bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of AK3L1 as a New Lung Cancer Tumor Progression‐associated Gene by Comparative Genomic and Proteomic Analysis
Authors: Chiu Tony , Chang Jeng‐Shou , Jan Yi‐Hua , Lai Tsung‐ching , Hsiao Michael
Published: June 2021

Lung cancer is the leading cause of cancer deaths worldwide. The challenge of finding a cure for lung cancer is to offer insights into metastasis in their developing stages. In this study we wished to identify new lung cancer metastasis‐associated biomarkers by utilizing global cDNA microarray and 2D‐gel proteomic analysis in metastatic lung cancer cells. We further validated our candidate pool by lung cancer patient tissue array analysis. In addition, we produced lentiviruses with specific shRNA to test their effects on lung cancer cell migration and invasion potentials. Our results showed 16 genes up‐regulated and 29 genes down‐regulated in invasive CL1–5 cell line. Of the 16 genes upregulated in CL1–5, we identified and characterized a novel gene AK3L1 as a lung cancer metastasis‐associated gene. Overexpression of AK3L1 protein was found primarily in late stage lung cancer and in patient with distal metastasis and significantly associated with dismal outcome by immunohistochemistry analysis of archival paraffin samples. Furthermore, knockdown of AK3L1 gene expression was found to sufficiently inhibit tumor invasion/migration potentials in CL1–5 and other lung cancer cell lines in vitro and in vivo. The possible mechanisms of AK3L1 with tumor involvement and metastasis were further explored. In conclusions, we have identified a new lung cancer progression gene AK3L1 by global RNA microarray and proteomic analysis, we have also validated this gene functions and mechanisms in vitro and in vivo. AK3L1 may be of value as a new biomarker in predicting the clinical outcome of lung cancers.
Muscle ring finger‐1 (MuRF1) regulates cardiac fatty acid and glucose metabolism via its interaction with PPARα
Authors: Rodriguez Jessica E. , Li Luge , Willis Monte S.
Published: June 2021

We recently identified that increasing the cardiac ubiquitin ligase Muscle Ring Finger‐1 (MuRF1) levels in cardiomyocytes experimentally resulted in a dose‐dependent inhibition of fatty acid oxidation and parallel increase in glucose oxidation. Since these changes in substrate utilization parallel those that occur during cardiac hypertrophy development, we set out to determine how MuRF1 regulated the members of the peroxisome proliferator‐activated receptor (PPAR) transcription factor family. Cardiomyocytes were co‐transfected with PPAR expression constructs, PPAR‐response element (PPRE)‐activity reporter constructs, and MuRF1. MuRF1 specifically inhibited PPARα activity (over 5 fold). MuRF1 transgenic hearts demonstrated decreases in PPARα activity detected by a decrease in PPAR bound to nuclear PPAR‐response elements. By immunoprecipitating MuRF1 or endogenous PPARα from cardiomyocytes, we identified specific binding of the reciprocal protein (PPARα or MuRF1, respectively). MuRF1 over‐expression displaced nuclear PPARα compared to adenovirus control cells by immunofluorescence. These studies identify a novel mechanism by which cardiac hypertrophy driven increases in MuRF1 might regulate characteristic decreases in fatty acid oxidation and increases in glucose oxidation by its post‐translational modification of PPARα to affect subcellular localization.
Effects of hyperlipidemia on adaptive responses to repeated zinc exposure
Authors: Dye Janice , Slade Ralph , McGee John K , Jaskot Richard H , Richards Judy H , LaGier Adriana J
Published: June 2021

Exposure of individuals with atherosclerosis/coronary heart disease (CHD) to near‐road air pollution correlates epidemiologically with deleterious health outcome. Cardiotoxicity purportedly involves generation of ROS and activation of local/systemic inflammatory pathways. This study used cholesterol‐fed rabbits with varying degrees of hyperlipidemia [none (N), mild (M), or severe (S)] to determine whether the degree of hyperlipidemia reduced their ability to endure repeated exposure to zinc (Zn, 16 g/kg instilled IT once/wk x 4 wk), a metallic subcomponent of near‐road air pollution. Data revealed that before and after Zn exposure, S‐rabbits had higher blood white cell counts, cholesterol, and LDL levels. After the 4th Zn exposure, S‐rabbits had flattened T waves and increased heart rates. At 24 h, S‐rabbits had changes in serum nitrite. At 48 h, M‐ and S‐rabbits had increased NAG and heterophils in lung lavage fluid, while lung gluthione (GSH) content was decreased. S‐rabbits also had decreased liver GSH; and reductions in liver GSH:GSSG ratios correlated with the degree of hyperlipidemia. Data suggest that health risk related to near‐road air pollution exposure in individuals with CHD involves a complex interplay of dyslipidemia, inflammation, and tissue specific antioxidant deficits that impede adaptation to additional pollutant‐induced oxidative stress. (Does not reflect EPA policy.)
The cytoplasmic tail of PECAM contains a recognition motif for trafficking into and out of the Lateral Border Recycling Compartment (LBRC)
Authors: Feng Gong , Muller William A.
Published: June 2021

