bluuebunny/crossref_metadata_2025 huggingface dataset:
MicroRNA Expression Profile in Bronchopulmonary Dysplasia
Authors: Bhaskaran Manoj , Wang Yang , Breshears Melanie A , Liu Lin
Published: June 2021

MicroRNAs (miRNAs) are small RNAs with a length of ~21‐24 nucleotides. They are endogenous in origin and regulate the expression of proteins post‐transcriptionally. miRNA expression profile in a disease provides valuable insights into the pathogenesis of that disease and possible therapeutic strategies. Bronchopulmonary dysplasia (BPD) is the chronic lung disease manifested in premature infants and has multifactorial etiology ranging from hyperoxia exposure to premature lung to neonatal therapeutics. Advancement in treating premature infants using new management and therapeutic strategies has changed the course of BPD pathogenesis. In this study we used a 'new' BPD model where the primary lesions have shifted from chronic inflammatory responses, as seen in 'old' BPD models, to actual dysplasia. Infant rats were exposed to 95% oxygen from postnatal day 3 to day 14. The time point selected showed changes in the lung that mimicked the new BPD pathology. Small RNA fraction was isolated and miRNA microarray was performed on BPD and control samples. We found that five miRNAs, miR‐342, miR‐335, miR‐150, miR‐126* and miR‐151* were down‐regulated and three miRNAs, miR‐21, miR‐141 and miR‐34a were up‐regulated in the BPD lung. Most of these miRNAs have target proteins vital to normal lung development and the study thus provide us with new insights into miRNA‐mediated regulation of BPD pathogenesis.
Assessing biology students' critical thinking abilities using a skills‐based classification model
Authors: Walton Kristen L.W. , Rybarczyk Brian J.
Published: June 2021

Our upper‐division course, Molecular Basis of Disease, requires students to synthesize content from disciplines ranging from physiology to molecular biology and genetics. Analysis of data from primary literature is emphasized to engage students with the scientific process. This requires students to know a considerable amount of technical information, experimental techniques, and basic biological concepts. We find that students' knowledge of experimental techniques such as RT‐PCR, western blot, and microarrays varies substantially. Therefore, we designed a pre‐ and post‐test instrument based on a model for classifying students' skills to measure their abilities at analyzing scientific data and improvement of these skills over time. The instrument addressed basic data observation abilities, identification of appropriate controls, interpretation of data from multiple experiments, and ability to predict results from experiments or propose follow‐up experiments. Students' scores on the pre‐test ranged from 40% to 90% correct, with synthesis and prediction questions the most frequently missed. Individual student gains in data analysis skills were determined by comparing pre‐test and post‐test scores. We propose that integration of primary literature in upper‐division courses can improve students' data analysis skills and promote analytical and critical thinking about scientific content.
The influence of pedagogy on test performance: preliminary data
Authors: Janssen Herbert Frederick , Osborne David Lee , Skeen Nicholas P
Published: June 2021

Progressive teaching styles in medical education have recommended changes for many years but change has been slow to materialize. Didactic lecture has retained a stronghold on medical education despite a century of recommendations that new approaches be implemented. The hypothesis of the current study assumed that presentation style would not alter the exam performance of students enrolled in the Joint Admission Medical Program (JAMP) of Texas. Test questions written to span the breadth of the cognitive domain were separated using a blinded stratified randomized approach. One exam was administered before class (pre exam), one after a typical didactic lecture (post didactic), and one after a class meeting where student working in groups discussed the material (post discussion). The results suggested that didactic lecture improved student performance on lower cognitive domain questions while group work improved performance on those questions written in a scenario format. Combining the two approaches reduced performance on the scenario style questions. Additional work is needed to better define the influence of pedagogy on test performance. Supported by FIPSE grant awarded to BYU (Bill Bradshaw and John Bell)
Science Faculty with Education Specialties: A Cross‐Disciplinary Research Study
Authors: Pelaez Nancy J , Bush Seth D , Rudd James A , Stevens Michael T , Tanner Kimberly D , Williams Kathy S
Published: June 2021

Efforts to improve science education in the US include university science departments hiring Science Faculty with Education Specialties (SFES), scientists who take on specialized roles in science education in their discipline. A cross‐disciplinary research study was conducted by surveying faculty who are SFES within the California State University. Two subpopulations were identified, those hired as SFES and those who transitioned into these roles. SFES reported more training in basic science than in science education. Many reported a lack of institutional support for scholarly activities that included efforts to improve K‐12 teacher preparation, reform undergraduate science education, conduct discipline‐specific science education research and/or basic science research. Almost 40% of SFES were seriously considering leaving their positions. Of those considering leaving, most were ready to leave their position, but not the field. Findings indicate that attention towards SFES training pathways, issues of professional isolation, and networks of support for scholarly activities would likely strengthen this model and its impact on science education. The career dynamics reported from this study of science faculty with interests in education point the way for developing this nascent career specialty.
Characterization of abnormal angiogenesis in B16BL6 melanoma
Authors: Kitahara Shuji , Itagaki Hiroko , Ezaki Taichi
Published: June 2021

