bluuebunny/crossref_metadata_2025 huggingface dataset:
KLF‐2‐dependent, shear stress‐induced expression of CD59: a novel cytoprotective mechanism against complement‐mediated injury in the vasculature
Authors: Mason Justin Charles , Kinderlerer Anne , Ali Faisal , Johns Michael , Leung Viola , Boyle Joseph , Evans Paul , Haskard Dorian O
Published: June 2021

Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress (LSS) led us to explore its effects on endothelial cell (EC) expression of complement inhibitory proteins CD59 and decay‐accelerating factor (DAF). Human umbilical vein and aortic EC were exposed to LSS (12 dynes/cm2), or disturbed flow (DF) (+/−5 dynes/cm2at 1Hz), in a parallel‐plate flow chamber. LSS induced a flow rate‐dependent increase in steady‐state CD59 mRNA, reaching 4‐fold at 12 dynes/cm2(p<0.01). Following 24–48h of LSS, cell surface CD59 was upregulated by 100% (p<0.01), while DAF expression was unchanged. The increase in CD59 following LSS was functionally significant, reducing C9 deposition and complement‐mediated lysis of flow‐conditioned EC by 50% (p<0.05). While CD59 induction was independent of PI‐3K, ERK1/2 and nitric oxide, an siRNA approach demonstrated dependence upon an ERK5/KLF2 signalling pathway. In contrast to LSS, DF failed to induce EC CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional LSS, when compared to atheroprone areas exposed to DF (p<0.05). We propose that upregulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement‐mediated injury, and protects from atherogenesis in regions of LSS.
Characterization of soluble epoxide hydrolase (sEH) inhibitors for use in vascular graft stenosis
Authors: Terry Christi M , Morisseau Christophe , Carlson Mary L , Sanders William G , Minami Shoko , Ulu Arzu , Hammock Bruce D , Cheung Alfred K
Published: June 2021

Vascular grafts often fail due to neointimal hyperplasia development. Vascular smooth muscle cells (SMC) and macrophages are likely key contributors to hyperplasia growth. Anti‐inflammatory epoxyeicosatrienoic acids (EETs) are derived from arachidonate by cytochrome (CYP) P450 enzymes but are catabolized by sEH. Thus, enhanced sEH activity that decreases EETs may contribute to graft hyperplasia.

In a pig arteriovenous graft model, we detected a 6‐fold decrease in CYP450 activity and a 4‐fold increase in sEH activity in the venous‐graft anastomatic tissue after graft placement. Consequently, EET levels decreased by 7‐fold. Further, there was intense tissue immunostaining for TNFα and phospho‐Iκb

Effects of inhibitors of sEH (sEHI) on human aortic SMC and macrophages were investigated. Polytetrafluoroethylene graft material stimulated the release of TNFα from mouse macrophage (RAW) cells. This release was inhibited by sEHI alone or in combination with EETs. sEHI also inhibited LPS‐induced chemotaxis of RAW cells. In SMC, sEHI prevented TNFα‐induced phosphorylation of Iκb, and PDGF‐induced SMC migration to the same extent as imatinib, a PDGF‐receptor antagonist.

These results suggest that sEH participates in vascular graft hyperplasia. Thus sEH inhibitors may be useful in preventing graft hyperplasia.
Chronic administration of the soluble epoxide hydrolase (sEH) inhibitor, AUDA, ameliorates end‐organ damage in stroke‐prone hypertensive rats (SHRSP)
Authors: Li Jing , Carroll Mairead A. , Chander Praveen N. , Li Yanna , Falck John R. , Hammock Bruce D. , Stier Charles T.
Published: June 2021

