effect of D4Z4 size on FSHD status. (Supported in part by NIH Grant NS048859)
Sinusoidal endothelial cell crosstalk with other liver cells
Authors: DeLeve Laurie D. , Wang Xiangdong
Published: June 2021

Hepatic sinusoidal endothelial cells (SEC) line the microcirculation and have a unique phenotype: open fenestrae clustered in sieve plates and lack of a basement membrane. In normal liver, the SEC phenotype is maintained by paracrine secretion of VEGF by hepatocytes and hepatic stellate cells (HSC) and, downstream, VEGF‐stimulated autocrine production of nitric oxide (NO) by the SEC. VEGF‐stimulated NO production by SEC also promotes HSC quiescence.

Capillarization, loss of SEC fenestration and formation of a basement membrane, precedes liver fibrosis and seems to be permissive for the final common step in fibrosis: activation of HSC. If loss of the differentiated SEC phenotype precedes and permits fibrosis, characterizing dysregulation of cell‐cell crosstalk in liver disease becomes important in understanding liver fibrosis. Progressive loss of paracrine secretion of VEGF, loss of SEC VEGF receptors, and decline in SEC NO production occurs in parallel with increasing severity of liver disease in experimental models of steatosis, steatohepatitis and cirrhosis. Further elucidation of the interplay between hepatocytes, HSC and SEC may provide therapeutic strategies to prevent fibrosis.
A subset of poly(A) factors is required for histone pre‐mRNA processing in D. melanogaster
Authors: Steiniger Mindy , Sullivan Kelly D. , Marzluff William F.
Published: June 2021

Metazoan histone pre‐mRNAs end in a stem‐loop (SL) structure rather than a poly(A) tail. Formation of histone mRNAs requires cleavage directed by the SL, which binds SLBP, and a histone downstream element, which is recognized by U7 snRNP. In D. melanogaster, poly(A) signals located downstream of the normal histone pre‐mRNA cleavage sites are utilized when cleavage is inhibited. Knockdown of CPSF73, CPSF100, and Symplekin results in polyadenylation of histone mRNA, while depletion of CstF50, 64 and 77, CPSF 30 and 160 does not affect processing, suggesting that CPSF73, CPSF100 and Symplekin comprise the core of a common cleavage factor. Depletion of Symplekin, CPSF73, or CPSF100 results in reciprocal co‐depletion of the other two factors. Each of these factors coimmunoprecipitates the other two. Lsm11 and SLBP also coIP with CPSF73 and Symplekin, indicating that the U7 snRNP and SLBP may recruit the cleavage complex. ChIP experiments show that both Symplekin and CPSF73 associate with D. melanogaster H1 and H3 genes, while CstF50 does not. Depletion of SLBP by RNAi results in association of RNA Pol II, CPSF73, Symplekin and CstF50 with the region between the H1 and H3 genes. These results suggest that CstF is not associated with the histone cleavage factor in vivo and support a model in which there is a distinct cleavage factor for histone pre‐mRNA processing which shares only some components with the poly(A) cleavage factor.
A Functional Interaction between the Evolutionarily Conserved Zinc Finger Poly(A) RNA‐binding Protein, Nab2, and the Exosome Links mRNA 3′‐End Processing/Export with mRNA Quality Control
Authors: Fasken Milo , Corbett Anita H
Published: June 2021