[Aim] Since the progression and metastasis of malignant tumors depend on their local microcirculation, we sought to characterize the tumor angiogenesis in a highly metastatic mouse melanoma model, B16BL6 (B16), injected with Matrigel into syngeneic C57BL/6 mice. [Materials & Methods] B16 cells in Matrigel were injected into the dosal subcutis of mice. After various times, the tumor baring mice were injected FITC‐conjugated tomato lectin intravenously to label all blood vessels 10 min before being perfused with 4% PFA. The tumor tissues were removed and either frozen or epon‐embedded for subsequent analyses. For the 3‐dimentional imaging by the confocal laser‐scanning microscopy and transmission electron microscopy, cryosections were made and the imunofluorescent staining for various markers was performed. [Results & Discussion] We found that B16 with Matrigel grew significantly faster than B16 alone and was accompanied by altered tumor angiogenesis. The tumor vessels grew vigorously in the opposite direction to the tumor without invading into the tumor mass until at least day 10. In addition, the vascular branching did not result from sprouting as was seen in B16 alone without Matrigel, but resulted from vascular splitting due to either compression from outside of the vessels or septum formation by endothelial cells. This phenomenon was characteristic of B16 cells, but not of other metastatic tumor cells tested, such as a Lewis lung carcinoma cell line and a hybridoma cell line (ASH‐1). The reduction of various angiogenic factors from Matrigel did not affect the angiogenic patterns and tumor growth. However, the denaturation of Matrigel by repeated freezing and thawing did abolish the peculiar angiogenic patterns of B16 with intact Matrigel. We conclude that the tumor vessels may vigorously alter their angiogenic patterns depending on the local microenvironment.
PI3K and RhoA/ROCK pathways play a critical role in hypoxia‐induced ATP exocytosis and ATP‐induced angiogenic responses in pulmonary artery vasa vasorum endothelial cells
Authors: Gerasimovskaya Evgenia , Woodward Heather , Riddle Suzette , Anwar Adil , Stenmark Kurt R
Published: June 2021

We recently reported that angiogenic vasa vasorum expansion occurs in the pulmonary artery adventitia of chronically hypoxic calves, and that extracellular ATP exerts dramatic angiogenic effects on vasa vasorum endothelial cells (VVEC) in vitro. In this study we tested the hypothesis that VVEC may represent a source of extracellular ATP in the PA wall, and that PI3K/Rho/ROCK pathway contributes to hypoxia‐induced ATP release and angiogenic activation of VVEC. We found that hypoxic exposure (1‐3% O2, 10‐60 min) resulted in a 3‐8 fold increases in ATP accumulation in VVEC conditioned media in a time‐ and O2‐dependent manner. Inhibitors of vesicular transport (monensin, brefeldin A, and N‐ethylmaleimide) attenuated this response by 40‐72%, suggesting that in VVEC, ATP release occurs, at least in part, through regulated exocytosis. Both hypoxia and ATP activated PI3K and RhoA by several‐fold and induced translocation of RhoA and ROCKII from the cytosolic to the membrane fraction. Using pharmacologic and genetic approaches, we demonstrated that PI3K, RhoA and ROCKII play a key role in hypoxia‐induced ATP release and in ATP‐induced proliferation, migration and tube formation of VVEC. Together, our findings provide novel evidence for the molecular mechanisms that link hypoxia‐induced increases in extracellular ATP and the vasa vasorum neovascularization observed in the PA wall of chronically hypoxic animals.
Soluble VEGF isoforms are required for the maintenance of the retinal pigment epithelium (RPE)‐choriocapillaris complex in the adult
Authors: Saintgeniez Magali , Kurihara Tomoki , Sekiyama Eiichi , Maldonado Angel , D'Amore Patricia
Published: June 2021

VEGF in mice is produced as three isoforms, VEGF120, VEGF164 and VEGF188, that differ in their ability to bind heparan sulfate proteoglycan. RPE normally produces only the more soluble isoforms, VEGF120 and VEGF164. We investigated the effect of the absence of soluble VEGF isoforms during early eye development and in the adult. While eye development of mice expressing only VEGF188 (VEGF188/188 mice) appeared normal, TEM analysis revealed age‐dependent defects of the RPE/choroid complex. VEGF188/188 mice displayed changes in the RPE and choroids, characterized by choriocapillaris loss and RPE dysfunction. These degenerative lesions progressed with age with the formation of drusen‐like deposits, RPE apoptosis and choroidal vessel atrophy. These defects were associated with a deficit in visual function evidenced by ERG. The absence of eye defects in embryonic and postnatal VEGF188/188 mice indicate that soluble VEGF isoforms are not required for proper eye development. However, the age‐dependent degenerations observed in VEGF188/188 mice suggest a role for VEGF signaling in the maintenance of the RPE‐choroidal complex. We speculate that in VEGF188/188 mice, Bruch's membrane compromises the diffusion of RPE‐derived VEGF. Reduced VEGF signaling in the choriocapillaris may contribute to the observed abnormalities that recapitulate many of the clinical features of dry AMD.
Activation of Hypoxia‐Inducible Factor‐1 by Sphingosine‐1‐Phosphate in vascular cells
Authors: Michaud Dumont Maude , Gratton Jean‐Philippe , Richard Darren
Published: June 2021

Sphingosine‐1‐phosphate(S1P) is a bioactive phospholipid playing a key role in angiogenesis by promoting a variety of vascular cell events. Hypoxia‐inducible factor‐1(HIF‐1), a transcriptional activator of many genes essential for adaptation to lowered oxygen levels, is also a major angiogenic inducer. Interestingly, S1P and HIF‐1 are both implicated in different cardiovascular diseases and in tumor progression. Recent studies have shown that non‐hypoxic stimuli can activate HIF‐1 in oxygenated conditions. Here, we attempt to determine whether S1P can modulate the vascular activation of HIF‐1. We show that treatment of cultured vascular cells with S1P strongly increases HIF‐1α but not HIF‐2α protein levels, through VHL‐independent stabilization of HIF‐1α. Using pharmacological inhibitors we show that elevation of HIF‐1α expression occurs through the S1P2 receptor. We demonstrate, that the HIF‐1 nuclear dimer formed upon S1P stimulation, is transcriptionally active and specifically binds to hypoxia‐responsive elements. Moreover, in smooth muscle cells, S1P regulates genes via HIF‐1, since the transcripts are diminished when HIF‐1α is silenced. Our results therefore identify S1P as a potent inducer of HIF‐1. We believe that the discovery of novel activators of HIF‐1, such as S1P, and comprehension of subsequent downstream events will have a strong impact on different aspects of vascular biology.