We have shown that treatment with the sEH inhibitor, AUDA (12‐(3‐adamantan‐1‐yl‐ureido) dodecanoic acid) for 10 days can markedly ameliorate the early salt‐sensitive pathologic changes in SHRSP (Li et al., Frontiers in Bioscience, 2008). In the present study, we examined whether long‐term treatment with AUDA (6 weeks) would protect against the development of severe end‐organ damage in these rats. Fifteen male SHRSP were maintained on stroke‐prone rodent diet and 1% NaCl drinking solution starting at 7 weeks of age. Eight SHRSP received AUDA (10 mg/Kg) once daily by gavage and 5 littermates served as controls. After 6 weeks of treatment, all of the control SHRSP developed proteinuria and exhibited histological evidence of severe widespread nephrosclerosis. In contrast, 5 AUDA‐treated SHRSP showed no proteinuria or renal damage and 3 AUDA‐treated SHRSP showed proteinuria but reduced renal damage relative to controls. Overall, proteinuria was reduced (142±25 vs 54±23 mg/d, P<0.05) and renal cortical cytochrome P450 2C11 protein expression was increased (P<0.05) and there was a highly significant inverse correlation between these parameters (P<0.0005). Pre‐terminal systolic blood pressure did not differ between the groups. These data suggest that the sEH inhibition as an intervention to maintain or increase EET levels may serveas a therapeutic approach to ameliorate salt‐sensitive end‐organ damage in SHRSP.
Dual Role of Soluble Epoxide Hydrolase in Regulating Cholesterol
Authors: Enayetallah Ahmed , Luo Belinda , Luria Ayala , Hammock Bruce , Grant David
Published: June 2021

Soluble Epoxide Hydrolase (sEH) is becoming an attractive potential therapeutic target in cardiovascular diseases due to the vasoactive and anti‐inflammatory properties of its fatty acid epoxide substrates. Furthermore, recent genetic studies suggest a potential role for sEH in cholesterol metabolism and atherosclerosis. Here we evaluated the effect of sEH on the cholesterol profile in mice and HepG2 cells. Targeted disruption of ephx2 in mice and the use of sEH hydrolase inhibitors suggest that both the C‐terminal hydrolase and the N‐terminal phosphatase are involved in regulating cholesterol. The results in mice are further supported by studies in HepG2 cells stably expressing various sEH constructs possessing either N‐terminal phosphatase activity or C‐terminal hydrolase activity. Whereas the N‐terminal phosphatase was found to increase cholesterol levels significantly, the C‐terminal hydrolase had a balancing cholesterol lowering effect. In conclusion, we propose the N‐terminal phosphatase of sEH as a potential therapeutic target in dyslipidemia and suggest that hyperlipidemia may be a potential side effect of inhibiting the C‐terminal hydrolase.
The effect of beta‐glucan pretreatment on TNF production in vivo
Authors: Newsome Courtni Takiyah , LeBlanc Brian , Reichner Jonathan S.
Published: June 2021

The soluble beta‐glucan preparation PGG‐glucan has been shown to prime antimicrobial functions, including increasing the oxidative burst response, microbicidal activity, and the chemotaxis of leukocytes, without eliciting the production of proinflammatory cytokines. In vitro experiments using primary macrophages and macrophage‐like cell lines have demonstrated that pretreatment with PGG‐glucan can reduce the production of TNF message and protein upon LPS stimulation. The purpose of this study was to examine the effect of PGG‐glucan pretreatment on LPS‐induced TNF protein production in vivo. The hypothesis is that PGG‐glucan pretreatment will result in a reduction of LPS‐induced TNF‐protein levels. To test this hypothesis, CD‐1 mice were pretreated with a single dose of PGG‐glucan 40 hours prior to stimulation with LPS. Following LPS stimulation, serum TNF protein levels were measured by ELISA. Our data suggest that PGG‐glucan pretreatment of mice can attenuate the LPS‐induced TNF production in serum. These results indicate that PGG‐glucan can stimulate cellular immune function while inducing suppression of proinflammatory cytokines in vitro and in vivo. These data and future studies may identify a pharmacological intervention that allows cells of the innate immune system to be primed while not overproducing inflammatory cytokines.