A novel membrane compartment in endothelial cells (EC), the LBRC, contains PECAM, CD99, and JAM‐A‐‐molecules that participate in leukocyte transendothelial migration (TEM). Membrane trafficking from the LBRC to surround the leukocyte is a critical event in the TEM. How a subset of molecules is selected to enter the LBRC from the cell borders is not known. Since Y663F mutation of PECAMâ€™s cytoplasmic tail disrupts its LBRC trafficking ability without interfering with PECAM signaling, we hypothesized that the cytoplasmic tail of PECAM might contain a motif for the trafficking of PECAM into and out of the LBRC. To test this we constructed chimeras with the extracellular domain of CD25, a membrane protein normally not expressed on EC, and the cytoplasmic domain of PECAM. When transfected into EC, wild‐type CD25 shows diffuse distribution on the plasma membrane. However, CD25 bearing the cytoplasmic domain of PECAM localizes to the cell borders. Furthermore, this chimera constitutively recycles from the LBRC to the cell junctions with the same kinetics as PECAM. Our data suggest the cytoplasmic tail of PECAM contains a recognition motif for trafficking into and out of the LBRC. Support: NIH R01 HL046489.
Adaptations in placental nutrient supply capacity to meet fetal demand: implications for intrauterine programming
Authors: Sibley Colin Peter
Published: June 2021

Fetal growth is absolutely dependent on supply of nutrients from mother via the placenta. A variety of human and mouse data together suggest that placental capacity to supply nutrient is adaptable in relation to fetal demand. (1) In normal human pregnancy the activity, per mg membrane protein, of the System A amino acid transporter in the microvillous plasma membrane (MVM) of the placental syncytiotrophoblast is inversely related to the birthweight and size of baby. (2) In mice with a deletion of the placental transcript of the insulin‐like growth factor 2 (igf2) gene, placental weight is reduced compared to wild type mice at e16 (term e20) but fetal weight is not reduced at this time, perhaps because System A expression and activity, per g placenta, is higher in the knockout conceptuses. (3) In the same knockout mice fetal calcium accretion is reduced compared to wild type at e17, but returns to normal by e19 apparently through an increased activity/expression of placental calcium transport mechanisms. (4) Most recently, we find apparent adaptations in placental transporter and vascular reactivity in pregnancies complicated by obesity and pre‐eclampsia respectively. The variable nature of the placental transfer adaptations in different situations, as found in these different studies, will variably affect the mix of nutrient transferred and so potentially variably programme fetal homeostatic mechanisms.
GENOME‐WIDE DISTRIBUTIONS OF RNA POL II CTD PHOSPHORYLATION AND CTD‐ASSOCIATED TERMINATION FACTORS IN BUDDING YEAST
Authors: Bentley David
Published: June 2021

Many of the processes that contribute to RNP biogenesis including RNA processing and packaging take place at the gene, co‐transcriptionally. The target for RNA binding and processing factors is therefore not a full‐length RNA molecule, but a transcription elongation complex (TEC) comprising the growing nascent RNA and RNA polymerase on a chromatin template. RNA polymerase II traverses transcribed genes is association with passenger proteins that can bind to the nascent RNA and/or the C‐terminal heptad repeat domain (CTD), a conserved signature feature of the large subunit. Dynamics of CTD phosphorylation during initiation, elongation and termination of transcription are thought to direct binding and release of associated proteins that recognize different phospho‐isoforms of the heptad repeats (YS2PTS5PS7). We surveyed CTD phosphorylation on Ser2, 5 and 7 residues of pol II engaged on genes in budding yeast by ChIP‐ChIP. As expected Ser5‐P is enriched relative to Ser2‐P at 5′ ends where pol II initiates. Conversely Ser2‐P is enriched relative to Ser5‐P downstream of ORF's as pol II terminates. Changes in Ser5‐P:Ser2‐P were mostly confined to 5′ and 3′ ends rather than within genes. Localization of the CTD‐associated transcription termination factors Nrd1 and Pcf11 correlated with patterns of Ser5/7 and Ser 2 phosphorylation respectively. Surprisingly Pcf11 can also bind tRNA genes transcribed by RNA pol III.
A simple lab exercise to challenge student's ability to predict changes in the functional state of the cardiovascular system
Authors: Appleyard Robert
Published: June 2021

Students routinely learn how separate properties comprising the functional state of the cardiovascular system will individually affect blood pressure, cardiac output, and tissue perfusion. The challenge is learning to integrate such compartmentalized understanding to interpret how the overall state adjusts based upon observed changes in dependent hemodynamic variables. A simple lab exercise facilitates integration of compartmentalized learning. A student rides a stationary bike while the workload is increased in steps with heart rate and blood pressure measured at each step. Students must then apply their knowledge of vascular hemodynamics and cardiac load to estimate changes in cardiac output and peripheral resistance from the observed changes in heart rate and blood pressure. They next explain how arteriolar vascular tone must be altered so as to maintain relatively stable diastolic arterial pressure while meeting the need for increased perfusion to the working muscle, followed by discussion of the expected changes in myocardial contractility and venous capacitance necessary to support increased circulatory flow. Control of state properties by autonomic reflexes is then discussed. As the intended outcome, students gain insight into the orchestrated changes in system properties that must occur as the body adjusts to moderate exercise, by predicting the new system state based upon observed changes in heart rate and blood pressure.
Study of Multiple Renal Arteries Using Corrosion Casts and CT Angiography
Authors: Zahoi Delia Elena , Miclaus Gratian Dragoslav , Alexa Aurora , Sztika Dorina , Farca Mioara
Published: June 2021

This study evaluated the vascular territories of multiple renal arteries according to their trajects and distances between their origins.

