bluuebunny/crossref_metadata_2025 huggingface dataset:
The diversity and distribution of lymphatic endothelial cell markers as identified by monoclonal antibodies
Authors: Ezaki Taichi , Shimizu Kazuhiko , Morikawa Shunichi , Kitahara Shuji
Published: June 2021

Recently, various novel lymphatic endothelial cell (LEC) markers have been discovered and our knowledge about lymphatic system has been accumulated. However, the distribution of each marker antigen does not necessarily coincide with each other. This may even cause a great confusion for the differentiation of LECs from other cell lineages, such as blood endothelial cells (BECs), lymphoid cells, and so on. In this study we focused on the diversity of LEC markers by the immunohistochemical characterization of the antigen distribution with monoclonal antibodies (mAbs). We have produced a novel mAb, LA102, reacting predominantly with mouse LECs (Ezaki et al., 2006) by the immunization with adjuvant‐induced benign mouse lymphangioma cells. It recognizes an about 26 kD protein antigen. We compared its immunoreactivity with those of other antibodies to LEC markers and found that the distribution of the markers varied depending on their tissue localization or various pathological conditions. This work was mainly supported by a Grant‐in‐Aid for Scientific Research (B) (No.19390053) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Role of Ca2+/Calmodulin‐dependent Protein Kinase II (CaMKII) in Endothelial Cells
Authors: Wang Zhen , Ginnan Roman G. , Singer Harold A.
Published: June 2021

Endothelial cell (EC) barrier function and angiogenesis are partially dependent upon increases of intracellular free Ca2+ through G‐protein coupled receptor (GPCR) agonists such as thrombin and vascular endothelial growth factor (VEGF). The goal of these studies is to fully characterize the endogenous CaMKII isoform(s) in endothelial cells, and to test the hypothesis that the multifunctional Ca2+/calmodulin‐dependent serine/threonine protein kinase II (CaMKII), responds to thrombin‐ and VEGF‐stimulated Ca2+ signals and regulates EC barrier function and angiogenesis. For the 1st time, by RT‐PCR and western blotting, we successfully characterized the predominant CaMKII isoform expressed in cultured human umbilical vein endothelial cells (HUVEC) is the delta 6 (δ6) isoform, a novel δ‐isoform lacking the 21aa c‐terminal domain. We utilized siRNA approach to suppress CaMKIIδ6 protein expression and found it attenuated, only low dose (2.5nM) but not high dose (10nM), of thrombin‐induced increase of EC permeability. These data suggest a role of CaMKIIδ6 in mediating changes in barrier function in response to physiological relevant doses of thrombin. By using the same approach, we found that CaMKIIδ6 is pivotal in regulating VEGF induced HUVEC migration through Boyden Chamber. This work was supported by NIH grant to H.A.S (R01 HL‐049426).

Grant Funding Source

NIH grant to H.A.S (R01 HL‐049426).
AMP‐activated protein kinase activation stimulates heme oxygenase‐1 gene expression in human vascular endothelium
Authors: Liu Xiao‐ming , Peyton Kelly J. , Wang Hong , Korthuis Ronald J. , Durante William
Published: June 2021

AMP‐activated protein kinase (AMPK) is an energy sensing enzyme that is activated in response to changes in cellular metabolism. Recent studies indicate that AMPK inhibits endothelial cell (EC) activation and apoptosis, but the underlying mechanisms remain unclear. Since heme oxygenase‐1 (HO‐1) is a critical vasoprotective protein, the present study investigated whether AMPK activation induces HO‐1 expression in cultured human umbilical vein ECs. Treatment of ECs with the AMPK activators 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR; 0.1‐1mM) or metformin (0.5‐2mM) stimulated a concentration‐ and time‐dependent increase in HO‐1 protein. A significant rise in HO‐1 protein was first detected after 4 hours, peaked at 8 hours, and persisted for 24 hours after AMPK activation. AICAR‐mediated increases in HO‐1 protein were preceded by significant elevations in HO‐1 mRNA beginning 1 hour after AICAR treatment. The induction of HO‐1 by AICAR was inhibited by the AMPK inhibitor compound C, the transcriptional inhibitor actinomycin D, and the antioxidant N‐acetyl‐L‐cysteine. In conclusion, this study demonstrates that AMPK stimulates HO‐1 gene expression in human ECs via a transcriptional and redox‐sensitive pathway. The ability of AMPK to induce HO‐1 may provide an important mechanism by which this kinase preserves EC function during periods of metabolic stress. Supported by NIH and AHA.
Protein arginine methyltransferase 5 (PRMT5) contributes positively to the induction of the E‐selectin gene in endothelial cells (EC) by binding to HOXA9
Authors: Bandyopadhyay Smarajit , Harris Daniel P. , Lause Gregory , Money Angela , DiCorleto Paul E.
Published: June 2021

Multiple transcription factors, including the homeobox protein HOXA9, participate in cytokine induction of the EC‐leukocyte adhesion molecule E‐selectin in EC. HOX proteins often require other activators/co‐activators to affect rates of transcription. We have recently reported that HOXA9 associates with the type II protein arginine methyltransferase PRMT5 in TNF‐a‐activated EC. Although the cellular distribution and the protein level of PRMT5 were unaltered in EC in response to TNF, nuclear PRMT5 transiently associated with HOXA9. The kinetics of this binding was consistent with the association of these proteins with the E‐selectin promoter, as shown by chromatin immunoprecipitation (ChIP) assays. Specific depletion of PRMT5 protein by siRNA led to a significant decrease in both E‐selectin promoter activity and E‐selectin gene induction by TNF. Binding of HOXA9 to its recognition sequence was required for PRMT5 recruitment to the promoter, but not vice‐versa. Moreover, TNF‐dependent transient simultaneous association of both PRMT5 and a symmetrically dimethylated arginine (SDMA)‐containing protein was readily detected by ChIP. This latter protein appears to be distinct from HOXA9. These results demonstrate that HOXA9 recruits PRMT5 to the E‐selectin promoter where it acts as a transcriptional co‐activator. (NIH HL29582)
CaMKII mediates Ang‐II induced vascular smooth muscle cell hypertrophy by a pathway involving HDAC4/MEF2
Authors: Li Hui , Li Weiwei , Gupta Arun , Anderson Mark E. , Grumbach Isabella M
Published: June 2021