This work is supported by the Canadian Institutes of Health Research
Kruppel Like Factor 15 is a Critical Regulator of Angiotensin II Mediated Vascular Remodeling
Authors: Lu Yuan , Haldar Saptarsi , Hao Caili , Cui Yingjie , Doughman Yongqiu , Watanabe Michiko , Jain Mukesh K
Published: June 2021

Kruppel Like Factor 15 (KLF15) is a transcription factor expressed in smooth muscle (VSMC) from a diverse array of vascular beds. However, the functions of KLF15 in VSMC are poorly understood. The aim of this study was to study the role of KLF15 in Angiotensin II (Ang II) mediated vascular remodeling. We employed a model in which Ang II was infused via subcutaneous osmotic minipumps at 1440ug/kg/day for 2 weeks in wild type (WT) and KLF15‐/‐ mice (age 10‐12 weeks). KLF15 expression was significantly reduced in the thoracic aorta (TA) of WT mice stimulated with Ang II. While WT mice developed aortic medial hypertrophy and perivascular fibrosis in response to Ang II, KLF15 ‐/‐ mice developed a severe degenerative aortopathy characterized by gross outward remodeling, excessive vascular inflammation, elastin lamellar disruption, reactive oxygen specious (ROS) accumulation, and apoptotic SMC loss. KLF15 ‐/‐ aorta have exaggerated expression of monocyte chemoattractant protein‐1(MCP‐1) and matrix metalloproteinase‐3 (MMP3) as well as exaggerated accumulation of p53 and its target genes (p21, bax). Promoter and ChIP studies demonstrate that KLF15 can inhibit MEF2 activity and its occupancy of the MCP‐1 promoter. Our results support a critical role for KLF15 in the regulation of Ang II mediated vascular inflammation and remodeling.

Supported by NIH R01HL084154
IN VITRO CAPILLARY ENDOTHELIAL TUBES AS A MODEL SYSTEM TO STUDY CYTOTOXICITY OF DIESEL PARTICLES
Authors: Chao Ming Wei , Koslosky John , Po Iris P , Gerecke Donald R , Svoboda Kathy KH , Laumbach Robert , Gordon Marion K
Published: June 2021

Epidemiological studies report acute exposure to diesel exhaust particles (DEP) correlates with an increase in myocardial infarctions within 48 hr. Inhaled DEP gain access to the bloodstream, but how is unknown. We have used human umbilical vein endothelial cells (HUVECs) assembled into in vitro capillary tubes to study the action of DEP. Increasing concentrations of DEP (1‐100 µg/ml) at sizes aound 2.5 um (PM2.5) increase tube cell death, as determined by LDH and TUNEL assays. DEP can be found on tubes and in the lumen of tubes in culture. Endothelial cell‐cell adherens junctional components redistribute away from the plasma membrane after a 24 hr DEP exposure, as assessed by confocal microscopy with vascular endothelial cadherin antibody. Westerns of culture medium demonstrated increased secretion of VEGF‐A with increasing doses of DEP, favoring vascular permeability. Endothelial capillary tubes produce nitrite oxide (NO) and reactive oxygen species (ROS) in response to DEP, and heme oxygenase 1 (HO‐1) and thioredoxin reductase are upregulated . Furthermore, inflammatory markers IL‐1 beta; and IL‐6 are increased in a dose dependent manner as assessed by ELISA. In summary, our in vitro capillary endothelial tube data suggest that DEP make capillaries leaky by disrupting endothelial adherens junctions and inducing secretion of VEGF‐A. The production of potentially damaging ROS is also induced by DEP.

Grant Funding Source

NIEHS P30ES005022.
FREQUENCY OF CORONARY ARTERY PREDOMINANCE IN HUMAN HEARTS
Authors: Swamy Kaki Bale
Published: June 2021

Materials & Methods

30 hearts were collected from the cadavers .They were carefully dissected and examined for the type of pattern of coronary vasculature. A dye was also injected for visualization of the coronary blood vessels and observed that how many hearts are of right predominance and how many are of left predominance. Like wise 460 coronary angiograms were also studied and noted the fact that how many hearts belongs to "Right predominance and how many hearts are of "left predominance".

Summary& results

The study with 30 cadaver hearts revealed that 90% of hearts are of right coronary predominance and 10% of them showed left predominance. From 460 angiograms of the live hearts of the patients attending the hospitals showed 85% ( 391) of right coronary artery predominance while 15% ( 69 ) of left coronary artery predominance. Among the smokers, 37% had Single vessel disease (SVD), 29% had Double vessel disease (DVD),24% had Triple vessel disease (TVD) and 10% of them had no significant cardiac disease. Among the hypertensive patients had 38% SVD, 29% DVD, 22% TVD and 11% had no significant cardiac disease. Among the diabetic patients had 38% SVD, 28% DVD, 25% TVD and 9% had no significant cardiac disease.