The materials used were 200 renal corrosion casts, made by injecting the vascular and ductal elements with a plastic compound and corroding the parenchyma with hydrochloric acid, and 100 CT angiographies conducted with a Siemens Somatom Sensation 64 CT.

There was a 22% frequency of multiple renal arteries. The CT angiographies were used to analyze the distances between the arteries' origin points and their trajects. The distances varied between 0.1–5.7 cm and the trajects of the additional arteries, in comparison to that of the main artery was parallel (above or below) in 42% of cases, ascending in 31% of cases, descending in 19% of cases and crossing in 9% of cases. Arteries only crossed when the distance between their origins was less than 0.8cm (9.53% of cases). The corrosion casts were used to determine intraparencymatous division and vascular territories. The superior renal artery is the main one in 76.2% of cases, the middle one in 2.38% of cases and the inferior one in 16.66% of cases. In 4.76% of cases, there are two main arteries – superior and middle or superior and inferior (equal occurrences).

Combining the two methods of study allows a more complex analysis of morphological parameters, the trajects and vascular territories in the cases where multiple renal arteries are present.
A detailed anatomical study of the papillary muscles and chordae tendineae of the left ventricle in perfusion fixed human hearts
Authors: Bateman Michael Geoffrey , Russell Cori E , Chan Brian Y , Iaizzo Paul A
Published: June 2021

Classical cardiac terminology describes the existence of two papillary muscles in the left ventricle; the anterolateral and the posteromedial. However, individual hearts can display multiple papillary muscles in either region and cardiac pathologies can greatly alter their sizes and shapes. Perfusion fixed human hearts (n=75) are employed for this study due to the unique attributes of their preservation whereby 10% formalin is continuously pumped into the great vessels fixing each heart in an end diastolic state. Endocscopic video footage and MRI stacks have been used to establish the position and size of the papillary muscles relative to the septal wall. Following this a smaller sample size will be dissected and the anatomical features of the papillary muscles and chordae will be recorded. To date, we have observed only a fraction of hearts in the library that elicit the classically defined anatomy and believe that an appropriate terminology can be used to greatly benefit the design and development of left sided trans‐catheter delivered systems and devices. Support for this project has been provided by the Dept. of Surgery and the IEM at the University of Minnesota.
AORTO‐ AND CAVAL‐RENAL CORROSION CASTS MADE BY IN SITU INJECTION
Authors: MATUSZ PETRU L , BORDEI PETRU , SAPTE ELENA , HOINOIU BOGDAN
Published: June 2021

In situ injecting of the abdominal portion of the aorta and of the inferior vena cava allows exposure of the extra‐ and intraparenchymal portions of the renal arteries and veins. We describe the modality of obtaining aorto‐ and caval‐renal corrosion casts by in situ injecting. We used 30 retroperitoneal specimens in pig, injected with fluid methyl methacrylate (Technovit 7143) into the abdominal portion of the aorta and the inferior vena cava. After plastic polymerization, excision of the vascular wall was performed. Subsequently, we extracted the aorto‐ and caval‐renal blocks, and the renal parenchyma was corroded with Natrium Hydroxide. In 10 casts we injected also the pyelo‐ureteral system on one side. The initial injection of the pyelo‐ureteral system allows exposure of the real intraparenchymal distribution of the segmental arteries. The injection of only the abdominal portion of the aorta leads to flattening of the intraparenchymal arterial planes. The initial injecting of the inferior vena cava leads to flattening of the abdominal portion of the aorta due to contact with the left renal vein. (Supported by CNMP 42097/2008).
Variation of the Chorda Tympani in the Infratemporal Fossa
Authors: Dare Folabo , Ruiz Maria , Crawford Tafline C.
Published: June 2021

The chorda tympani is a branch of the facial nerve returning taste sensation from the tongue's anterior two‐thirds and conveying secretory parasympathetics to the submandibular ganglion. While it is well known to be at risk of injury as it traverses the facial canal, its anatomical potential for injury in the infratemporal fossa remains largely unexplored. Damage to the chorda tympani has been reported during dental procedures requiring inferior alveolar nerve block. The exact mechanism of injury is unknown, although several possibilities exist, including direct trauma during injection and possible neurotoxic effects of the anesthetic. We examined variation in the chorda tympani in the infratemporal fossa to assess whether particular morphological variants might be at greater risk of injury during routine dental anesthesia. Variation of the chorda tympani and lingual, mylohyoid, and inferior alveolar nerves were examined. Discrete traits and metrics of the distance the chorda tympani traverses from the petrotympanic fissure to its union with the lingual nerve were recorded for 26 cadavers. On average, the chorda tympani extends 50.6% of the lingual nerve's length in the infratemporal fossa. Few individuals exhibit a chorda tympani extending more than 66% (n=2) or less than 33% (n=1) of this distance. Variants are discussed in light of their potential for injury during routine dental procedures.
An anatomic study of the genicular arterial anastomosis – a dissection and 3D model
Authors: Sabalbal Maher , McAlister Vivian
Published: June 2021

What is it about the arterial supply to the lower limb that allows tissues to remain perfused despite a diseased superficial femoral artery? It is well known in the literature that the profunda femoris artery has great anastomotic possibilities as it passes down the thigh. This has implications during vascular surgeries when a diseased arterial segment needs to be bypassed. The arterial anatomy of this anastomosis has not been well‐documented since the late 1800s. This study combines dissection with 3D imaging to delineate the arteries involved in the genicular anastomosis. Data from the Visible Human Project was utilized in the 3D imaging software, AMIRA, and a stereoscopic model of this arterial circulation was created. Moreover, CT angiograms of the lower limb were used in the imaging software OSIRIX to reconstruct a similar 3D model. Finally, arterial supply to the lower limb was dissected in 10 cadavers from the inguinal ligament to the point of trifurcation of the popliteal artery. Arteries involved in the genicular anastomosis were photographed and documented. Describing the specific arteries involved in the genicular anastomosis may benefit vascular surgeons concerned with lower limb salvage.