Introduction

The calcium/calmodulin‐dependent kinase II has been implicated in VSM growth and is activated by Ang‐II. Ang‐II activates the HDAC4/MEF2 dependent gene transcription which is important in muscle growth and hypertrophy. CaMKII specifically phosphorylates HDAC4 S467,632. We assessed the hypothesis that CaMKII mediates Ang‐II induced smooth muscle hypertrophy by a pathway involving HDAC4/MEF2.

Methods and Results

The medial hypertrophy induced by Ang‐II over 10 days in C57Bl/6 mice was significantly reduced when the CaMKII inhibitor KN93 was given daily i.p.

Infection of rat aortic smooth muscle cells with adenovirus expressing CaMKIIδ2 resulted in a 108% increase in 3H‐Leucine uptake with Ang‐II compared to 50% (p<0.05) increase in controls, whereas overexpression of the CaMKII peptide inhibitor CaMKIIN resulted in a 14 % increase.

The phosphorylation of HDAC4 in response to Ang‐II was increased with the overexpression of CaMKIIδ2. CaMKII overexpression resulted in increased nuclear export of HDAC4, MEF2‐promoter activity and nucleoprotein complex formation in EMSA.

HDAC4 overexpression led to decreased MEF2‐promoter activity and hypertrophy in response to Ang‐II. The dominant negative mutant HDAC4 S467,632A abrogated the activation.

Conclusion

CaMKII mediates Ang‐II induced VSM hypertrophy by derepressing HDAC4 and activating MEF2.

Supported by: AHA, Carver Trust.
Dimerization of LFA‐1/ICAM‐1 bond provides a spatio‐temporal cue in PMN recruitment
Authors: Dixit Neha , Tsuzuki Yuki , Simon Scott I.
Published: June 2021

Polymorphonuclear leukocyte (PMN) adhesion to the inflamed endothelium is in part mediated by LFA‐1 which recognizes its ligand ICAM‐1. It has long been known that ICAM‐1 rapidly forms homodimers and this conformation supports more stable bonds to high affinity LFA‐1 with lifetimes that are 10‐fold longer in duration and exhibit greater dynamic strength than monomeric ICAM‐1. We hypothesize that dimerization of the LFA‐1 bond to ICAM‐1 results in outside‐in signaling that serves as a gatekeeper in the transition from PMN arrest to a migratory phenotype. Imaging PMN rolling on an inflammatory mimetic substrate expressing E‐selectin and ICAM‐1 under shear flow revealed that calcium flux was markedly higher upon arrest on dimeric as compared to monomeric ICAM‐1. Immunofluorescence revealed colocalization of cytoplasmic F‐actin, Talin, Rap‐1, and phospho‐Src within regions correlating with high affinity clusters of LFA‐1. In summary, these data reveal that at a minimum, dimerization of LFA‐1/ICAM‐1 bonds elicits the assembly of a macromolecular complex that provides a key navigational step in the transition from PMN arrest to a migratory phenotype. This work was supported by AI47294 to S.I.S.
Gr.T. Popa in USA ‐ A hypothesis explaining his concept of upward blood flow through the hypothalamo‐pituitary portal system
Authors: Chirculescu Andy Remus Marius , Chirculescu Mihaela , Morris John F.
Published: June 2021

The Romanian scientist Gr.T. Popa and Unna Fielding described the hypothalamo‐pituitary portal system, by 3D reconstruction of the vessels, in 1930. That started a debate on the direction of blood flow, which he postulated to be upward toward the hypothalamus. Why, then, did Popa propose an ascending direction of blood flow through the portal veins, although he made no dynamic study on microcirculation, but only analysed serial sections of the region? One possible explanation could be that a Rockefeller grant in Chicago, USA (1925‐26) gave him the opportunity to train and act as a demonstrator in embryology in C.R. Moore's lab. There it is quite likely that he would be impressed by the classical embryology concept, stated by Wolff, that any organ ascending or descending during ontogenesis keeps its original neurovascular supply and carries it to its final location. He would also have been aware that Rathke's pouch grows upward to meet the infundibular diverticulum. Therefore, when he came to London in 1930, he doubtless remembered this principle of development, and this led him to presume that blood continues to flow from the superior hypophyseal artery upward, from the pituitary to the hypothalamus, through the portal veins which he discovered.