Supported by NIHGM66194
The role of BMP signaling in endoderm patterning
Authors: Cho Ken W , Blitz Ira , Sun Aixu , Javier Anna
Published: June 2021

Bone morphogenetic proteins (BMPs) are members of the TGF beta superfamily of secreted signaling molecules and play critical roles in innumerable biological processes from the subdivision of tissue types during early embryogenesis to the formation of limbs, internal organs, and bone formation. While it is relatively clear how BMP signal transduction works, relatively little is known regarding the mechanism of target gene regulation by BMPs. We have recently identified a core BMP‐responsive element (BRE) that responds specifically to BMP signaling and a critical phylogenetically‐conserved transcription factor (Schnurri‐related zinc finger protein) mediating BMP signaling. Using these tools, we have recently constructed various transgenic animals (mice, frogs and zebrafish) and examined the activities of BMP signaling in developing embryos and adult tissues. We report on how BMP signaling may be involved in the patterning of endoderm.
Genetic analysis of the somatic cell niche and male germline stem cell development
Authors: Kumar T. Rajendra
Published: June 2021

Male germline stem cells colonize testis tubules and are positioned on the basement membrane. These either self‐renew or commit to spermatogonial progenitors which undergo mitotic and meiotic divisions and finally produce spermatozoa. The stem cells are surrounded by somatic Sertoli cells that from a microenvironment or niche. The interstitial Leydig cells also influence male germ cell development. Sertoli and Leydig cells are sensitive to gonadotropins, FSH (acts on Sertoli cells) and LH (acts on Leydig cells) that are heterodimers (a common α, and hormone‐specific β). We have generated mice lacking either FSHβ or LHβ, and hence FSH or LH. FSHβ null mice are hypogonadal, have reduced number of germline stem cell progenitors, qualitatively normal, but quantitatively reduced spermatogenesis and normal fertility. Genetic removal of cyclin D2, a cell cycle regulator, over the FSHβ null background caused more severe hypogonadism, aberrant Sertoli cell maturation and defects in spermatogenesis and complete infertility. LHβ null mice are hypogonadal, have low testosterone, defective steroidogenesis and are infertile. Defects in Leydig cells, Sertoli cells, Sertoli‐germ cell junctions and a block in spermatogenesis are observed. Thus, these mouse models allow us to developmentally track the properties of somatic cell niche, and how it influences male germline stem cell self‐renewal and differentiation.
Reconstructed Skin by the Self‐Assembly Approach: Is the Stem Cell Niche Present In Vitro?
Authors: Lavoie Amélie , Claudie Paquet , Larouche Danielle , Fugère Claudia , Martel Israel , Fradette Julie , Germain Lucie
Published: June 2021

Since stem cells are responsible for the continuous generation of the stratified epithelium, their isolation and preservation upon culture in tissue‐engineered skin (TES) is suitable to allow the long‐term regeneration of these tissues after grafting. The objective of the present study is to evaluate the dynamic of stem cells and their division in TES produced by the self‐assembly approach. The slow‐cycling property of stem cells was used to identify them with bromodeoxyuridine (BrDU) in TES produced by the self‐assembly approach. TES cultured at the air‐liquid interface were treated with BrdU for a 7‐day‐pulse period, followed by a 21‐day‐chase period. Epithelial cells of the TES were dissociated by enzymatic digestion and analyzed by flow cytometer or on tissue sections by imunofluorescence with antibodies raised against BrdU and keratin 19 (K19). Analysis revealed that basal cells have retained the nuclei BrdU labelling after the 21‐day‐chase period and most of these label‐retaining cells expressed the K19. This observation was corroborated by FACS analysis. These results suggest TES reconstructed by the self‐assembly approach provide a three‐dimensional environment that allows the preservation of stem cells in vitro and we conclude that it is suitable for grafting.