Conclusion

The right coronary artery is the predominant artery in most cases according to this study.
Effective detection of histochemical reaction products by phase contrast electron microscopy
Authors: Usuda Nobuteru , Atsuzawa Kimie , Nagayama Kuniaki
Published: June 2021

Effort has been made for sensitive detection of reaction products in histochemistry at electron microscopic level. Phase contrast electron microscopy utilizing phase plates has been considered suitable for high contrast observation of weak phase objects. This novel technique was newly applied to histochemically stained strong phase objects of osmificated biological specimens. Sections of various thicknesses, specifically stained for the Golgi apparatus by the ZIO technique using the heavy metals Zn and Os, were observed with a phase contrast electron microscope in Zernike and Hilbert imaging modes. Quantitative analysis of image contrast in real space and the power spectrum in Fourier space showed a high contrast gain even for strong phase objects. This result indicates that phase contrast electron microscopy is useful for effective detection of reaction products in histochemistry.
Periostin gene expression is regulated by the bHLH transcription factor TWIST2 in human skin fibroblasts
Authors: Franco Hector , Casasnovas José J , Cadilla Carmen Lydia
Published: June 2021

Periostin (POSTN), a cell adhesion protein involved in connective tissue development and maintenance, is regulated positively by the basic Helix‐Loop‐Helix (bHLH) transcription factor TWIST2. Two naturally occurring nonsense mutations of TWIST2 (Q119X and Q65X) were found in Puerto Rican and Omani Setleis Syndrome (SS) patients. The Yeast‐Two‐Hybrid assay was used to assay the dimerization properties of TWIST2 mutants. Wildtype and Q119X forms of TWIST2 form homodimers as well as heterodimers with E12 (a ubiquitous bHLH factor), while the Q65X TWIST2 mutant lacks ability to dimerize with itself and with E12. Microarray analysis of human skin fibroblasts suggests that TWIST2 seems to act as a positive regulator more frequently than as a repressor in human skin fibroblasts. POSTN is down‐regulated in SS patient fibroblasts when compared to normal controls, as confirmed by real time RT‐PCR. RNAi and TWIST2 over‐expression techniques provide evidence that POSTN is positively regulated by TWIST2. Knockdown of TWIST2 shows a decrease of POSTN expression over a 72hr time course in fibroblastic cells. Similarly, TWIST2 over‐expressed in these same cells results in increased POSTN expression over a similar time course. Overexpression of Twist2 in SS patient skin fibroblasts caused an increase in POSTN gene expression. This research is supported by MBRS RISE Grant R25GM061838, and RCMI Grant G12RR03051.
An NF‐kB‐regulated micro‐RNA‐146a‐mediated inflammatory circuit in Alzheimer's disease brain
Authors: LUKIW WALTER J , CUI JIAN‐GUO , ZHAO YUHAI
Published: June 2021

Human brains utilize discrete populations of micro RNA (miRNA) that normally support homeostatic brain gene expression functions. The abundance of these miRNAs are significantly altered in neurological disorders such as Alzheimer's disease when compared to age‐matched controls. Here we provide evidence in Alzheimer's disease brains of a specific up‐regulation of an NF‐kB‐sensitive miRNA‐146a highly complementary to the 3' un‐translated region (3'UTR) of complement factor H (CFH), an important repressor of the brain's inflammatory response. Up‐regulation of miRNA‐146a coupled to down‐regulation of CFH was observed in both Alzheimer's disease brain and in IL‐1β, Aβ42 and/or oxidatively‐stressed human neural cells in primary culture. Transfection experiments using an NF‐kB‐containing pre‐miRNA‐146a promoter‐luciferase reporter construct in stressed human neural cells showed significant up‐regulation of luciferase activity that paralleled decreases in CFH gene expression. Parallel treatment of these stressed cells with the NF‐kB inhibitors pyrollidine dithiocarbamate (PDTC) or the resveratrol analog CAY10512 abrogated this response. Incubation of an antisense oligonucleotide to miRNA‐146a (anti‐miRNA‐146a; AM146a) was found to restore CFH expression levels. These data indicate that NF‐kB‐sensitive miRNA‐146a‐mediated modulation of CFH gene expression may in part regulate an inflammatory response in Alzheimer's disease brain and in cultured cell models of AD, and underscores the potential for anti‐miRNAs as an effective therapeutic strategy against pathogenic inflammatory signaling.
Clustering of cis‐acting regulatory sequences predicts maternal transcript regulation in nematode embryos
Authors: Ryder Sean P , Farley Brian M , Pagano John M , Stefani Robert
Published: June 2021

Post‐transcriptional regulation of gene expression governs patterning events in nematode embryogenesis. Several RNA‐binding proteins guide these events by regulating the translation and/or stability of critical maternal transcripts. They are inherited to different extents in the daughters of each round of division, suggesting that their relative concentration controls which mRNAs are activated or destroyed in a given cell. In order to predict the network of transcripts regulated by each protein, we determined the sequence specificity of three of them using quantitative methods. Each protein binds to a unique consensus varying between 6‐8 nucleotides. The binding sites are more abundant than predicted based on the observed dinucleotide frequency. The limited specificity, combined with the prevalence of binding sites, suggests that the presence of a single site is not sufficient to predict a regulatory relationship. In contrast, a map of the consensus sequences to a database of maternal transcripts reveals several mRNAs that contain binding site clusters. There is excellent correlation between the presence of binding site clusters and regulated expression in the germline and the embryo. Thus, we predict that cooperative and/or competitive association of multiple RNA‐binding proteins is required to select a specific maternal transcript for spatial and temporal regulation.
Human αB‐Crystallin suppresses the aggregation in vitro of partially folded intermediates of human γC‐, γD‐, and γS‐Crystallins
Authors: Acosta Ligia Isabel , King Jonathan
Published: June 2021