ARMC is a Senior visiting fellow of ASGBI in Dept. of Physiology, Anatomy & Genetics, University of Oxford, UK.
Expression of Foxo transcription factors during osteoblast differentiation and cell death
Authors: Berry Fred , Mirzayans Farideh
Published: June 2021

Foxo transcription factors regulate the differentiation of adipocytes and myoblasts. However less is known regarding their role in the differentiation of other mesenchyme‐derived cell types such as osteoblasts and chondrocytes. Here we examined the expression levels of Foxo protein and mRNA during osteo‐ and chondrogenesis in vitro. Levels of Foxo1a and Foxo4 protein remained relatively unchanged throughout osteoblast differentiation of MC3T3 cells. Low levels of Foxo3a protein were detected in undifferentiated MC3T3 cells however, Foxo3a increased 1 day after the onset of differentiation. Foxo3a mRNA and protein levels also increased following BMP2 treatment of mouse primary limb bud micromass cultures. Since Foxo proteins play critical roles in mediating cell survival following oxidative stress, we assayed the expression of Foxo proteins following H2O2 treatment. MC3T3 apoptosis was detected by caspase 3 cleavage in cells treated H2O2 and resulted in a decrease in Foxo1a. Finally the affect of modulating Sirt1 activity and Foxo protein function, on survivial of MC3T3‐E1 cells was examined. H2O2‐mediated apoptosis increased in MC3T3‐E1 cells when treated with the Sirt1 agonists, while treatment with Sirt1 inhibitors decreased apoptosis. Together these findings indicate the Foxo3a and Foxo1a participate specific processes governing differentiation and apoptosis of osteoblast cells in vitro.
Morphologic growth of the hominoid choanae: Implications for understanding functional integration of the basicranium and respiratory tract.
Authors: Pagano Anthony S , Yuan Derek Y , Laitman Jeffrey T
Published: June 2021

The internal nares (choanae) lie at the nexus of key respiratory tract functions, yet their comparative and functional morphology in hominoids has been poorly studied. We examined the growth of the choanae and adjacent facial and basicranial regions in crania of extant hominoids (Homo sapiens, n= 130; Pan troglodytes, n= 32; Gorilla gorilla n= 22; Pongo pygmaeus, n= 17; Symphalangus syndactylus, n= 19; Hylobates hoolock, n= 39) from eruption of the first through third permanent maxillary molars. Shape variation was quantified using 3D landmarks and analyzed with generalized procrustes analysis (GPA). Results show that ontogenetically the relative size and proportions of the choanae remained stable in all observed species. Humans are characterized by shorter, broader choanae than pongids, but not hylobatids, whose choanae are nearly twice as wide as they are tall. Among all hominoids, choanal area is negatively (and significantly; p<.01) correlated with the area of the piriform aperture, flexion at the spheno‐occipital synchondrosis, and facial kyphosis. These results imply a functional relationship between the dimensions of the osseous nasopharynx and overall basicranial flexion.

Grant Funding Source

n/a
Evaluating the relationship between thoracic form and individual rib cross‐sectional shape
Authors: Eaves‐Johnson K. Lindsay
Published: June 2021

Within the context of human evolution, differences in rib cross‐sectional shape have been explained as a means of preserving equivalent pulmonary function in differently shaped thoraces during respiratory excursions (Jellema et al., 1993.). Relatively flat ribs should then be associated with a narrower trunk (modern), whereas rounded ribs should accompany a thorax that is transversely broad ("archaic"). This study tests whether the more modest variation in thoracic shape within living humans related to climatic adaptation may also elicit these differences. To test the influence of thoracic form on individual rib cross‐sectional shape, external shape data was collected from the 4th and 8th ribs of males from within two ecogeographically distinct populations: Inuit (n=20) and Zulu (n=20). Overall thoracic shape at each level was characterized using measures modified from Franciscus and Churchill (2002), and external rib shaft molding was used to capture cross‐sectional shape. The 2‐D outline of each cross‐section was acquired using TPS software (Rohlf, 2007). As predicted, the Inuits, with their wider torso, are determined to have statistically significantly rounder ribs, accounted for by greater medio‐lateral dimensions, though not greater cross‐sectional area. Results are presented in the context of climatic adaptation, as well as previous considerations regarding rib shape and resistance to bending.

Research funded with generous support from University of Iowa Student Government, UI Graduate Student Senate, Center for Global and Regional Environmental Research, the Stanley ‐UI Foundation Support Organization.

Grant Funding Source

University of Iowa Student Government, UI Graduate Student Senate, Center for Global and Regional Environmental Research, the Stanley â€“UI Foundation Support Organization
The association between patella alta and structural features of osteoarthritis on MRI: The Multicenter Osteoarthritis (MOST) Study
Authors: Stefanik Joshua Jon , Zhu Yanyan , Gross K Douglas , Clancy Margaret , Zumwalt Ann , Lynch John , Felson David
Published: June 2021

Patella alta (PA) is present when the patella position is high relative to the femoral trochlea, and is a known risk factor for patellar dislocation and anterior knee pain in young persons. PA is defined as present when the Insall‐Salvati ratio (ISR), length of the patellar tendon (LT) divided by length of the patella (LP), is = 1.2. The purpose of this study was to determine, in middle aged and older persons at risk for knee osteoarthritis, the association between PA and patellofemoral joint (PFJ) cartilage morphology and bone marrow lesions (BMLs). We measured the ISR on the lateral radiograph in 443 subjects from a cohort study of persons aged 50‐79 years. Cartilage morphology and BMLs were graded on MRI using a semi‐quantitative scale (0‐6, 0‐3 respectively, with 0 representing absence of pathology) in the medial and lateral PFJ. Using logistic regression, we determined the association between the ISR and cartilage morphology and BMLs, adjusting for age, sex, and BMI. The mean ISR was 1.095 and 113 subjects' (26%) ISR was = 1.2. Compared with subjects who did not have PA, those with PA had 1.82 (95% CI 1.26, 2.63) and 2.38 (1.59, 3.58) times the odds of lateral PFJ cartilage damage and BMLs, respectively. We are currently investigating the biomechanical mechanism for the changes seen in bone and cartilage in subjects with PA. This research was supported by the NIH and Arthritis Foundation.