Grant Funding Source: Internal
Fetal malformations in mice caused by tap water exposure
Authors: Hrubec Terry C , Ferguson Elizabeth E , Mallela Murali K , Keller Vanessa E
Published: June 2021

In May of 2006, we began to observe neural tube defects (NTDs ‐ a common birth defect resulting in spina bifida and anencephaly) in untreated mice. The longer the mice were housed, the greater the incidence of defects. The cause of these defects was eventually traced to the municipal tap water provided to the mice. Rearing mice for 2 generations (F2 mice) on distilled deionized (DDI) water eliminated the NTDs. When the mice were placed back on tap water from 4 different municipalities, their offspring developed NTDs again. We hypothesized that the contaminant in the tap water could induce other fetal malformations as well. To test the hypothesis, F2 mice were exposed to tap water or DDI for 8 weeks before breeding and throughout gestation. Gestational day 17 embryos were examined for eye, tail, limb, palate, and craniofacial malformations. The embryos were then cleared and stained to assess axial skeleton malformations such as number of ribs and vertebrae, fused, transitional, hemi, and supernumary vertebrae/ribs. Tap water exposure significantly increased cleft palates (5.0% vs. 1.6%), rib malformations (68% vs. 30%) and number of resorptions (7.5% vs. 5.0%) compared to DDI. Analysis of the tap water is underway to identify the teratogen and possible risks to humans. This work was funded through the Harvey Peter's Foundation; Institute for Critical Technology and Applied Science (ICTAS); and NIH grant K01RR16241.
Functionality of bundle branches in developing hearts of Cx40 deficient mice
Authors: Benes Jiri , Sankova Barbora , Sedmera David
Published: June 2021

Connexin40 (Cx40) is a major gap junction protein that is strongly expressed in His‐Purkinje system. We focused on the impact of Cx40 deficiency on development of murine His‐Purkinje system, specifically on the function of bundle branches (BB). We used optical mapping of ventricular activation at different embryonic stages of Cx40 deficient mice and compared them with data from Cx40(+/−) and WT mice.

We found that RBB function, evidenced by presence of epicardial breakthrough near the apex of the right ventricle, was not affected by Cx40 deficiency at the early developmental stages (12.5 and 14.5 dpc). In contrast, LBB function was significantly impaired by Cx40 deficiency and its development seemed to be delayed (Chi square, p>0.05, at 14.5dpc). This finding differs from previous studies on adult Cx40 deficient mice, where RBB function was predominantly impaired (slowed conduction up to RBB block). We speculate that this discrepancy is due to developmental changes in Cx40 and Cx43 levels in working and conduting myocardium as well as differences in absolute conduction velocity, tissue insulation and geometry.
Biometric iris recognition system evaluation
Authors: Latman Neal S , Herb Emily
Published: June 2021

The automatic, computerized recognition of an individual based on measurements of their unique anatomical, physiological, or behavioral characteristics is referred to as “biometrics.” The iris of the eye appears to satisfy the requirements for a good biometric system. The purpose of this study was to evaluate a biometric iris‐scanning recognition system: “Mobile‐Eyes” (Retica Systems, Inc., Waltham, MA.).

The enrollment, verification, and identification applications were evaluated for accuracy and reliability on 277 subjects. Subjects' age range was 18 to 86 years; 36 had a current medical eye condition; 37 had a surgically corrected prior eye condition. A sub‐set of 35 subjects had drug‐induced nystagmus. There were 2,675 verification and identification attempts.

The Mobil‐Eyes successfully enrolled 100% of subjects on first attempt. All subjects were successfully verified and identified on the day of enrollment. None of the current or prior eye conditions prevented enrollment, verification, or identification. All of the subjects with drug‐induced nystagmus were successfully verified and identified. There were no false verifications or false identifications.

In conclusion, the Mobile‐Eyes biometric iris recognition system appeared to exhibit accurate and reliable enrollment, verification, and identification applications in this study.
Ultrasound‐guided Suprascapular Nerve Blocks: An Anatomical Pilot Study
Authors: Barry Christin , Parnes Gregory , Vilnits Yefim , Stone Michael , Marquez Samuel
Published: June 2021