α‐crystallin, a small heat shock protein chaperone, is one of the ubiquitous crystallins in the vertebrate lens, along with the ??‐crystallins. It is a polydisperse complex of ~ 800 kD consisting of two subunits (~20 kD) αA‐ and αB‐crystallin (αA‐ and αB‐crys). Its chaperone activity involves suppressing aggregation by binding aggregation‐prone species. Aggregates isolated from mature‐onset cataracts, the major cause of blindness worldwide, contain damaged and misfolded forms of ??‐crystallins. Human ?D‐ and ?C‐crystallin (H?D‐ and H?C‐crys) are stable, long‐lived ?‐crystallins in the lens nucleus, while ?S‐crystallin (H?S‐crys) is abundant in the lens cortex. All three ?‐crystallins can refold in vitro to their native state after unfolding in high concentrations of GdnHCl. Refolding unfolded H?C‐, H?D‐, or H?S‐crys to very low GdnHCl concentrations (< 1 M) resulted in partially folded intermediates partitioning between productive refolding and aggregation pathways. H?D‐, H?C‐ or H?S‐Crys protein was allowed to refold and aggregate in the presence of HαB‐Crys at different monomer‐to‐monomer ratios. H?D‐ and H?C‐Crys aggregation was suppressed to similar levels, whereas H?S‐Crys aggregation was not suppressed as well under identical conditions in assays measuring solution turbidity at 350 nm. These results provide a model for how α‐crystallin interacts with aggregation‐prone substrates in vivo
Gene expression and functional characterization of two multicopper oxidase genes in the malaria mosquito, Anopheles gambiae
Authors: Lang Minglin , Gorman Maureen J. , Gardner Stewart G. , Yungeberg Sandi L. , Kanost Michael R.
Published: June 2021

The multicopper oxidase (MCO) family of enzymes includes laccases, which oxidize a broad range of substrates including diphenols, and several oxidases with specific substrates such as iron or copper. MCOs have been implicated in a diverse set of physiological processes in fungi, plants and bacteria; however, little is known about the function of MCOs in invertebrates. Our goal is to determine the functions of MCOs in the malaria vector mosquito, Anopheles gambiae, by analyzing gene expression patterns, enzyme activity and the effect of gene silencing. MCO1 and MCO3 were upregulated in the midguts and Malpighian tubules of adult females following a blood meal, and they were upregulated in other tissues in response to an immune challenge. These results suggest that MCO1 and MCO3 may function in detoxification and/or immunity, possibly by oxidizing ferric iron. We have purified recombinant MCO1 and MCO3 and have begun to test their activity with a set of possible substrates including o‐ and p‐ diphenols, iron, tannin and flavonoids. Both enzymes oxidize diphenols, but their substrate specificities are not identical. RNAi‐mediated gene silencing will be used to determine whether the enzymes function in detoxifying iron or in defense against microbes. This work was supported by NIH Grants R03AI057816 and R01AI070864.
Biochemical Characterization of Toxoplasma gondii Mitochondrial Type II NADH Dehydrogenase Isoform I as a potential Drug Target
Authors: Lin San San , Kerscher Stefan , Bohne Wolfgang
Published: June 2021

Mitochondria of apicomplexan parasites such as Plasmodium falciparum and Toxoplasma gondiidisplay significant differences compared to human mitochondria, which can exploited for drug development. T. gondii lacks complex I in the respiratory chain, but expresses two "alternative" NADH dehydrogenases, which are localized to the mitochondrion as shown by epitope tagging. The absence of such "alternative" NADH dehydrogenases in the human host defines these enzymes as promising drug targets. Functional expression of TgNDH2‐I in the yeast Yarrowia lipolytica as an internal enzyme, with the active site facing the mitochondrial matrix, permitted growth in the presence of the complex I inhibitor DQA. Bisubstrate kinetics of TgNDH2‐I measured within Y. lipolytica mitochondrial membrane preparations were in accordance with a ping‐pong mechanism. Using inhibition kinetics we demonstrated here that 1‐hydroxy‐2‐alkyl‐4(1)quinolones with long alkyl chains of C(12) (HDQ) and C(14) are high affinity inhibitors for TgNDH2‐I, while compounds with shorter side chains, C(5) and C(6) displayed significantly higher IC50 values. The efficiency of the various quinolone derivatives to inhibit TgNDH2‐I enzyme activity mirrors their potential to inhibit parasite replication in vitro, suggesting that derivatives with a long acyl site chain are promising new compounds against apicomplexan parasites.
Characterization of HIV‐1 subtype C I50V and I50L proteases
Authors: Bracho‐Sanchez Edith Rosvely , Fernandez Marty , Koch Sandra , Marzahn Melissa , Coman Roxana M , Dunn Ben M
Published: June 2021

Last year alone there were approximately 2.5 million new HIV infections; contributing to the total estimate of 33.2 million people infected with HIV worldwide.

HIV is classified into different groups, types, subtypes, and recombinant forms. Subtype C alone accounts for 56% of infections. The HIV protease (PR) is crucial in the replication of the virus as it cleaves precursor viral proteins into their mature forms, an essential step in viral maturation. In addition to subtypes and recombinant forms, variability of the HIV PR arises from drug resistance development, as drugs currently used in clinical treatment of HIV include protease inhibitors (PIs).

Our studies have focused on introducing the mutations I50L and I50V to the HIV‐1 subtype C PR. These mutations arise upon clinical treatment with the PIs atazanavir and amprenavir, respectively. The mutant PRs have been characterized by both kinetic and crystallographic studies. The HIV‐1 C I50V PR showed an eighteen fold increase in Ki for amprenavir, and an even greater increase in Ki for lopinavir. The HIV‐1 C I50L PR on the other hand, showed a thirteen fold increase in Ki for the PI atazanavir, and a nine fold increase for lopinavir.