Grant Funding Source

NIH and Arthritis Foundation
Dancing with the devil? Teaming with deans to take Anatomy to a bold new future.
Authors: Laitman Jeffrey Todd , Reidenberg Joy S , Lerner Susan , Friedman Erica
Published: June 2021

For decades, anatomists ran a solo ballet, meticulously choreographing their courses to maximize factual learning. Others ‐ particularly those annoying deans ‐ were kept as far removed as possible. This "silo‐esque" approach frequently precluded Anatomy from evolving as an integrative engine for promoting creativity and partnerships. We report our approach at Mount Sinai in developing a "new" 21st century Anatomy course, one that has from the "blueprint" stage teamed anatomists, surgeons, radiologists ‐ and deans ‐ to maximize its inherent strengths and relevance. Our approach has emphasized anatomical learning (particularly, as it is appreciated via newer and ubiquitous lenses of imaging) within the context of incorporating concepts and educational principles of lifelong physician learning: professional behavior, integrity; communication skills; moving from "me to we," team skills and team responsibility; individual responsibility of time and stress management; and self and group assessments. Our team's interactive journey has resulted in a very different anatomy experience than each of us alone might have expected, but one that we believe will robustly enhance our students' growth, learning, and enjoyment (no prohibition against the latter!) and result in physicians who are team players, self‐reflective, and outstanding educators.
Microstructural basis of elastic properties of cortical bone in baboon mandibles
Authors: Wang Qian , Ashley Dennis W. , Dechow Paul C.
Published: June 2021

Successful bone regeneration in reconstructive surgery should go beyond reproducing shape and structure to restore biological and mechanical function as well as long‐term integration with surrounding native tissues. Recent work has demonstrated that the three‐dimensional material properties of cortical bone vary throughout craniofacial skeletons. However, how the bone microstructure is related to the mechanical properties of bone at the tissue level throughout the skull needs systematic investigation. Cortical samples from symphyseal, corporal and ramal areas of a baboon mandible were studied using micro‐CT and ultrasonic techniques. Results demonstrated that there were significant variations in elastic properties, porosity, and morphology of osteonal canals throughout the baboon mandible, and the principal orientation of osteonal canals agreed well with the axis of maximum stiffness, and further with the long axis of the bone in three functional areas. These findings on the relationships between bone material properties and microstructure, and their relationships to the skeletal form and function at the organ level will greatly enhance our ability to address questions derived from both evolutional and craniofacial medicine fields about the development and biomechanics of skulls. Supported by NSF HOMINID grants (BCS 0725141, 0725183), MEDCEN, and Mercer Seed.
Biomechanical characteristics of cortical bone regenerate after mandibular distraction osteogenesis in dogs
Authors: Zapata Uriel , Kontogiorgos Elias , Elsalanty Mohammed E , Dechow Paul C , Opperman Lynne A
Published: June 2021

Objectives

Biomechanical quality of the newly formed cortical bone after mandibular distraction is critical for assessing distraction successfully. This study examined bone density, ultrasound speed, and directions of maximum stiffness in distracted canine mandibles. The hypothesis tested was that because of the orientation of the distraction loads, cortical bone will be stronger in the distraction direction.

Methods

Five adult foxhound dogs had a distraction device placed to close a 35 mm mandibular defect. After 7 days of latency, the device was activated 1 mm/day for 5 weeks. After a healing period of 12 weeks animals were sacrificed. 35 cylindrical cortical bone specimens were tested to determine the relationship between the specimen diameter and the minimum time delay from the longitudinal ultrasonic pulse through the specimen in the direction of maximum stiffness. In an additional test, densities were obtained.

Results

Density was significantly less in the regenerate bone (1721.7±177.8 mg/cm3) than in the control cortical bone (1974.9±87.2 mg/cm3). There is significantly reduced maximum ultrasonic speed in the regenerate bone specimens (4.0±0.4 mm/ μs), compared to the control bone (4.3±0.1 mm/µs).

Conclusions

The mechanical quality of the regenerate bone was lower on the side of the device placement within the time frame of the experiment.

Grant Funding Source

NIH/NIDCR DE017259‐01 and a grant from COLCIENCIAS (Bogotá, Colombia)
Dental phenotype of a mouse model expressing a truncated form of ameloblastin
Authors: Wazen Rima Marie , Moffatt Pierre , Zalzal Sylvia Francis , Yamada Yoshihiko , Nanci Antonio
Published: June 2021

Ameloblastin (AMBN) is the second most abundant protein produced by ameloblasts and is found mainly in forming enamel. Our aim was to characterize the tooth phenotype in a mouse model lacking expression of full‐length AMBN (AMBN−FL). Since after building enamel ameloblasts and associated cells participate in the formation of the gingival attachment to the tooth (junctional epithelium, JE), we have also examined its integrity. Materials and

Methods

Mandibles from normal and AMBN−FL mice were processed for morphological analyses and immunohistochemical detection of AMBN. Freeze‐dried enamel organs and enamel were prepared for molecular and biochemical analyses.

Results

Our data show that absence of full‐length AMBN causes structural changes at the level of the enamel organ, shows displatic enamel layer, and loss of integrity of the JE. Immunohistochemical staining indicated that the enamel organ in these mice continued to produce an AMBN‐related peptide that is translated from a truncated RNA missing exons 5 and 6. The protein was deduced to have 295 amino acids and an apparent molecular weight of 32‐35 kDa.