The use of localized direct nerve blocks for anesthesia instead of pre‐operative anesthetic procedures based on intubation has shown greater successful surgical outcomes. Peripheral nerve blocks have been used to achieve local anesthesia but the risk of direct puncture of the nerve has been documented. This study utilized ultrasound on donor bodies to visualize the suprascapular nerve and to safely guide the “anesthetic” saturation delivery to the surrounding tissue without damaging the nerve (n=14). Using a 5–2 MHz transducer placed the sagittal plane, the scapular spine was visualized. The ultrasound transducer was then placed in a transverse orientation to the scapular spine. Moving the transducer superior from the scapular spine, the suprascapular notch was visualized in the supraspinatous fossa. Real‐time imaging was used for injection of methyl blue via a 20 gauge syringe into the suprascapular notch. During real‐time clinical application of this technique, the suprascapular artery could be visualized and avoided by utilizing ultrasound color‐flow. Dissection of the donor shoulder region was then completed to confirm accurate dispensation of the methyl blue to the suprascapular nerve confirming the technique to be viable. Advantages of using ultrasound guidance for this technique include limiting radiation exposure to the patient, being cost‐effective compared with general anesthesia protocols, and having clinical applications in chronic and acute pain management including osteoarthritis of the glenohumeral joint.
Regulation of the Transcription of the UbD Promoter and Identification of ISRE Sequence
Authors: Oliva Joan , Bardag‐Gorce Fawzia , French Samuel W.
Published: June 2021

Mallory‐Denk bodies (MDBs) are found in the liver of patients with alcoholic and chronic nonalcoholic liver disease, and human hepatocellular carcinoma (HCC). DDC is used as a model to induce the formation of MDBs in mouse liver. Previous studies showed that DDC induced the expression of UbD. The mechanism of transcriptional regulation of the gene UbD is unknown, but previous publication showed that inflammatory cytokines played a key role. The understanding of this regulation is very important because UbD is a liver preneoplastic marker, over expressed in 70–90% of the HCC. In presence of serum in the cell culture media, the mRNA of the specific immunoproteasome genes and UbD were down regulated by PS‐341. In the absence of serum, the TNFa and IFNg cotreatment increased the expression of 3 immunoproteaomes proteins and the expression of UbD. Similar results were found using luciferase reporter gene downstream of the UbD promoter. In the presence of the p53 consensus sequence, the cytokine cotreatment didn't induce the activity of the promoter. In the absence of p53 sequence, the cytokine cotreatment induced the activity of promoter. The analysis of the UbD promoter sequence reveals the presence of an ISRE sequence (Interferon Sequence Responsive Element) in the activated promoter by cytokine cotreatment. TNFa and IFNg increased the expression of UbD through the ISRE sequence present on the UbD promoter. The identification of the cytokines pathway activating UbD expression could help to determine a way to down regulate it and, thus, to prevent the HCC formation.

The study was supported by NIH/NIAAA grants 8116 and the Alcohol Center Grant on Liver and Pancreas P50‐011999, including the morphology core.
UBC9‐mediated sumoylation of PR‐Set7 is involved in regulation of gene repression
Authors: Spektor Tanya Magazinnik , Rice Judd Christopher
Published: June 2021

The PR‐Set7/KMT5A histone methyltransferase and its mark, histone H4 lysine 20 monomethylation (H4K20me1), have been implicated in transcriptional repression, however, the exact molecular mechanism leading to this biological outcome remains unclear. Biochemical purification of PR‐Set7 reveals that it is part of a large multi‐protein complex. We hypothesized that identification of PR‐Set7‐associated proteins would provide important insights into the role of PR‐Set7 in gene repression. Yeast two‐hybrid screen revealed a novel interaction between PR‐Set7 and the sumoylation‐conjugation enzyme, UBC9. Additional studies demonstrated that direct interaction with UBC9 is mediated by the N‐terminal (non‐catalytic) domain of PR‐Set7. Furthermore, we discovered that PR‐Set7 is covalently modified specifically with SUMO‐1 and that this occurs in an ARIP3, E3 ligase‐dependent manner in vivo. Importantly, depletion of UBC9 in cells resulted in the derepression of PR‐Set7 target genes strongly suggesting that sumoylation by UBC9 is required for the normal repressive effects of the PR‐Set7 H4K20 monomethyltransferase.
Expression of DNA methyltransferases and their variants are altered in male IUGR rat liver
Authors: Cox Holly D , Burshears Bridget , Callaway Christopher W , Yu Xing , McKnight Robert A , Lane Robert H
Published: June 2021

Uteroplacental insufficiency during fetal development can lead to intrauterine growth restriction (IUGR). IUGR is associated with gene‐specific epigenetic modifications, such as DNA methylation. DNA methyltransferases (DNMTs) mediate cell differentiation during embryonic development through the establishment of DNA methylation patterns. Relative expression levels of DNMT1, 3a, and 3b, as well as their splice variants, are regulated during development, and may be altered in IUGR. Previously we have shown a 50% decrease in total DNMT1 mRNA in IUGR liver relative to control, with no change observed at day 21. We have now examined expression of DNMT1, 3a, and 3b splice variants at postnatal day 0 and day 21 by real‐ time RT‐PCR and Western blot. Preliminary Western blot analysis suggests a 42% decrease in total DNMT1 protein as well as a change in DNMT1 isoform expression at day 0 in male IUGR liver. Expression of DNMT3a2 was also decreased by 50% at day 0 in male IUGR liver. At day 21, we observed a 3‐fold increase in DNMT3b3 mRNA levels in male IUGR liver relative to control, but no change at day 0. DNMT3b3 is the major variant found in the liver and has been shown to be catalytically active. Changes in the relative DNMT levels and isoform expression may explain the methylation changes observed in IUGR liver.
Temporally and Functionally Mapping the SV40 Epigenome
Authors: Milavetz Barry , Gefroh Amanda , Balakrishnan Lata
Published: June 2021