Quality control of mRNA processing and export ensures that only properly processed transcripts reach the cytoplasm. A key player in RNA quality control is the exosome, a riboexonuclease complex with a critical catalytic subunit, Dis3, that degrades or trims RNA. In S. cerevisiae, the essential zinc finger poly(A) RNA binding protein, Nab2, plays important roles in the control of mRNA poly(A) tail length and export. The N‐terminal domain of Nab2 (Nab2‐N), which is critical for Nab2 function, forms a five alpha‐helix bundle with a proline‐tryptophan‐isoleucine (PWI)‐like fold found in several RNA‐binding proteins including ZC3H14, a putative human orthologue of Nab2. Expression of a Nab2 deletion mutant, Nab2 δN, causes slow growth, extended poly(A) tails and a block to poly(A) RNA export. To gain insight into the role of Nab2‐N and the function of this evolutionarily conserved protein family, we performed a transposon mutagenesis screen on nab2 δN cells to identify extragenic suppressors that rescue nab2 δN cell growth. This screen identified transposon insertions in two genes that encode essential components of the exosome, DIS3 and RRP4, revealing that Nab2 genetically interacts with the exosome and suggesting that Nab2 may physically associate with this riboexonuclease machine. Thus, Nab2 may recruit the exosome to modulate poly(A) tail length and target improperly processed mRNA for degradation.
RNA damage and degradation under oxidative stress
Authors: Liu Min , Li Zhongwei
Published: June 2021

RNA is extensively damaged under oxidative stress (OS) and becomes dysfunctional. Oxidized RNAs are presumably degraded by selective surveillance mechanisms which control the quality of cellular RNA. We have identified many proteins in Escherichia coli that bind oxidized RNA at high affinity. In this work, we have studied the role of 138 proteins, including the ones binding oxidized RNA and others function in RNA metabolism, in supporting cell viability under OS. Nearly 40 proteins showed protective effect against H2O2 treatment, including RNases, RNA helicases, poly(A) polymerase, and enzymes involved in RNA modification. Mutants lacking an RNase and another enzyme in combination are frequently more sensitive to H2O2. Under OS, cells defective in RNA degradation contain elevated 8‐hydroxyguanosine, an oxidized RNA lesion, as determined by HPLC. Ribosomal RNAs account for 80% of total RNA and are normally stable, presumably carrying the majority of oxidized nucleotides. RNA profile analysis using Agilent Bioanalyzer revealed increased fragmentation of rRNA in some of the strains upon H2O2 treatment. These data suggest that a number of proteins function in removing damaged rRNA. The specific roles of individual activities in targeting, fragmenting and degrading oxidized RNA are studied in details. This project is supported by FAU Integrative Biology Ph.D. Program to M.L. and NIH grant S06 GM073621 to Z.L.
Membrane protein quality control in post‐Golgi compartments
Authors: Apaja Pirjo , Lukacs Gergely L
Published: June 2021

Elimination of non‐native polypeptides is critical for protein quality control (QC) and fundamental to preserve cellular proteostasis. The "peripheral" QC of cell surface membrane proteins (MP) remains poorly understood. To uncover the principles of MPQC in post‐Golgi compartments, we engineered chimeric CD4 molecules containing wt and temperature‐sensitive mutant l domain. The mutant was accumulated at the cell surface and unfolded at the non‐permissive temperature. Increased internalization and impaired recycling accounts for the metabolic destabilization of the unfolded chimera at the cell surface. The postendocytic sorting of the internalized chimeras was conformational‐dependent; unfolding redirected the chimera from constitutive recycling towards lysosomal degradation. Ubiquitination plays a pivotal role in the non‐native chimera degradation since; a) the chimera ubiquitination was enhanced several fold upon unfolding, 2) depletion of ubiquitin‐dependent endosomal sorting machinery (Hrs, Stam‐2 and Tsg101) delayed lysosomal delivery and 3) physical and functional association of the misfolded variant with an E3 Ub‐ligase could be identified. We propose a working model for the peripheral QC of MPs with a central role in cellular proteostasis and the etiology of conformational diseases. Supported by CIHR.
Molecular Cloning and Biochemical Characterization of Manduca sexta Serpin‐7, a Regulator of Prophenoloxidase Activation
Authors: Suwanchaichinda Chansak , Ochieng Rose , Zhuang Shufei , Kanost Michael R.
Published: June 2021