Conclusion

Our study shows that AMBN influences both cells and matrix events. Identification of the portion of AMBN protein still produced in this AMBN−FL mouse model provides new insights on the functional domains that may be implicated in these activities. Supported by the CIHR.
The role of p53 loss in human cancer progression and therapeutic response
Authors: Weiss Michele B , Vitolo Michele I , Mohseni Morassa , Park Ben Ho , Bachman Kurtis E , Weber David J
Published: June 2021

Understanding the roles somatic mutations play in cancer progression is a critical step towards the development of novel treatment strategies. The p53 tumor suppressor gene is the most commonly mutated gene in human cancer. A non‐tumorigenic, human cell line model system to study p53 loss in the absence of other oncogenic changes has not been available. Such a model system is key to investigating the possible direct causal connection that p53 deficiency may have in human cancers and to discovering the potential therapeutic effects of such loss. To evaluate the relevance of p53 loss, we have generated a novel in vitro model system using somatic cell knock‐out technology. This knock‐out technology, involving targeted homologous recombination, has enabled us to delete p53 in the non‐tumorigenic epithelial cell line, MCF10A. The initial hypothesis to be tested is that loss of p53 will cause a non‐tumorigenic cell line to display a more oncogenic phenotype. Recently, the homozygous deletion of p53 in four MCF10A clones was confirmed. While MCF10A cells have the appropriate G1/S and G2/M checkpoint responses upon DNA damage, the p53‐/‐ clones display a defective G1/S phase checkpoint response. Further characterization of each clone is being performed to assess the utility of p53 loss as a potential biomarker to predict the therapeutic response of cells treated with current chemotherapeutic regimens.
Revealing the DNA Structural Determinants for ATM Activation
Authors: Zou Lee
Published: June 2021

Both the ATM and the ATR checkpoint kinases play important roles in the cellular response to double‐strand DNA breaks (DSBs). A number of protein factors implicated in the activation of ATM and ATR at DSBs have been identified and characterized. Nonetheless, how exactly DNA ends contribute to the activation of these kinases was unclear.

To investigate the role of DNA in the activation of human ATM kinase, we have developed a new ATM activation assay using cellular extracts and defined DNA structures. Using this assay, we have systematically analyzed how the DNA structures associated with DSBs contribute to ATM activation. Our analysis has revealed that the activation of ATM is regulated by multiple structural components of DNA breaks. We found that distinct DNA structures are involved in the in initiation and enhancement of ATM activation. Surprisingly, the activation of ATM is inhibited by specific structures of DSBs, suggesting that ATM activation is not an irreversible event but a dynamic process regulated by the changes of DNA breaks. In addition, we have identified the key DNA structures that differentially regulate ATM and ATR at DSBs. These results have shed new light on how specific DNA structures contribute to the activation of ATM and ATR, and how the DNA‐damage specificities of ATM and ATR are determined at DSBs.
Demonstration of a highly efficient fungal transformation method used to target four genes involved in conidial pigment biosynthesis
Authors: Chang Perng‐Kuang , Scharfenstein Leslie Lloyd
Published: June 2021

The development of a highly efficient gene‐targeting approach is a prerequisite to the success of functional genomics of Aspergillus flavus, an aflatoxin‐producing fungus of great economic importance. To this end, the ku70 gene, a gene of the nonhomologous end‐joining pathway was deleted to increase the homologous recombination frequency. The pyrG gene in the resulting strain was disrupted by a gene knock‐in strategy for using the A. parasiticus pyrG gene as a selectable marker for transformation. The gene‐targeting frequencies of the A. flavus br1, br2, wA and ygA genes, homologous to genes reported to be involved in conidial pigment biosynthesis in A. fumigatus or A. nidulans, were examined in the A. flavus ?ku70?pyrG strain. The br1 gene is located about 100 kb from the clustered br2 and wA genes on chromosome IV, and the ygA gene is located on chromosome I. The gene‐targeting frequencies were achieved at 80 to 100% as confirmed by PCR screenings of altered genomic patterns of the transformants. Disruption of br2 and wA resulted in mutants of white and olive‐green conidial color, respectively. The br1 and ygA mutants retained the parental strain's yellowish green conidial color. This system substantially reduces the time and workload necessary to obtain knockout mutants. It will expedite the progresses in the functional genomic studies of A. flavus.
Double‐strand break repair functions of 53BP1
Authors: Hartlerode Andrea , Schotta Gunnar , Jenuwein Thomas , Scully Ralph
Published: June 2021

Establishing the earliest events of DNA repair around a double‐strand break (DSB) is critical to our understanding of how alterations in DNA repair proteins influence human disease. Following DNA damage, a number of DSB response factors associate with chromatin containing phosphorylated histone H2AX ("γH2AX") near the break, facilitating repair via two major pathways, homologous recombination (HR) and nonhomologous end‐joining (NHEJ). In a screen for potential mediators of H2AX HR functions, we found that the DNA damage response protein 53BP1 primarily mediates not HR, but XRCC4‐dependent NHEJ. It has been proposed that 53BP1 accumulation on γH2AX chromatin requires interaction of the 53BP1 tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). To test this hypothesis, we examined DSB repair in cells genetically deleted for both of the H4K20 histone methyltransferases, Suv4‐20h1 and Suv4‐20h2. These double null cells have dramatically reduced, although not completely absent, levels of H4K20 dimethylation that are not inducible by IR. Our data suggest that H4K20me2 alone is not responsible for recruiting 53BP1 to perform its NHEJ function.
The Yeast High Mobility Group Protein HMO1 Facilitates DNA End‐Joining and Functions in DNA Double Strand Break Repair
Authors: Xiao Lijuan , Williams Alan , Grove Anne
Published: June 2021