This work was supported by the CIHR. L.G. is the holder of a Canadian research chair on stem cells and tissue engineering from CIHR.
Structure‐based inhibitors of DNA repair
Authors: Ellenberger Tome , Cotner‐Gohara Elizabeth , Oberman Robert , Kim In‐Kwon , Nolan William , Tomkinson Alan , Scharer Orlando D.
Published: June 2021

Damage‐specific DNA repair pathways are well understood as independent entities whereas little is known about how different repair activities are marshaled and integrated in a coordinated cellular response to DNA damage. Crosstalk between repair pathways, in the form of functional or physical interactions of constituent repair factors, can affect normal sensitivity to DNA damaging agents or modulate the consequences of genomic instability caused by the specific loss of a repair function. Tumor cells that are compromised in one DNA repair pathway may be particularly susceptible to killing by inhibitors of another DNA repair pathway, as evidenced by the sensitivity of BRCA1‐deficient tumors to inhibitors of PARP, the poly(ADP‐ribose) polymerase. We are developing small molecule inhibitors of protein interactions and enzymatic activities underlying nucleotide excision repair and DNA crosslink repair. Reversible, site‐specific inhibitors of DNA repair can serve as chemical genetic tools to explore functional connections between repair pathways and to validate candidate targets for the development of therapies that sensitize tumors to DNA damaging drugs. Our results with inhibitors of mammalian DNA ligases and the structure‐specific endonuclease ERCC1‐XPF will be discussed.
Structural and mechanistic studies of AlkB
Authors: Hunt John Francis , Yu Bomina , Ergel Burce , Pastore Chiara , Liebisch Gregory , Edstrom William , Gibney Brian , Friesner Richard , Farid Ramy
Published: June 2021

Recently, there has been rapid progress in understanding the molecular functions of DNA repair enzymes in the AlkB superfamily, which are present in many eubacteria and all higher eukaryotes. These enzymes have been demonstrated to catalyze direct repair of adenine and cytosine bases that have been alkylated on endocyclic nitrogen atoms, a toxic DNA modification mediated by both endogenous and environmental mutagens. Elucidation of the chemical reaction catalyzed by AlkB has led to progress in understanding the physiological functions of the homologous enzymes, which include repair of damaged mRNA and tRNA molecules that reduce translation efficiency in addition to repair of mutagenic lesions in DNA. However, numerous features of AlkB superfamily enzymes remain to be characterized, including the biological functions of the 8 distinct sequence homologues conserved in mammalian organisms. This presentation will summarize the results of coordinated structural and mechanistic investigations of AlkB homologues. X‐ray crystallographic studies have provided insight into the catalytic reaction cycle of E. coli AlkB as well as the structural mechanisms underlying its ability to repair damage to chemically diverse nucleotide bases. These studies suggest a functional coupling between protein dynamics and enzyme turnover which is being investigated using experimental and computational techniques. Computational methods have also been used to identify inhibitors of AlkB which are being applied to studies of the physiological functions of the homologues human enzymes.
Mammalian pioneer translation initiation complex and mRNA decay
Authors: Maquat Lynne E. , Isken Olaf , Matsuda Daiki , Sato Hanae , Woeller Collynn
Published: June 2021

Nonsense‐mediated mRNA decay (NMD) is an mRNA surveillance pathway that largely functions to control the quality of gene expression. NMD in mammalian cells generally occurs when translation terminates sufficiently upstream of a splicing generated exon‐exon junction during a pioneer round of translation. The pioneer round utilizes newly synthesized mRNA that is bound by the cap binding protein (CBP) heterodimer CBP80‐CBP20 and, if the mRNA is a product of pre‐mRNA splicing, one or more exon junction complexes (EJCs). Ultimately, the pioneer translation initiation complex is remodeled so that CBP80‐CBP20 is replaced by eukaryotic translation initiation factor (eIF)4E and the EJCs are lost. The eIF4E‐bound translation initiation complex is not detectably targeted for NMD.

We will present characterizations of the pioneer translation initiation complex. For example, CBP80 promotes NMD by interacting directly with the Upf1 NMD factor and increasing the interaction between Upf1 and Upf21. We will also discuss mRNP rearrangements that occur when translation terminates in a way that elicits NMD. Comparisons of NMD to Staufen1‐mediated mRNA decay (SMD)2 will also be made. Unlike NMD, SMD functions to conditionally decrease the expression of genes encoding mRNAs that bind Staufen1 downstream of a termination codon3. Considering that the majority of cellular mRNA is bound by eIF4E and only a small fraction is bound by CBP80‐CBP20, our finding that SMD targets mRNA undergoing pioneer and subsequent rounds of translation makes sense1.
New Directions in Actin‐Based Motility of Intracellular Bacterial Pathogens
Authors: Theriot Julie
Published: June 2021