Serine protease inhibitors (serpins) are involved in various physiological reactions in humans and insects. Six serpins, SPN1‐SPN6, have been identified and characterized in the tobacco hornworm Manduca sexta. In this study, we obtained a complete DNA sequence of another Manduca serpin gene, SPN7. The putative mature SPN7 protein was 385 amino acid residues long (MW 42.90 kDa, pI 5.32) with a predicted 15 residue signal sequence. Multiple sequence alignment of the reactive center loop region of the M. sexta serpins indicated that SPN7 contained Arg and Ile residues at P1′‐P1 positions, similar to SPN4 and 5. RT‐PCR results demonstrated an up‐regulation of SPN7 expression in the fat body at 24 h after injection of larvae with Micrococcus. The recombinant SPN7 protein was expressed in E. coli and purified using nickel‐affinity chromatography. Addition of recombinant SPN7 significantly inhibited both 1) spontaneous melanization of the 5th instar larval plasma, and 2) phenoloxidase activity of the plasma challenged by Micrococcus bacteria. SPN7 inhibited the enzymatic activity of the active prophenoloxidase‐activating protease‐3 by forming a stable serpin‐protease complex as indicated by SDS‐PAGE under reducing conditions. SPN7 inhibited bovine trypsin but not chymotrypsin. We conclude that SPN7 is inhibitory and likely involved in regulation of innate immunity in the insect. (supported by NIH grant GM41247)
Activation of plasminogen by Bacillus anthracis spores and proteases results in down‐regulation of thrombin‐activatable fibrinolysis inhibitor
Authors: Chung Myung‐Chul , Tonry Jessica H. , Jorgensen Shelley C. , Narayanan Aarthi , Bailey Charles L. , Popov Serguei G.
Published: June 2021

Plasminogen is a serine protease zymogen released by the liver and activated by several proteases to regulate mainly fibrinolysis. In this study we examined whether plasminogen activation is involved in Bacillus anthracis pathogenesis. At first, we explored whether secreted anthrax metalloproteases Npr599 and immune inhibitor A (InhA) are involved in pro‐urokinase (pro‐uPA) activation, which leads to plasminogen activation. Exposure of human pro‐uPA to Npr599 and InhA resulted in its proteolytic activation. Western blot analysis performed on liver samples from InhA‐injected mice showed a significant rise in uPA and plasminogen activation when compared to uninfected liver controls. In addition, plasmin(ogen) efficiently bound to spore and cell surfaces in a lysine‐dependent manner. GroEL, enolase, elongation factor Tu and GAPDH were identified as candidate spore surface plasminogen receptors. Spore coated plasmin, as well as Npr599 and InhA degraded thrombin‐activable fibrinolysis inhibitor (TAFI), a plasma zymogen that inhibits fibrinolysis. Moreover, active and inactive TAFI antigen levels were significantly decreased in mouse liver samples without a decrease in gene expression after challenge with the toxigenic Sterne spores and InhA. Taken together, our data suggest that B. anthracis infection down‐regulates TAFI via plasminogen activation and may lead to unregulated fibrinolysis.
Peptide binding affects the conformation and ATPase activity of the Bacterial AAA+ domain 2 of ClpA
Authors: De Donatis Gian Marco , Piszczek Grzegorz , Maurizi Michael R
Published: June 2021