The Saccharomyces cerevisiae high mobility group protein HMO1 has two DNA binding domains, box A and box B, and a lysine‐rich C‐terminal tail. Among other functions, HMO1 has been implicated in mutagenesis control. Our in vitro assay shows that HMO1 promotes DNA end‐joining in presence of T4 DNA ligase. Analysis of truncated HMO1 variants shows that enhanced DNA end‐joining is critically dependent on the C‐terminal domain, but that the box A domain is dispensable. Since interactions between box A and the C‐terminal domain are thought to be required for DNA bending, DNA end‐joining may involve a binding mode that does not depend on significant changes in DNA topology. Notably, transformation of an hmo1δ strain and its isogenic HMO1 parent strain with linearized plasmid DNA shows significantly reduced transformation efficiency for the hmo1δ strain, consistent with a role for HMO1 in DNA double strand break repair by non‐homologous end‐joining. While these data are consistent with an accessory role of HMO1 in apposition of DNA ends in preparation for association with the non‐homologous end‐joining apparatus, the reported physical interaction between HMO1 and components of various chromatin remodeling complexes suggests that an indirect role in chromatin remodeling at double strand break sites is also possible.
Immune functions of liver sinusoidal endothelial cells
Authors: Knolle Percy A.
Published: June 2021

Immune regulation in the liver is biased towards tolerance rather than immunity. Antigen‐presenting LSEC induce tolerance in CD8 T cells and rely on crosstalk leading to exchange of tolerogenic signals through B7‐H1 and PD1. We investigated whether LSEC underwent functional maturation. We found that LSEC express pattern recognition receptors (PRR), which upon activation cause increased expression of CD106, but there was no functional maturation of LSEC. Comparing the gene expression profile of LPS‐stimulated DC with LSEC, it became evident that sentinel function in LSEC elicited a fundamentally different gene expression program. So we next investigated the influence of a viral infection (MCMV). Although LSEC were infected and allowed for viral gene expression, IFNb release prevented viral replication. Viral infection improved T cell stimulation and caused phenotypic maturation of LSEC that now elicited full CD8 effector T cell differentiation in the absence of costimulatory molecules. Bioinformatic analysis of the gene expression profile revealed that complex regulation of gene expression was correlated with immunogenic maturation. Our results identify a novel principle of CD8 T cell differentiation in the absence of well‐known costimulatory signals and will thus shed new light on development of organ‐specific autoimmunity.
Aging and the Hepatic Sinusoidal Endothelium
Authors: Le Couteur David G
Published: June 2021

Old age in many species including humans is associated with substantial thickening and defenestration of the liver sinusoidal endothelial cell, sporadic deposition of collagen and basal lamina in the extracellular space of Disse and increased numbers of fat engorged, non‐activated stellate cells. Associated immunohistochemical age‐related changes include peri‐sinusoidal upregulation of von Willebrands factor, VEGFR‐2, collagen I and IV and ICAM‐1. There is also a small reduction in the fractal dimension of the microvascular architecture but without any change in vascular dispersion. On the other hand, unlike most liver disease, there is no change in expression of alpha‐SMA, desmin or VEGF, reduced expression of caveolin‐1 and stellate cells are not activated. These age‐related changes have been termed pseudocapillarization in order to differentiate them from capillarization seen in cirrhosis. Pseudocapillarization is reversed by caloric restriction indicating it is likely to be a primary aging change. There is an associated impairment of endocytosis. In aged rats the loss of fenestrations leads to impaired transfer of lipoproteins from the sinusoid into the space of Disse. This provides a novel mechanism and therapeutic target for impaired chylomicron remnant clearance and post‐prandial hyperlipidemia associated with old age.
The bHLH protein Twist2 regulates expression of the CHRDL1 (Chordin‐like 1) gene in human skin fibroblasts
Authors: Casasnovas Jose J , Franco Hector L , Cadilla Carmen L
Published: June 2021

Puerto Rican Setleis Syndrome (SS) patients harbor a nonsense mutation in Gln 119 of TWIST2 and have craniofacial abnormalities. This mutation causes truncation of the C‐terminal, which is required for transactivation. Microarray analysis and real time RT‐PCR of normal and patient skin fibroblasts revealed 287 differentially regulated genes, where the chordin‐like 1 (CHRDL1) gene being up‐regulated in SS patients. CHRDL1 is a bone morphogenic protein (BMP) antagonist which is expressed in mesenchymal and neural tissues. Knockdown of Twist2 by siRNA in SV40‐transformed human fibroblasts showed increased levels of CHRDL1 transcripts 48 hrs after transfection. Additionally, transfection of wild type Twist2 in SS patient fibroblasts reduced CHRDL1 expression. In silico analysis of 3000 bp upstream from the transcription start site of the CHRDL1 gene identified putative TWIST binding sites on positions ‐2663, ‐2344, ‐1292, ‐1055, and ‐247. DNA sequence analysis carried out using mVISTA showed conservation of these sites. Electrophoresis mobility shift assay (EMSA) showed specific binding activity of Twist1 and Twist2 homodimers to the ‐2663 putative site while the Q119X protein weakly bound the ‐2663 putative site. This difference in binding could account for CHRDL1 being upregulated in patients. The MBRS RISE R25GM061838 and RCMI G12RR03051 grants provided support for this work.
Diverse splicing patterns of exonized Alu elements in human tissues
Authors: Lin Lan , Shen Shihao , Tye Anne , Cai James J , Jiang Peng , Davidson Beverly L , Xing Yi
Published: June 2021