We have also conducted crystallization studies of both mutant proteases complexed with amprenavir, atazanavir, lopinavir, and topinavir under conditions previously published by our lab. The HIV‐1 C I50V and I50L proteases both diffracted at resolutions of around 2Å.

Kinetic data from our studies will hopefully impact clinical treatment of patients with the HIV‐1 C virus. In addition, structural data obtained from the crystallographic studies will have an effect on future drug design.
Novel Scaffolds For Inducing Allosteric Inhibition of Coagulation Enzymes
Authors: Desai Umesh R
Published: June 2021

We have discovered novel scaffolds, called sulfated DHPs, which possess very interesting anticoagulant properties. In clotting assays, sulfated DHPs prolong PT at concentrations 2‐6‐fold below that of the clinically used LMWH enoxaparin, while in the APTT assay they required 2‐6‐fold higher concentration. Whole blood studies using thromboelastography and hemostasis analysis system reveal that sulphated DHPs inhibit clotting with potency only 18‐30‐fold weaker than enoxaparin. The new anticoagulants inhibit thrombin and factors Xa and XIa of the coagulation cascade with nM potency. Mechanistically, this potent inhibition arises primarily from direct inhibition of the enzymes through an allosteric disruption of the catalytic apparatus. Competitive binding studies show that the sulfated DHPs interact with exosite II of thrombin, a site not known to be associated with inhibition. Studies using A549 lung and HepG2 liver cell lines show no induction of toxicity by sulfated DHPs at concentrations as high as 50 mg/L. Rational drug design based on the sulphated DHP scaffold led to the synthesis of a small sulfated molecule, which exhibits good inhibition of thrombin & factor Xa. The novel mechanism of action and the new scaffold of these molecules suggest a strong possibility for discovering new therapeutically effective structures.
Alternative fuel metabolism in lung cancer by metabolic phenotyping
Authors: Okar David Anthony , Sadiq Ahad A , Kratzke Marian , Kratzke Robert
Published: June 2021

High glycolytic rate and proportioning of pyruvate to lactate (fermentation), even in the absence of hypoxia, are characteristic of transformed cells. This Glycolytic Phenotype (GP) is most beneficial at the leading edge of a developing tumor. In the trailing edge, cancer cells compete among themselves and the GP offers no clear advantage. We hypothesize that interior cells will benefit from an ability to mobilize alternative fuels, especially in response to glucose deprivation. To identify which alternative fuels human lung cancer cells can oxidize in the face of glucose deprivation we used alternative fuel microarray screening of H441, H522, H2009 and non‐malignant HBEC cultures. The results clearly demonstrate differences in alternative fuel use among lung cancer cell lines, as well as in comparison to the HBEC cultures. This strongly suggests the existence of cancer‐specific alternative fuel phenotypes (AFPs) and supports our hypothesis. Parallel studies indicate that differential changes lactate metabolism in response to glucose depletion are a critical aspect of the AFPs observed in lung cancer cell lines. The results encourage our continued effort to identify cancer‐specific regulatory mechanisms that work to provide the neoplasm the ability to readjust fuel metabolism in response to signals from the microenvironment.
EGFR ligand‐mediated activation: insights from a quantitative study in mammalian membranes
Authors: Li Edwin , Placone Jesse , Hristova Kalina
Published: June 2021

The activation of receptor tyrosine kinases occurs via lateral dimerization in the plasma membrane, which enables the cross‐phosphorylation of the two receptors in the dimer. Ligands are important regulators of this process, exerting their regulation by stabilizing the dimers. Activation is further regulated by various cellular processes which are not very well understood. A question arises if the complex process of RTK activation in mammalian membranes can be modeled, and predicted based on the laws of mass action, as a function of receptor and ligand concentration. Here we investigate whether this physicochemical approach is successful in describing the activation of EGFR in the presence of EGF and TGF‐α . We show that the apparent deactivation constants for EGF and TGF‐α are 1.08±0.33 x 103 nM3 and 2.17±0.59 x 103 nM3, respectively. These are the first quantitative measurements of the potency of EGF and TGF‐α in EGFR activation, and are consistent with previous binding measurements. We further show that measurements of active receptor fraction are very well described by an apparent equilibrium between inactive monomers and active dimers, suggesting that a physicochemical description of ligand binding and phosphorylation is sufficient to describe and predict EGFR activation data based on Western blotting.
Conformational specificity in allosteric signaling: high‐throughput measurement of modular secondary structural changes within human Eg5 kinesin
Authors: Kim Sunyoung , Buckley Rebecca , Learman Sarah , Richard Jessica , Kim Elizabeth , Wojcik Edward , Walker Richard
Published: June 2021

The motor domain of human Eg5 kinesin (HsEg5) displays an allosteric pocket at the extracellular surface; binding of monastrol or S‐trityl‐L‐cysteine (STC) to this allosteric site, created by the L5 loop, and subsequent distal conformational changes impairs ATP hydrolysis and cellular function. We examined the roles of E116 and E118 in the L5 loop in allosteric signaling. First, there is a positional correlation on the outcome of amino acid substitutions, irrespective of its nature. Single‐site substitution of E118 increased basal ATP hydrolysis rates of Eg5, whereas substitution of E116 resulted in lower ATPase rates. Thus, sequence variation at residues 116 and 118 of kinesin L5 loop can drive upregulation and downregulation of ATP hydrolysis, respectively. From high‐throughput measurements of the secondary structure composition of proteins in solution, vibrational signatures from upregulated proteins were distinct from the net changes measured from downregulated proteins. Moreover, spectral changes found for E116 variants were similar to those of wildtype Eg5 with monastrol or STC. Therefore, allosteric inhibition by amino acid substitution or by small molecules both result in convergent steady‐state changes to the secondary structure of the Eg5 motor domain in solution, and these rapid methods will provide insight into how long‐range structural changes impact motor function.
Mice bearing a targeted inactivation of nBmp2 show decreased muscle strength
Authors: Schmidt Alina D , Loganathan Sampath K , Adam Mike M , Nichols Caitlin A , Mayo Jaime L , Felin Jenny E , Capecchi Mario R , Barrow Jeffery R , Bridgewater Laura C
Published: June 2021