Actin‐based motility by the intracellular bacterial pathogen Listeria monocytogenes is typically persistent and unidirectional, with the rod‐shaped bacteria moving nearly parallel to their long axis, while tracing out gently curving paths that can form circles, sine waves, figure eights, or spirals. We have taken two different approaches to understanding the shapes of these trajectories. First, we find that a simple kinematic model for motion that incorporates two different torque terms (for rotations both parallel to and perpendicular to the long axis of the bacterium) along with the speed can predict many of the observed bacterial trajectories, even those with very complex shapes. Second, we have explored the physical forces that govern the trajectories of movement for artificial particles resembling the bacteria in size and shape. Our results suggest that the polar comet tail orientation on moving wild‐type L. monocytogenes is due to polar protein expression and not to any mechanical or physical preference for movement parallel to the bacterial long axis, and that simple models for slight imbalances of force and torque are sufficient to explain many complex aspects of bacterial movement.
The role of Lipid Chaperones in Integrating Lipid Signals with Inflammatory and Metabolic Responses
Authors: Hotamisligil Gokhan S
Published: June 2021

The mechanistic core of a cluster of diseases associated with obesity appears to reside at the interface of metabolic and inflammatory pathways. Hence, molecules that coordinately control metabolic and inflammatory responses are of great importance for the pathogenesis and therapeutic strategies. We have recently identified two related adipocyte/macrophage fatty acid binding proteins (FABP), aP2 and mal1, as key coordinators of inflammatory and metabolic responses through their integrated actions in adipocytes and macrophages. FABP‐deficiency has a profound impact on lipid homeostasis and provides protection against all pathological components of metabolic syndrome through distinct actions on adipocytes and macrophages. We have reported that macrophage FABPs play a dominant role in atherosclerosis. Recently, we examined the impact of bone marrow‐derived cells in systemic glucose metabolism and demonstrated that FABP action in the stroma is more critical in regulation of systemic insulin sensitivity and hepatosteosis, although bone marrow derived cells also contribute to inflammatory changes in adipose tissue and insulin action. In macrophages, FABPs coordinate lipotoxic responses through a lipid chaperoning mechanisms involving a transcriptional program targeting lipid metabolism. In adipocytes, the principal action of FABPs appears to be through regulation of specific lipid signals that act as endocrine signals at distant sites. Here, I will present these new insights into the mechanism of action of FABPs in different target cells to integrate inflammatory and metabolic pathways and regulate systemic metabolic homeostasis. Finally, the development of an orally active synthetic inhibitor of aP2 and the role of this fatty acid binding protein in human metabolic disease will be discussed.
Utero exposure to methamphetamine: Risks and adverse outcomes in offspring.
Authors: Chirwa Sanika Samuel
Published: June 2021

Sporadic reports suggest that babies born to mothers who abuse methamphetamine during pregnancy are predicted to suffer from severe, irreversible brain damage, including reduced intelligence and social skills. These studies are difficult to interpret, however, because of maternal use of other drugs. We have developed and rigorously characterized a guinea pig animal model that can be used to study developmental outcomes such as cognition and behavioral status of subjects exposed to methamphetamine before birth. The animal model allows us to translate our current fundamental examination of the mechanisms underlying memory consolidation to testing how these processes are altered by in utero exposure to drugs of abuse, in particular methamphetamine. This talk will present data we have obtained using this animal model that is beginning to define the consequences of in utero exposure to methamphetamine per se during critical stages of fetal development on intrinsic neural properties, synaptic plasticity and memory persistence as measured by electrophysiological and behavioral paradigms in the offspring. The relevance to methamphetamine use by females of child‐bearing age as a rising major public health concern in terms of the long‐term risk to the offspring will be addressed.