ClpA consists of 3 domains, an N‐terminal and two ATP‐binding domains, D1 and D2. ClpA recognizes specific motifs on the termini of proteins and mediates unfolding and translocation to the protease, ClpP. We have performed sedimentation velocity measurements on ClpA in the absence and presence of 30‐fold excess of SsrA peptide, a C‐terminal tag that targets proteins to ClpXP and ClpAP, and we have analyzed the kinetics of ATP hydrolysis of ClpA mutants in the presence or absence of SsrA. Ultracentrifugation of ClpA in the presence of a non‐hydrolysable ATP analog, ATP ?×S, gave a distribution of sedimentation coefficients that becomes narrower in the presence of SsrA. Similar effect persists with a mutant lacking the N terminal domain, while the effect is not present in mutant lacking the D2, indicating that the effect of SsrA can be ascribed to the presence of D2. Stimulation of ATP hydrolysis mediated by SsrA binding was observed only with wild type ClpA or mutants with a functional D2 domain. Changes in activity upon SsrA binding to mixed hexamers of wild type ClpA and ClpA with inactive D2 revealed that, differently from the absence of SsrA, a single subunit with a mutated D2 exerted a negative allosteric effect, completely blocking the activity of the other subunits. Our results indicate that SsrA binding reduces conformation flexibility in the ClpA hexamer and affects interactions and catalytic properties of the D2 domains.
Functional Annotation of Unknown Enzymes within the Amidohydrolase Superfamily
Authors: Cummings Jennifer Ann , Xu Chengfu , Nguyen Tinh , Fedorov Elena V. , Fedorov Alexander A. , Almo Steven C. , Raushel Frank M.
Published: June 2021

Approximately one third of the genes for the completely sequenced bacterial genomes have an unknown, uncertain, or incorrect functional annotation. This suggests that a significant fraction of the metabolic landscape remains to be discovered. The relationship between substrate specificity and the 3‐dimensional structures of two N‐acyl‐D‐amino acid deacetylases and 5 renal dipeptidase (RDP)‐like proteins from the amidohydrolase superfamily has been examined. Compound libraries of N‐succinyl‐ and N‐acetyl‐ derivatives of L‐ and D‐ amino acids were used to screen two putative deacetylases, Bb3285 and Gox1177, for catalytic activity. The analysis revealed that Bb3285 only liberated D‐Glu, but Gox1177 hydrolyzed the N‐acetyl‐derivatives of neutral and hydrophobic D‐amino acids. The best substrates of Bb3285 and Gox1177 were N‐formyl‐D‐Glu and N‐acetyl‐D‐Leu, respectively. The crystal structure of Bb3285 was determined and comparison with a deacetylase from A. faecalis reveals that the specificity of Bb3285 is due to an arginine located on a loop that varies between the two structures. The same screening methods have been applied to five microbial renal dipeptidase‐like proteins (Sco3058, Gox2272, Cc2746, Lmo2462 and Bh2271) using 55 dipeptide libraries. The renal dipeptidases hydrolyzed a wide range of dipeptides, but respectable substrates have been identified for these enzymes with kcat/KM values on order of 104 M−1s−1 for Bh2271 and 105 M−1s−1 for the other four. The best substrates of Lmo2462, Bh2271, Sco3058, Gox2272 and Cc2746 are L‐Leu‐D‐Ala, L‐Leu‐D‐Ala, L‐Arg‐D/L‐Asp, L‐Asn‐D‐Glu and L‐Arg‐D‐Asp, respectively. The crystal structures of 2 microbial renal dipeptidases, Sco3058 and Gox2272, have been determined. Supported by NIH.
Expression and characterization of active francisella tularensis FabI enzyme, a potential antibacterial target
Authors: Bohn Kelsey C , Chmielowski Jennifer N , Wen Lisa , Huang Jenq‐Kuen , Timsina Yam N , Chand Phani C , Pathak Ashish K
Published: June 2021

Francisella tularensis (F. tularensis) has been identified by the United States Centers for Disease Control (CDC) as one of the top six bacteria that could potentially be used as biological weapons. This research focuses on an enzyme of fatty acid biosynthesis in F. tularensis, namely FabI. FabI is an enoyl‐ACP reductase which catalyzes the last reduction step in each elongation cycle of type‐II fatty acid synthesis. The enzyme has been identified as a potential target for antibacterial drug development. In this research, the gene encoding of FabI was custom synthesized and cloned in pET15b expression vector. The enzyme was over‐expressed in E. coli, purified using Ni‐NTA affinity column, and its activity assayed spectrophotometrically. The pure FabI was tested with a set of indole‐2‐carboxylic acid compounds for their ability to inhibit FabI. We have discovered several new compounds with promising activity against F. tularensis FabI. The best result was obtained with indole‐2‐carboxylic acid analog WIUAKP‐031. The compound at 37.5 μM inhibited 98% of the FabI enzyme with IC50 value of 6 μM. The availability of the functional F. tularensis FabI will greatly aid in structural studies of this essential bacterial enzyme and facilitate the identification of small molecule inhibitors of type II fatty acid synthase.