An important growth factor in the body, bone morphogenetic protein (Bmp2) contributes to mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. We have recently discovered a novel variant of this protein in the nucleus (nBmp2). This protein is produced by translation from a downstream alternative start codon and is localized to the nucleus by means of a bipartite nuclear localization signal. To determine the function of nBmp2, a mouse was engineered in which the conventional Bmp2 growth factor is still expressed normally, but the nuclear variant, nBmp2, cannot be translocated to the nucleus. The mice appear phenotypically normal at birth and are fertile. A series of flexed limb‐hang strength tests, however, revealed that mutant mice have significantly reduced strength compared to wild type mice. Western blot analysis performed on mitochondrial extracts showed increased amounts of cytochrome C in mutant compared to wild type muscle mitochondria. These results suggest that nBmp2 may affect muscle strength by playing a role in mitochondrial function. This work was supported by NIH grant AR48839 and by an award from the Fulton Family Foundation to promote undergraduate research.
Raf kinase inhibitory protein protects cells against locostatin‐mediated inhibition of migration
Authors: Shemon Anne Nataley , Clark Mathew C , Heil Gary , Granovsky Alexey , Eves Eva , Zeng Lingchun , Imamoto Akira , Koide Shohei , Rosner Marsha R
Published: June 2021

Raf Kinase Inhibitory Protein (RKIP), a member of the Phosphatidylethanolamine Binding Protein family, is a negative regulator of the Raf‐1/MAPK pathway. Recently, locostatin was reported to bind RKIP and inhibit cell migration by a mechanism involving Raf kinase and RKIP. We have analyzed the interaction of RKIP with locostatin and examined the biological consequences of locostatin binding to RKIP. NMR studies [SK1]showed that a locostatin precursor binds the conserved PE binding pocket of RKIP with no effect on its association with Raf‐1, nor phosphorylation by PKC at a site that prevents RKIP interaction with Raf‐1. Moreover, exposure of wild type, RKIP‐depleted HeLa cells or RKIP‐deficient MEFs[SK2] to locostatin had no effect on MAP kinase activation. However, locostatin induced cytoskeletal defects and spindle abnormalities and resulted in inhibition of cell migration following wounding which was exacerbated in RKIP deficient MEFs. These results show that locostatin binds to the RKIP ligand‐binding pocket but alters cytoskeletal structure and inhibits migration independently of RKIP and Raf/MAPK signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.[SK3][SK1]should we use the past tense for our results?[SK2]is "MEFs" a common term?[SK3]I like this conclusion!
Hepatic CYP3A protein suppression by proteasomal inhibitors is due to induction of ER‐stress, activation of the eIF2‐alpha kinase PERK leading to translational arrest
Authors: Acharya Poulomi , Correia Maria Almira
Published: June 2021

Hepatic cytochromes P450 3A (CYP3A) are endoplasmic reticulum (ER)‐proteins, responsible for xenobiotic metabolism. They are degraded by the ubiquitin‐dependent 26S proteasome. Consistent with this, we have shown that proteasomal inhibitors MG132 and MG262 stabilize CYP3A proteins. However, more recently MG132 was reported to suppress CYP3A due to an NF?B‐mediated regulation of protein stability. Because MG132 concentration used in those studies was 10‐fold higher than that required for CYP3A stabilization, we examined the effect of MG132 (0‐300 µM) concentration‐dependent proteasomal inhibition on CYP3A turnover in cultured primary rat hepatocytes. We found a biphasic MG132‐concentration effect on CYP3A turnover: Stabilization at 5‐10 µM with marked suppression at >100 µM. Because such CYP3A suppression could be due to MG132‐induced ER‐stress, we monitored the activity of PERK, the ER‐stress‐activated eIF2α kinase. Indeed, we found a significant (5‐fold) MG132 concentration‐dependent PERK autophosphorylation, along with a 8‐fold increase in eIF2α‐phosphorylation. Pulse‐chase and CYP3A immunoprecipitation analyses documented the dramatic translational arrest of total hepatic protein as well as CYP3A protein syntheses; thereby confirming that at high concentrations, MG132 is indeed cytotoxic and can suppress CYP3A synthesis.

Supported by NIH grants GM44037, DK26506 and DK26743.
Oxygen‐Dependent Degradation of Yeast SREBP Requires the N‐End Rule E3 Ligase Ubr1
Authors: Espenshade Peter , Lee Chih‐Yung S , Hughes Bridget T , Stewart Emerson V
Published: June 2021

Eukaryotic cells experience fluctuating environmental oxygen concentrations and elicit a physiological response to allow the organism or tissue to adapt. Sre1, the fission yeast Sterol Regulatory Element Binding Protein, is an ER membrane‐bound transcription factor that responds to changes in oxygen‐dependent sterol synthesis as an indirect measure of oxygen availability. Under low oxygen, Sre1 is proteolytically cleaved and the released N‐terminal transcription factor (Sre1N) activates gene expression essential for hypoxic growth. In addition, Sre1N turnover is regulated by oxygen. Ofd1, a prolyl 4‐hydroxylase family member, accelerates Sre1N degradation in the presence of oxygen. However, unlike the prolyl 4‐hydroxylases that regulate mammalian hypoxia inducible factor (HIF), Ofd1 uses multiple domains to regulate Sre1N degradation by oxygen; the Ofd1 N‐terminal dioxygenase domain is required for oxygen sensing and the Ofd1 C‐terminal domain accelerates Sre1N degradation. Proteasomal degradation of Sre1N requires the E2 ubiquitin conjugating enzyme Rhp6 and the N‐end rule E3 ligase Ubr1. Genetic experiments position Ofd1 in this pathway upstream of Ubr1. Thus, oxygen regulates Sre1 activity at two points to mediate adaptation to hypoxic stress: (1) proteolytic cleavage and (2) transcription factor degradation.