Histone modifications are thought to play a significant role in regulating the life cycle of Simian Virus 40 (SV40). In order to determine the specific function of these modifications in the biological events occurring during a viral infection, we are mapping the SV40 epigenome. Specifically we are using modified Chromatin Immunoprecipitation (ChIP) procedures to determine which forms of histone modifications are present in the functional forms of SV40 minichromosomes present during an infection. To date we have analyzed minichromosomes for the presence of unmodified H3, hyperacetylated H3, H3K4me1, H3K4me2, H3K4me3, H3K9me1, H3K9me2, H3K9me3, unmodified H4, hyperacetylated H4, H4K20me1, H4K20me2, and H4K20me3. While the studies are ongoing, we have already observed several examples of specific associations between a histone modification and a time during infection or form of SV40 minichromosome. Our results suggest that there is significant specificity with respect to the combination of modified histones present in the various forms of SV40 minichromosomes present during the course of an infection. These results also demonstrate that SV40 will be a useful model to study epigenetic regulation.

This work was funded by the National Institutes of Health grant GM074811 and AI070193 to B.M.
The Effects of Histone Modifications on Lens Cell Denucleation
Authors: Sheehan Robert Patrick , Duncan Melinda
Published: June 2021

The lens of the eye contains both epithelial and fiber cells, with fibers cells deriving from the equatorial epithelium. When these cells differentiate, the nucleus and cellular organelles are broken down to facilitate transparency. However, small fragments of DNA can remain in fully mature lens fiber cells, although their structure is unknown. We found that histone H3, trimethylated on Lysine 9, which is associated with compact heterochromatin, co‐localizes with these DNA fragments. In contrast, the DNA remnants are not associated with either histone H3 Lysine 9 acetylation or histone H4 lysine 8 acetylation which is predominant in the more accessible euchromatin. These data suggest that the DNA fragments persisting in mature lens fiber cells are entirely composed of heterochromatin, perhaps because their highly compacted state prevented access of the nucleases responsible for DNA degradation during lens fiber cell differentiation. Supported by Howard Hughes Medical Institute and the National Eye Institute.
Catalytic activity of HDAC8 and HDAC11
Authors: Joseph Caleb Georgesson , Fuchter Matthew , Fierke Carol
Published: June 2021

Histone deacetylases (HDACs) catalyze the deacetylation of acetylated lysine residues on histone proteins. In general, increased HDAC activity results in the silencing of genes, and HDAC inhibitors have been implicated in growth arrest, differentiation, and apoptosis, making them attractive anti‐cancer drugs. The catalytic mechanism of HDACs proposed from the crystal structure involves two conserved histidines in the active site behaving as a general acid‐base pair. Metal‐dependent HDAC isozymes have been divided into two classes, based on sequence homology. HDAC11, a recently discovered isozyme, is not yet characterized but contains conserved residues present in both classes of HDACs. Human HDAC11 has been recombinantly expressed in E. coli. The catalytic activity of this isozyme for deacetylating the peptide substrate (R‐H‐K(Ac)‐K(Ac)‐fluorophore) is 10‐fold lower than that of HDAC8. The activity of HDAC11 is activated by metal ions; the activity of Co2+‐substituted HDAC11 is 15‐fold larger than the activity of the zinc‐bound enzyme, comparable to the metal dependent activation of HDAC8. HDAC11 activity also varies with pH and monovalent cation (Na+, K+) concentration. In HDAC8, Arginine 33 is positioned in a cavity that is perpendicular to the substrate‐binding site in HDAC8. Computational studies implicate this residue in modulating the dissociation of the acetate product from the active site. Removal of this side chain in the Arg33Ala mutation decreases catalytic activity by 1000‐fold and eliminates inhibition by acetate.
Functional Interaction of piGST and Peroxiredoxin 6 (Prdx6) in Cultured Cells
Authors: Zhou Suiping , Lien Yu‐chin , Shuvaeva Tea , Feinstein Sheldon I. , Fisher Aron B.
Published: June 2021

Prdx6 is a moonlighting protein that has both PLA2 and peroxidase activities. Partial purification of native enzyme from bovine lung revealed the presence of πGST in an active preparation and we have observed that purified recombinant Prdx6 from E coli generally lacks peroxidase activity unless incubated with GSH‐loaded πGST. We provided direct evidence for the formation of a complex between Prdx6 and πGST in vitro (Biochemistry, 2006, Vol. 45, p360), and showed that delivery of recombinant Prdx6 into cells (MCF7) that lack πGST did not increase the phospholipid hydroperoxide peroxidase activity unless πGST was co‐delivered (PNAS, 2004, Vol. 101, p3780). These data indicate an in vitro interaction between Prdx6 and πGST that is required for the peroxidase activity of Prdx6. To evaluate this functional interaction in vivo, we used the Duolink Proximity Ligation Assay to explore the interaction of πGST and Prdx6 in vivo. There is a dramatic increase in interaction between endogenous πGST and Prdx6 in mouse pulmonary microvascular endothelial cells (MPMVEC) upon their treatment with tert‐butyl hydroperoxide. Furthermore, transfection of MCF7 cells with a vector expressing πGST increases the phospholipid hydroperoxide peroxidase activity in the cells, presumably due to activation of Prdx6 by πGST. These in vivo studies provide evidence that πGST functionally interacts with Prdx6 in the intact cell.
Identifying putative Ornithine Decarboxylase gene sequence in Phytomonas Jma
Authors: Ponce Alvaro Patricio Villarreal , Carrillo Carolina
Published: June 2021