Supported in parts by seed grants from University Research Council, Western Illinois University.
Crystal structure of histo‐aspartic protease (HAP) from Plasmodium falciparum
Authors: BHAUMIK PRASENJIT , Xiao Huogen , Parr Charity , Yada Rickey , Gustchina Alla , Wlodawer Alexander
Published: June 2021

Histo‐aspartic protease (HAP) from Plasmodium falciparum is involved in hemoglobin degradation by the parasite, and thus offers a promising target for antimalarial drug development. The crystal structures of the recombinant HAP, as apoenzyme and complexes with two inhibitors, pepstatin A and KNI‐10006, have been solved at 2.5, 3.3, and 3.0 Åresolution, respectively. In the apoenzyme crystals HAP forms a tight dimer, not seen before in any aspartic proteases, with the flaps that cover the active sites (residues 70‐83) adopting an open conformation. Unexpectedly, the active site of the apoenzyme was found to contain a zinc ion tightly bound to the two active site residues, His32 and Asp215 from one monomer, and to Glu278A from the other monomer, with the coordination resembling its counterparts in metalloproteases. The flap is closed in the structure of pepstatin A complex and Lys76, uniquely present at the tip of the flap in HAP, interacts with the inhibitor. The observed mode of binding of pepstatin A in the HAP active site disproves the previously proposed hypothesis that HAP is a serine protease. The binding mode of KNI‐10006 to HAP is very unusual compared to other members of the KNI series binding to aspartic proteases. The novel features of the active site of HAP should allow designing specific inhibitors that could be developed into antimalarial drugs.
Glycosylation of cyclooxygenase‐2 (COX‐2) influences effectiveness of COX‐2 inhibitors
Authors: Graham Kamara Whitney , Sevigny Mary B. , Gabriel Bianca S
Published: June 2021

Cyclooxygenase‐2 (COX‐2) is an enzyme that catalyzes the rate‐limiting step in the prostanoid synthesis pathway, which plays an important role in a variety of physiological and pathophysiological processes including inflammation and cancer. COX‐2 exists as two major glycoforms of 72 and 74kDa, the latter resulting from an additional oligosaccharide chain at amino acid residue Asn580. The purpose of this study is to determine if this additional glycosylation affects the inhibitory ability of various COX‐2 inhibitors. COS‐1 cells were transiently transfected with either the wild‐type or Asn580‐mutant COX‐2 gene. Subsets of both cell groups were treated with various concentrations of either aspirin, flurbiprofen, ibuprofen, or celecoxib. After addition of the COX‐2 substrate arachidonic acid to inhibitor‐treated and untreated (control) cells, media was collected and subjected to an ELISA which measured levels of the downstream product prostaglandin E2. Results indicate that, at low concentrations, both aspirin and flurbiprofen are more effective at inhibiting the activity of the 72 than the 74 kDa glycoform, whereas ibuprofen and celecoxib are more effective at inhibiting the 74 kDa glycoform. This indicates that glycosylation of COX‐2 at Asn580 influences the efficacy of COX‐2 inhibitors.