Research was supported by the NIH‐HL‐077588 and NIH‐AI‐072186.
Mannosylphospho dolichol synthase (DPMS) overexpression enhances capillary endothelial cell proliferation
Authors: Zhang Zhenbo , Banerjee Aditi , Baksi Krishna , Banerjee Dipak K
Published: June 2021

DPMS an Mr31 kDa protein catalyzes a biochemical step in the ER critical for N‐glycosylation, O‐glycosylation, and C‐mannosylation of proteins as well as for the synthesis of GPI anchor. DPMS is essential for the viability of yeast and its deficiency has been found associated with variants of congenital disorder of glycosylation (CDG). We have shown recently that phosphorylation activation of DPMS accelerates the lipid‐linked oligosaccharide (LLO) synthesis, turnover, protein N‐glycosylation and consequently the angiogenesis. cDNA cloning and sequencing of DPMS from capillary endothelial cells confirmed the presence of a PKA motif RKIIS. Angiogenesis, the proliferation and differentiation of capillary endothelial cells is essential for breast tumor growth and metastasis. We therefore asked, if DPMS could be an activator of the angiogenic switch. To answer this question we have transfected capillary endothelial cells with a DPMS overexpressing plasmid in pEGFP‐N1 vector and isolated a stable cell line. The expression of DPMS in the clone has been confirmed by fluorescence microscopy, western blotting and qRT‐PCR. These cells when subjected to a proliferation assay we observed that DPMS overexpressing clone proliferates faster than the parental cells or cells transfected with the vector (pEGFP‐N1) alone. On the other hand, silencing the DPMS with siRNA reduced cellular proliferation confirming our hypothesis. Supported in part by grants U54‐CA096297 and Susan G. Komen for Cure BCTR0600582.
Periodate oxidation of sea urchin hyalin(s): A glycobiological study towards understanding of hyalin's function as a specific cell adhesion molecule
Authors: Ghazarian Haike , Hutchins‐Carroll Virginia , Coyle‐Thompson Catherine , Metzenberg Stan , Razinia Ziba , Dalrymple William , Carroll Edward J. , Oppenheimer Steven B.
Published: June 2021

Hyalin (Hy), a set of large glycoproteins containing 2‐3% carbohydrate appears to be a specific cell adhesion molecule in the sea urchin embryo model. Sodium (meta) periodate (PI) can selectively degrade carbohydrate by oxidizing the C‐C bonds between vicinal diols. Untreated Strongylocentrotus purpuratus Hy blocked the adhesion of the tip of the archenteron to the roof of the blastocoel (the specific adhesive interaction under study) in two assays: (1) a microplate assay using live Lytechinus pictus embryos and (2) using dissected pieces of the archenteron and blastocoel roof. Hy cross reacts between these two species. PI treated Hy did not block this interaction in either assay. The microplate analysis, with over 16,000 L. pictus embryos, indicated that 6 hr PI treated Hy (0.17‐0.34 mg treated Hy/ml low calcium artificial sea water ‐LCSW) did not block the cellular interaction, while untreated Hy (0.17‐0.34 mg untreated Hy/ml LCSW) did. 76.4‐77.2% ± 2% of the embryos in PI treated Hy possessed complete archenterons, while 2.7‐7.6% ± 12% possessed complete archenterons in the untreated Hy samples with a p value of less than 0.001. Similar results were obtained using the dissected pieces assay. While this study suggests that Hy carbohydrate is involved in Hy function, further studies are needed to determine if PI is also affecting Hy in other ways (Supported by NIH NIGMS SCORE (S0648680), MARC, RISE, the Joseph Drown Foundation and the Sidney Stern Memorial Trust).
Metabolic profiling of Helicobacter pylori glycosylation
Authors: Koenigs Maria B. , Dube Danielle H.
Published: June 2021

Helicobacter pylori is a pathogenic bacterium that causes duodenal ulcers and some gastric cancers. Recent studies have revealed that H. pylori contain a unique glycan that is linked directly to its virulence. Here we set out to further explore the glycobiology of H. pylori by profiling this pathogen's glycome. We employed a technique termed metabolic oligosaccharide engineering that has been utilized extensively to profile glycosylation of mammalian cells. H. pylori were grown in liquid culture supplemented with the unnatural azide‐containing sugar peracetylated N‐azidoacetylglucosamine (Ac4GlcNAz). H. pylori cells and lysates were then probed via Staudinger ligation to assess azide incorporation. Western blotting reveals that azides are metabolically incorporated into several intracellular H. pylori proteins. Further experiments reveal that azide‐dependent signal is diminished by beta‐elimination but not by treatment with peptide‐N‐glycosidase‐F, suggesting that the azide is metabolically incorporated into O‐linked glycans. Future work will further establish the metabolic fate of Ac4GlcNAz in H. pylori and the identity of labeled proteins. Support was provided by the HHMI and the Dreyfus Foundation.