Exonization of Alu elements is a major mechanism for birth of new exons in primate genomes. Prior analyses of expressed sequence tags show that almost all Alu‐derived exons are alternatively spliced, and the vast majority of these exons have low transcript inclusion levels. In this work, we provide genomic and experimental evidence for diverse splicing patterns of exonized Alu elements in human tissues. Using Exon array data of 330 Alu‐derived exons in 11 human tissues, and detailed RT‐PCR analyses of 38 exons, we show that some Alu‐derived exons are constitutively spliced in a broad range of human tissues, and some display strong tissue‐specific switch in their transcript inclusion levels. Most of such exons are derived from ancient Alu elements in the genome. In SEPN1, mutations of which are linked to a form of congenital muscular dystrophy, the muscle‐specific inclusion of an Alu‐derived exon may be important for regulating SEPN1 activity in muscle. Realtime qPCR analysis of this SEPN1 exon in macaque and chimpanzee tissues indicates human‐specific increase in its transcript inclusion level and muscle‐specificity after the divergence of humans and chimpanzees. Our results imply that some Alu exonization events may have acquired adaptive benefits during the evolution of primate transcriptomes.

Supported by the Hereditary Disease Foundation (to YX and BLD) and Roy J. Carver Trust (to BLD).
A plant 5S rRNA mimic regulates alternative splicing of transcription factor IIIA pre‐mRNAs
Authors: Hammond Ming Chen , Wachter Andreas , Breaker Ronald R.
Published: June 2021

Transcription factor IIIA (TFIIIA) is required for eukaryotic synthesis of 5S ribosomal RNA by RNA polymerase III. Here we report the discovery of a structured RNA element with striking resemblance to 5S rRNA that is conserved within TFIIIA precursor mRNAs (pre‐mRNAs) from diverse plant lineages. TFIIIA protein expression is controlled by alternative splicing of the exon containing the plant 5S rRNA mimic (P5SM). P5SM triggers exon skipping upon binding of ribosomal protein L5, a natural partner of 5S rRNA, which demonstrates the functional adaptation of its structural mimicry. Since the exon‐skipped splice product encodes full‐length TFIIIA protein, these results reveal a ribosomal protein‐mRNA interaction that is involved in 5S rRNA synthesis and has implications for cross‐coordination of ribosomal components. This study also provides insight into the origin and function of a newfound class of structured RNA that regulates alternative splicing.

M. C. H. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund, and A. W. held a postdoctoral fellowship from the German Research Foundation. The research also was supported by grants from the NIH (GM068819) and HHMI.
Changes in β‐secretase activity and regulation in aging and disease
Authors: Holler Christopher J , Webb Robin L. , Ahmed Rachel R. , Li Feng , Beckett Tina L. , Murphy M. Paul
Published: June 2021

Background

Alzheimer's disease (AD) is thought to be causally related to the production of amyloid‐β peptide (Aβ); β‐secretase is the rate‐limiting enzymatic activity in this process and is considered to be a prime target for therapeutics. β‐secretase may increase in AD, although it is unknown whether this effect is specific for BACE1, the major isoform of the enzyme in brain. Our lab is interested in β‐secretase activity and its regulatory factors; one candidate is cellular nucleic acid binding protein (CNBP).

Objective

To determine how BACE1 and BACE2 change in aging and disease as it relates to CNBP levels.

Methods

We have developed a fluorometic assay that measures BACE1 or BACE2 activity. Using this assay, we analyzed a set of control and late‐stage human AD cases (N=11).

Results

BACE1 specific activity was significantly increased in AD brain, whereas BACE2 specific activity was significantly decreased. We also observed that CNBP levels decreased by 17% in AD cases.

Conclusions

These data indicate that BACE1 and BACE2 may have opposing enzymatic effects in AD, and that CNBP may play a regulatory role. In order to gain a better understanding of how BACE1/2, and CNBP change during disease, we will expand our analysis to include earlier clinical times (mild‐cognitive impairment and pre‐clinical AD). These studies will help guide the future development of therapeutic treatments for AD. Supported by NIH NS058382.
Characterization of human mortalin
Authors: Luo Wen‐I , Dizin Eric , Yoon Taejin , Cowan James A.
Published: June 2021

Human mortalin, an Hsp70 chaperone that has been implicated in cancer and Parkinson's disease, is engaged in Fe‐S cluster biogenesis; however, the lack of isolable samples has prohibited its studies. Herein, we describe the cloning, expression, purification and biochemical characterization of mortalin. The purified recombinant mortalin possesses ATPase activity and a characteristic blue‐shift in the fluorescence emission maximum following ATP addition. This protein was characterized through spectroscopic methods: UV‐visible absorbance, fluorescence, and circular dichroism spectroscopies. Steady‐state and single turnover kinetic experiments were performed and compared with another Hsp70 protein, T. maritima DnaK. Both enzymes show slow ATP turnover (mortalin: khyd=0.00060 s−1; Tm DnaK: khyd=0.00036 s−1) and similar secondary structures (~30% alpha‐helix and 15% beta‐strand). While Tm DnaK displays Michaelis‐Menten behavior, mortalin shows a non‐Michaelian profile. This work demonstrates key similarities and differences with other chaperones that are of functional importance.
Chaperone Ability of Two Small Heat Shock Proteins from Maize
Authors: Klein Roger Davies , Bouchard Robert A. , Pett Virginia B.
Published: June 2021