Research supported by the Dept. of Natural Sciences and Mathematics at Dominican University of California.
A Novel Irreversible Peptide Inhibitor to Counteract Botulinum Neurotoxin A (BoNT/A) Poisoning In Vitro
Authors: Andres Devon K. , Keyser Brian , Zhang Peng , Benton Betty , Ray Prabhati , Ray Radharaman
Published: June 2021

BoNT/A light chain (LC) hydrolyzes the vesicle fusion protein SNAP‐25, thus blocking neuroexocytosis and causing flaccid paralysis. A potential therapeutic approach against BoNT/A poisoning is to inhibit the LC‐induced hydrolysis of SNAP‐25 using a short peptide inhibitor. A high affinity synthetic peptide inhibitor, N‐acetyl‐CRATKML‐amide, has been reported. We studied a derivative of N‐Acetyl‐CRATKML, peptide inhibitor #3 (PI3), for its antagonism of SNAP‐25 hydrolysis and neuroexocytosis inhibition due to BoNT/A in the human neuronal M17 cell line. Using SNAP‐25 hydrolysis and high KCl (80 mM) stimulated [3H]‐glycine release assays, we observed that PI3 significantly inhibited BoNT/A effects. We also observed, by Alexa 488 labeling, that PI3 permeates through M17 cell membranes. Additionally, using dialysis and SNAPtide assays, we found that PI3 is an irreversible BoNT/A inhibitor. To our knowledge, this is the first irreversible BoNT/A inhibitor reported. Disclaimer: The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army or the Department of Defense. This research was supported by the DTRA‐JSTO, Med. S&T Div.
Plasminogen Activator Inhibitor‐1 shows metal dependent stabilization
Authors: Thompson Lawrence , Peterson Cynthia
Published: June 2021

Plasminogen activator inhibitor‐1 (PAI‐1) is the major inhibitor of both urokinase (uPA) and tissue plasminogen activator (tPA). PAI‐1 forms a stable complex with these proteases in a suicide fashion. The active conformation of PAI‐1 is metastable and spontaneously converts to a latent form with a half‐life of 1.1 hours at 370C. The half‐life of the active conformation is extended to 1.5 hours when PAI‐1 is bound to the plasma protein vitronectin. However, we have evidence that this stabilization is metal dependent. Our collaborators have shown that human PAI‐1 (hPAI‐1) binds to immobilized metal affinity media charged with Mn, Co, Ni, Cu, or Zn. In this study, we measured the functional effects of metals on hPAI‐1 to determine the relevance of the hPAI‐1/metal interaction. hPAI‐1 was mixed with metal chloride in the absence or presence of added vitronectin. The decay in the amount of active PAI‐1 was measured over time by mixing it with tPA, and then measuring the amount of active/free tPA. Our results show that Mg, Ca, and Mn show modest increases in the life span of hPAI‐1 with and without vitronectin; however, Fe, Co, Ni, and Cu dramatically destabilize hPAI‐1 in the absence of vitronectin while Co, Ni, and Cu show a large stabilizing effect on hPAI‐1 in the presence of vitronectin.
Docosahexaenoic acid (DHA) alters the deformability of murine melanoma cells
Authors: Ullah Sarah Parveen , Williams Eugene
Published: June 2021

We examined the effects of DHA on the deformability of two variants of a murine melanoma cell line. Cells of the B16‐F1 variant are less metastatic than those of the aggressively metastatic B16‐F10 variant. We measured the deformability of cells by assessing their ability to survive forced passage through 8 ?m apertures in polycarbonate. A higher percentage of cells of the F10 variant survived passage (67% ± 5 SEM versus 59% ± 2 SEM, n =7). Dose response curves using oleic acid (OA) and DHA (both in ethanol) revealed that, unlike OA, DHA at concentrations of 0.30 mM induced cell death in both strains. The viability of cells of both strains exposed to 0.075 mM OA or DHA for 8 h remained above 90%. After a 6 h exposure to 0.075 mM DHA the phospholipids of B16‐F1 cells contained DHA as 20% of total fatty acids compared to 6% in control cells. Cells of both melanoma variants treated with DHA in this way showed significantly reduced ability to survive passage through 8 µm apertures. The reduction was statistically equal for both strains. Cells treated with OA or ethanol showed no such change. We conclude that the incorporation of DHA into phospholipids alters the deformability of the cells and that this effect is the same in cells with high or low metastatic capacities. This work was supported by Undergraduate Research Grants from the Henson School of Science and Technology at Salisbury University and by a Guerrieri Summer Research Award.
Biochemical characterization of Calponin homology domain of human IQGAP1
Authors: Kurella Vinodh Babu , Lecour Louis F , Worthylake David K
Published: June 2021