Ornithine Decarboxylase (ODC) is an enzyme responsible for the first step in polyamine synthesis. It catalyzes the conversion of ornithine into putrescine. Putrescine as well as other polyamines, such as cadaverine, spermidine and spermine, play an essential role in regulating both cell cycle progression and differentiation. Therefore it is of great interest to study different aspects of the polyamine synthesis pathway(s) in pathogens. Phytomonas Jma is closely related to parasitic protozoans. Its cells are flagellate and they infect various plants. In this study we cloned a DNA fragment that most probably corresponds to the gene coding for the P. Jma ornithine decarboxylase. P. Jma cDNA was generated using a poly T primer as a reverse primer and a well‐conserved sequence of the ornithine decarboxylase gene from Crithidia fasciculate as the forward primer. Four recombinant clones harboring fragments of 1000bp, 800bp, 200bp, and 150bp respectively were obtained. The recombinant clones were propagated in Escherichia coli cells and subjected to restriction mapping and nucleotide sequencing. Our preliminary results strongly suggest that one these recombinant clones includes the P. Jma ornithine decarboxylase gene.
A tangible 3D model of the human Adipocyte Plasma Membrane‐Associated Protein (APMAP)
Authors:   Galileo SMART team, Colton Shannon , Herman Tim , Giles Kurt , Koo Ben , Jeske Sabine , Hicks Michael , DeBella Stan
Published: June 2021

Though the human genome has been decoded, we still know relatively little about the function of many of our genes and gene products. One such example is the human protein encoded by chromosome 20, open reading frame 3, also known as adipocyte plasma membrane‐associated protein (APMAP). The functionally characterized protein to which APMAP shares the highest level of amino acid similarity is the plant protein strictosidine synthase, which functions as the precursor to the medically relevant indole alkaloid biosynthesis pathway. However, orthogonal sources of information suggest APMAP may function quite differently. Protein structure can often provide unique insights into molecular function that amino acid sequence alone cannot. Using the homology‐based threading program Modeller (Sali and Blundell. J. Mol. Biol. 234, 779–815, 1993.), a reasonable three‐dimensional model of APMAP was generated. Using rapid prototyping technology, we have built tangible 3D models of this protein. The SMART (Students Modeling A Research Topic) program was established by the Center of BioMolecular Modeling at the Milwaukee School of Engineering and involves partnerships between university researchers and local high schools. This work is funded by NIH‐NCRR‐SEPA and HHMI.
Filamentation protects the human fungal pathogen, Candida albicans, from programmed cell death
Authors: Laprade David , Austriaco Nicanor
Published: June 2021

Apoptosis, or programmed cell death, is a controlled form of cell suicide that occurs when cells are exposed to stressful conditions. Candida albicans is a dimorphic human fungal pathogen that proliferates in either a yeast blastospore form or a filamentous hyphal form. When cultured in media containing either amphotericin B or farnesol, this yeast undergoes programmed cell death. We now provide evidence that filamentation, the process that results in the production of the long, cellular protrusions characteristic of hyphal cells, protects this yeast from cell death death. This may be an evolutionary adaptation that allows hyphal cells in a colony to forage by utilizing nutrients released by their dying blastospore neighbors. (Supported by RI‐INBRE Grant # P20RR016457 from NCRR, NIH.)
Proanthocyanidins (Vaccinium macrocarpon) induce apoptosis in DU145 human prostate cancer cells
Authors: Hurta Robert , Scott Bradley , Patel Kunal , Gottschall‐Pass Katherine , Neto Catherine
Published: June 2021

Cranberry can slow the growth of DU145 cells by inhibiting cell proliferation and also by apoptosis induction. Apoptosis may occur either via the mitochondrial‐mediated intrinsic pathway or death‐receptor mediated extrinsic pathway. This study investigated how a proanthocyanidin‐enriched fraction (PAC) from cranberry may regulate this complex process. PAC contained proanthocyanidin oligomers (major ones appear to be dimers, trimers & tetramers) (with 2–8 epicatechin units, with at least one A‐type linkage between units). PAC treatment resulted in a dose dependent inhibition of Bcl‐2, Bax, and Bid protein levels coupled with an induction of tBid protein. PAC also inhibits Smac/DIABLO levels. PAR‐4 protein levels increased in response to PAC. PAC resulted in a dose dependent induction of cytochrome ‐C in total cellular extract and cytochrome‐C protein expression also increased in mitochondrial extracts. Increased PARP protein expression occurred in response to PAC (25 ug/mL). PAC inhibited Fas‐L, FADD, and TRAIL protein expression levels whereas Fas protein levels were not affected by PAC. These results suggest that PAC is inducing apoptosis in DU145 cells via the intrinsic pathway and ensures this occurs by specifically inhibiting the extrinsic pathway of apoptosis.(N.C.I.C‐ Canadian Cancer Society, P.E.I. Heath Research Program, Cranberry Institute (Wisconsin Board) funded)
Exploring Caspase Specificity with a Physical Three‐Dimensional Model
Authors:   Galileo SMART team, Colton Shannon , Herman Tim , Giles Kurt , Koo Ben , Jeske Sabine , Crawford Emily , DeBella Stan
Published: June 2021

Apoptosis, or programmed cell death, is critical for many facets of multicellular life, including embryonic development, immune system function, tumor formation and neurodegeneration. Destruction of an unneeded, damaged or infected cell is carried out by a set of proteases called caspases. Caspases act as cellular demolition experts, cleaving key proteins so that the cell is destroyed in a safe and efficient manner. Although structures of caspases have been solved, and details of their catalytic mechanism are known, much remains unexplained about how caspases recognize their specific set of substrates. In order to better understand caspase substrate recognition, we have used the rapid prototyping technology at the Milwaukee School of Engineering's Center for BioMolecular Modeling to construct a physical three‐dimensional model of caspase‐3 in complex with a short inhibitory peptide. Students have used this physical model to visually explore how caspase‐3 might interact with its substrates, and to generate testable hypotheses about how mutations on the surface of caspase‐3 could affect substrate specificity.