ZmHsp17.0‐CII and ZmHsp17.8‐CII are Class II small heat shock proteins (sHsps) found in maize (corn). These highly conserved Hsps protect client proteins from irreversible aggregation in times of stress. The sHsp was expressed as a fusion protein with a chitin‐binding domain on the C‐terminus that allows for chitin affinity column purification. Light scattering of the model substrate citrate synthase (CS) in solution was measured using a fluorescence spectrophotometer. Instrumental settings and improved processing of CS were devised to maximize reproducibility. After heat shock at 43 °C the light scattering increased over time as CS aggregated. By comparing the light scattering curves of CS alone with a solution containing CS plus varying amounts of ZmHsp17.0‐CII, a clear, consistent reduction in aggregation was seen for all molar ratios of sHsp monomer: CS monomer of 6:1 and greater. Preliminary results also indicate a significant difference between the aggregation suppression effects of ZmHsp17.0‐CII and 17.8. It has been determined that ZmHsp17.0‐CII and ZmHsp17.8‐CII are concentration‐dependant molecular chaperones that protect cellular proteins from aggregation during times of heat stress.

This work was supported in part by a grant to The College of Wooster from the Howard Hughes Medical Institute through the Undergraduate Science Education Program.
Use of cMet Crystal Structures to Identify Potential Drug‐Resistant Mutants
Authors: Meyer Sheryl Lynne , Pauletti Daniel , Husten Jean , Federov Alexander , Herbertz Torsten , Federov Elena , Underiner Ted L. , Wells Gregory J. , Weinberg Linda R. , Milkiewicz Karen L. , Dorsey Bruce D. , Almo Steven C. , Ator Mark A.
Published: June 2021

For Oncology drugs targeted to tyrosine kinases, drug‐resistance often occurs from selection of cells harboring mutations in the kinase domain of the target. The identification of potential drug‐resistant mutants in advance of patient treatment allows for early design of second‐generation drugs that retain selectivity for these mutants. Towards this goal, co‐crystal structures of two diverse inhibitors were generated. Based on these structures, 11 mutants at 5 positions within the ATP pocket were examined. The kinetic parameters for the mutants were compared to wild‐type (WT) for both the basal and activated enzyme forms. Numerous differences were observed, but most notably the activated M1229 and Y1230 mutants had elevated catalytic efficiencies relative to WT. Two distinct inhibitors were studied against this panel, the oxindole SU11274 and the diaminopyrimidine compound 1. Two mutants (I1084L, M1229I) had higher affinity than WT for both inhibitors in both conformations and the gatekeeper L1157I mutant had substantially lower affinity. SU11274 also had reduced affinity for Y1230 and D1228 mutants, whereas the shifts were much less pronounced with compound 1. These data expand the repertoire of mutations in the ATP pocket that retain or enhance enzymatic activity and show that these mutants can display divergent effects on the potency of inhibitors from different chemical series.
Analysis of hemolymph proteinase 16 from an insect, Manduca sexta
Authors: Christen Jayne , Kanost Michael R.
Published: June 2021

Hemolymph of the caterpillar, Manduca sexta, contains at least twenty‐five serine proteinases, some of which function in aspects of innate immune responses. These hemolymph proteinases are synthesized as zymogens and remain inactive until proteolytic cleavage occurs. Hemolymph proteinase 16 (HP16) is a 47 kDa protein that has a carboxyl‐terminal S1 family serine proteinase domain with predicted chymotrypsin/elastase‐like specificity. The amino‐terminal 170 residues of HP16 have no significant match to any characterized protein. The goal of this project was to study the expression of HP16 during larval development and in response to immune challenge. Western blot analysis of plasma from naïve insects showed that expression levels of HP16 were the highest during the wandering, prepupal, and pupal stages. Real‐time PCR analysis demonstrated an increase in HP16 transcript level in fat body of wandering stage insects. Fifth instar larvae that were immune challenged by injection of Escherichia coli or Micrococcus luteus showed an increase in HP16 expression levels in plasma and HP16 mRNA in fat body when compared to saline‐injected larvae. We have developed a baculovirus for the expression of recombinant HP16 and are currently working on purification of this protein for experiments to investigate its proteolytic activity and biological function. Supported by NIH grant GM41247.
Loss of Stearoyl‐CoA Desaturase activity increases ER stress and PKCζ mediated inhibition of Akt in response to saturated fatty acids in breast cancer cells.
Authors: Paton Chad Michael , Ntambi James M
Published: June 2021

Increased de novo lipogenesis is a hallmark of invasive tumors and is negatively correlated with survival. However, the end product is the saturated fatty acid (SFA) palmitate that is lipotoxic unless desaturated. Stearoyl‐CoA desaturase (SCD) converts SFA into monounsaturated fatty acids and is the rate limiting step in the storage of triglycerides and the generation of phospholipids. We demonstrate that SCD increases proliferation in HeLa cells in the presence of Cerulenin or exogenous SFA and that stearate decreased cell viability in a dose dependent fashion. While the product of SCD (oleate) can activate Akt, substrate (stearate or palmitate) treatment of cells exhibited a dose dependent increase in PARP cleavage and PKC? expression. We further probed the role of SCD‐1 in WT and SCD‐1 knockout MEFs and established that SFA prevents Akt translocation to lipid rafts. In the presence of a small molecule SCD‐1 inhibitor, palmitate treatment induced ER stress as evidenced by Xbp‐1 splicing. Density gradient analysis and IP experiments of palmitate treated cells showed a decrease in both PKC? and Akt lipid raft localization and an increase in TLR4/IKKβ complex formation. Finally, in transgenic MMTV‐PyMT mice, SCD‐1 deficiency reduced invasiveness of mammary gland tumors. Therefore, we conclude that SCD regulates MUFA mediated proliferation, prevents ER stress, and reduces tumor invasion.