Human IQGAP1 is a190 kDa molecular scaffold that contains several domains required for interaction with proteins such as F‐actin, calmodulin, Cdc42/Rac1, β‐catenin and E‐cadherin. Calponin homology domain (CHD) is a ~160 amino acid module located in the N terminal region of IQGAP1. The CHD belongs to the type 3 family of CH domain containing proteins such as actin‐binding proteins, calponin and Vav. IQGAP1 localizes to the cell cortex, binds to and crosslinks actin via its CHD. When this activity occurs at cadherin‐based cell‐cell junctions, adhesion is stabilized. Calmodulin a calcium sensor, negatively regulates the interaction of IQGAP1 with its binding partners. Preliminary calorimetric analysis suggests that Ca2+ /calmodulin but not apo‐calmodulin (calcium fee) binds to the N‐ terminal region of IQGAP1 containing the CHD. The mode of binding is apparently sequential with two calmodulin molecules binding to one IQGAP1 CHD. We have just obtained crystals of this CHD and are currently screening for crystals of a complex between Ca2+/calmodulin and CHD. It is expected that these structures will reveal conformational changes in the CHD necessary for complex formation and why Ca2+ /calmodulin but not apo‐calmodulin binds to the CHD.

Louisiana board of regents grant
Control of co‐translational protein targeting by self‐regulating GTPases
Authors: Shan Shu‐ou , Zhang Xin , Schaffitzel Christiane , Ban Nenad
Published: June 2021

Co‐translational targeting of proteins to cellular membranes is controlled spatially and temporally by two homologous GTPases in the signal recognition particle (SRP) and the SRP receptor (SR). Intriguingly, SRP and SR represent a growing number of noncanonical GTPases that do not employ the classical 'GTPase switch' mechanism. Instead, we showed that these GTPases undergo a series of discrete conformational changes during their interaction with one another, culminating in reciprocal GTPase activation. Importantly, each conformational change allows SRP and SR to provide a distinct point of regulation during protein targeting. Cargo loading on the SRP and membrane association of SR each accelerates SRP‐SR complex formation over 100‐fold, ensuring the efficient delivery of cargo to the membrane. Subsequent conformational changes in the GTPase complex weakens the affinity of SRP for the cargo, driving the unloading and delivery of cargo to the protein conducting channel. Finally, the cargo also delays GTPase activation in the SRP▸SR complex, creating a valuable time window that can improve the efficiency and fidelity of protein targeting. We propose that the SRP and SR represent a new class of 'self‐regulating' GTPases that can use their intrinsic conformational flexibility to provide the molecular driving force and to improve the fidelity of complex cellular processes.
Establishment of minimal requirements for sustained enterocyte migration in a wound healing model
Authors: Pazin Marina Viktoria , Rudnicki Jean Weston , Caplan Michael S , Jilling Tamas
Published: June 2021