The SMART (Students Modeling A Research Topic) program was established by the Center of BioMolecular Modeling at the Milwaukee School of Engineering and involves partnerships between university researchers and local high schools. This work is funded by NIH‐NCRR‐SEPA and HHMI.
The flavones induce breast cancer cell apoptosis via activation Foxo3a
Authors: Lin Chia‐Hung , Wan Lei
Published: June 2021

Flavonoids constitute the largest and most important group of polyphenolic compounds in plants. Flavonoids possess numerous beneficial biological properties including antiallergic, antiviral, anti‐inflammatory, antioxidation, and antitumor activities. More importantly, flavones are good candidates of antitumor agents by inducing apoptosis in various types of human cancers. In this study, we investigated the effects of the flavone, apigenin, and luteolin on the growth of breast cancer cells. The results showed that flavonoids significantly decreased the viability of MCF‐7 cells. Flavonoids‐induced apoptosis in MCF‐7 cells was confirmed by Hoechst33342 staining and induction of sub‐G1 phase by flow cytometry. Flavonoids‐treated MCF‐7 cells were arrested at G1and S phase, which was associated with a marked increment of the expression of p21and p27 protein. The induction of p21 and p27 appeared to be transcriptionally upregulated and was p53‐dependent. Next, we use microarray to determine the flavonoids‐induced‐apoptosis regulated genes. Our results revealed flavonoids will increase the expression level of FOXO3a. FOXO3a, one of large forkhead family members, play a pivotal role in the regulation of a myriad of cellular functions including cell cycle arrest and cell death from stress stimuli. To further investigate its mechanism of action, we treated MCF‐7 cells with flavones and showed increase in nuclear localization of FOXO3a, which coincided with decreased Akt signaling activity. Taken together, the data show that cell death of breast cancer cells in response to flavones is dependent upon FOXO3a activation resulting in Akt inhibition.
Defects in DNA Ligase I Trigger PCNA Ubiquitination at Lysine 107
Authors: Das‐Bradoo Sapna , Nguyen Hai Dang , Ricke Robin M. , Haworth Justin C. , Bielinsky Anja‐Katrin
Published: June 2021

The anti‐parallel nature of DNA permits continuous synthesis of DNA on the leading strand and discontinuous DNA synthesis on the lagging strand. On the lagging strand, Okazaki fragments are joined by the catalytic action of DNA ligase I. An individual with a DNA ligase I deficiency exhibited growth retardation, sunlight sensitivity and severe immunosuppression, likely due to accumulation of DNA damage. Surprisingly, little is known about the DNA damage response (DDR) in DNA ligase I‐deficient cells. We utilized Saccharomyces cerevisiae as a model system to address this question. We uncovered a novel pathway, which facilitates ubiquitination of proliferating cell nuclear antigen (PCNA) on lysine 107. Unlike PCNA ubiquitination at lysine 164 in response to UV irradiation, which triggers translesion synthesis and is dependent on Rad6 and Rad18, modification at lysine 107 requires the E2 variant Mms2 in conjunction with Ubc4 and Rad5. Surprisingly, DNA ligase I‐deficient cdc9‐1 cells that carry a PCNAK107R mutation are inviable, because they cannot activate a robust DDR. Our data show that depending on the type of damage that a cell encounters, PCNA is ubiquitinated at different lysine residues. We propose a “DNA Damage code” that allows cells to recognize different types of DNA damage through differential PCNA ubiquitination, allowing the cells to activate the appropriate repair response.
The large, noncoding OLE RNA is associated with membrane biochemistry
Authors: Block Kirsten Florence , Wallace Jason , Puerta‐Fernandez Elena , Breaker Ronald
Published: June 2021

OLE RNA is characterized by its ornate structure, large size, and distribution in anaerobic, extremophilic Gram‐positive bacteria. All organisms that carry OLE express a membrane‐spanning protein of unknown function, termed OLE‐Associated Protein (OAP). Through gel shift assays, we have demonstrated that Bacillus halodurans OLE and OAP form a specific ribonucleoprotein complex and isolated the probable binding site within the OLE molecule. We used RNA fluorescence in situ hybridization (RNA FISH) to visualize the location of OLE within the cell. In Escherichia coli cells carrying transgenic OLE and OAP, OLE is localized to the membrane only when OAP is co‐expressed. Northern blot analysis of B. halodurans shows that OLE is upregulated in the presence of the membrane stressor ethanol. Additionally, high‐throughput sequencing of wild‐type and OLE/OAP knockouts of B. halodurans reveals that OLE is extremely abundant in cells, and that lack of either OLE or OAP causes an upregulation in specific energy‐generating pathways. Together, these results suggest that OLE is involved in a function associated with membrane biochemistry. This research was supported by the Howard Hughes Medical Institute.