Rat small intestinal epithelial cells (IEC6)were monitored by live cell imaging to identify required components for sustained linear migration. Supplementation of HBSS with L‐glutamine, L‐arginine (4mM each), insulin (0.1mg/ml), sodium pyruvate (1mM) and FBS (5%), sustained linear migration for at least two hours at 0.38±0.04 ?m/min. Without sodium pyruvate, migration rate diminished to 0.26±0.02 ?m/min, but remained linear for 2h. Without FBS, cells migrated for 1 h at 0.34±0.07 ?m/min, but slowed to 0.03±0.002 µm/min in the second hour, correlating with severely reduced lamellipodia formation and with the emergence of actin bundles perpendicular to the direction of migration on the leading edge of most cells, as revealed by phalloidin‐AF488 staining of fixed cells. Fluorescence image analysis of IEC6 transfected with AKTPH‐GFP, i.e., a probe for PIP3, indicated a rhythmic accumulation of PIP3 at the leading edges of lamellipodia, which diminished when cells stopped migrating. It is yet to be determined which serum‐derived factors are responsible for the induction of lamellipodia formation and what is the role of PI3 kinase in this process. Supported: NIH HD37581, DK062960.
In Vivo Identification of Multiple Response Elements Mediating Thyroid Hormone Stimulation of Hepatic HMG‐CoA Reductase Transcription
Authors: Boone Lindsey R. , Ness Gene C.
Published: June 2021

Hepatic HMG‐CoA reductase (HMGR) is the enzyme that catalyzes the rate‐limiting step in cholesterol biosynthesis. Hypothyroidism is a known risk factor for cardiovascular disease. HMGR transcription is decreased in hypothyroid mammals. Treatment of hypothyroid (Hx) Sprague Dawley rats with triiodothyronine (T3) increased HMGR mRNA by approximately 2.7‐fold. We have utilized in vivo electroporation to introduce HMGR promoter‐luciferase constructs directly into the livers of Hx Sprague Dawley rats treated ±T3 in an effort to identify the regions of the HMGR promoter responsible for this T3‐mediated regulation. Electroporation of an HMGR promoter construct inclusive of the ‐325/+70 promoter region resulted in a 2.6‐fold induction of promoter activity in the T3‐treated animal. Mutations in the sterol response element (SRE), nuclear factor‐y site (NF‐Y), and the sequence AGGTCA at ‐316/‐321 identified the necessity of these elements for the T3 response. EMSAs with liver nuclear extracts from +T3 rats showed strong shifted bands with probes to each of these elements. Antibody supershift assays revealed that SREBP‐2 binds to the SRE, NF‐Y binds to the NF‐Y site, and USF‐2 binds to the AGGTCA element at ‐316/‐321. In vivo electroporation of siRNA to SREBP‐2 was shown to reduce HMGR mRNA in normal Sprague Dawley rats to an average of 60% of control. These data suggest that the SRE, NF‐Y site, ‐316/‐321 element and their corresponding binding proteins appear to mediate the T3 response. (This investigation was supported by NIH grant DK075414).
Cyclin‐dependent kinase‐mediated phosphorylation of yeast phosphatidate phosphatase
Authors: Morgan Jeanelle M , Carman George M
Published: June 2021

The yeast PAH1‐encoded phosphatidate (PA) phosphatase catalyzes the dephosphorylation of PA to yield diacylglycerol and Pi. The diacylglycerol generated in this reaction is used for the synthesis of the storage lipid triacylglycerol and for the synthesis of the membrane phospholipids phosphatidylcholine and phosphatidylethanolamine via the Kennedy Pathway. PA phosphatase is phosphorylated by cyclin‐dependent kinase (Cdk). Data indicate that Cdk phosphorylation down‐regulates PA phosphatase function. PA phosphatase is also dephosphorylated by the Nem1‐Spo7 complex, which results in an up‐regulation of PA phosphatase function. In this work, we examined the effects of phosphorylation and dephosphorylation of PA phosphatase on its cellular location, and on the composition of phospholipids and neutral lipids. In wild type cells, PA phosphatase is about equally distributed between the cytosolic and membrane fractions of the cell. A defect in Cdk phosphorylation of PA phosphatase caused an increase in the amount of enzyme associated with membranes, whereas a defect in the dephosphorylation of PA phosphatase caused a decrease in membrane association. Cdk phosphorylation of PA phosphatase caused a decrease in triacylglycerol content and an increase in phospholipid content. Conversely, dephosphorylation of the enzyme had the opposite effects. Supported by NIH grant GM 28140.