Supported by NIH grant MH 57067
A telomerase holoenzyme
Authors: Collins Kathleen
Published: June 2021

To gain understanding of the physiological mechanisms that direct telomerase biogenesis and regulation, we purified endogenously assembled Tetrahymena telomerase holoenzyme using epitope‐tagged TERT. Together, the results of molecular, genetic and biochemical assays define a holoenzyme complex containing TER, TERT, p75, p65, p45 and p19 subunits. Each subunit has an essential, non‐redundant function in telomerase‐mediated telomere maintenance. Genetic depletion of any subunit results in telomere shortening, but a division of labor among the subunits emerges from a comparison of their impact on telomerase holoenzyme biogenesis. One Tetrahymena telomerase holoenzyme subunit, p65, is an RNP biogenesis factor required for assembly of TER and TERT. Two other subunits, p75 and p45, act to allow telomerase RNP function at telomeres.

To understand how telomerase interacts with its telomere substrates, we have characterized substoichiometric interaction partners of Tetrahymena telomerase holoenzyme. New DNA binding proteins have been identified that are crucial for telomerase access to the telomere.
Restoring Protein Homeostasis to Ameliorate Human Disease
Authors: Kelly Jeffery W
Published: June 2021

The misfolding and/or misassembly of a given intracellular protein leads to gain of toxic function diseases, including Huntington's and Parkinson's, and loss of function diseases including cystic fibrosis and the lysosomal storage diseases. Intra‐ and extracellular protein misfolding and/or misassembly also appears to cause age onset neurodegenerative diseases known as the amyloidoses, including Alzheimer's and the numerous familial disorders such as the transthyretin amyloidoses. The talk will start with an overview of the mechanistic insight that we and others have acquired from studying loss and gain of function diseases of protein homeostasis. I will then focus on the restoration of protein homeostasis in diseases where age onset loss of control of proteostasis has occurred, using a new therapeutic strategy involving small molecules and biologicals that influence the biology of proteostasis. In other words, we aim to reprogram evolved and innate biological networks to repair damaged proteins associated with loss‐ or gain‐of‐toxic function diseases. Several examples of the power of proteostasis regulators will be discussed along with their mechanisms of action. This strategy is highly appealing as one proteostasis regulator can be used to ameliorate several diseases of similar origin.
Protein Folding and Misfolding in the Eukaryotic Cytosol
Authors: Frydman Judith
Published: June 2021

Molecular chaperones are required for the correct folding and assembly of cellular proteins. Because unfolded or partially folded polypeptide chains are known to have a strong tendency to aggregate, the high density of folding nascent chains emerging from polysomes requires that the growing polypeptide is effectively prevented from misfolding and aggregating, until a chain length suitable for productive folding has been synthesized. Molecular chaperones play an important role in this process. In the eukaryotic cytosol, a network of ribosome‐associated chaperones creates a protected folding environment that sequesters folding intermediates from the bulk cytosol and facilitates folding of the emerging nascent chains. These include Hsp70 family members as well as the ring‐shaped chaperonin TRiC/CCT and the prefoldin/GIMc complex. Molecular chaperones also play an important role preventing aggregation and targeting misfolded proteins for degradation. The mechanism of action and relative contribution of these chaperones to cellular folding and misfolding will be discussed.
Investigative learning in exercise physiology research: a liberal arts model
Authors: Stavrianeas Stasinos
Published: June 2021

The preparation of students to enter research, clinical, fitness, or management fields is a critical component of our efforts to promote a healthy lifestyle and curb the prevalence of obesity, diabetes, and cardiovascular disease. We report on the impact of investigative learning on exercise science education in a small, liberal arts college. An NSF award facilitated the acquisition of equipment for assessment of physical fitness and performance. Laboratories were redesigned with emphasis on student‐centered investigative activities. Impact was assessed via student surveys and the number and quality of independent research projects. Surveys revealed that 86% of the students found that the equipment improved their laboratory experience and 65% noted that the availability of equipment influenced their choice of research project; 92% indicated that the laboratories enhanced their understanding of the physiological processes during exercise. All students indicated that the experiences enhanced their confidence in designing and conducting experiments. During the last 2 years 8 student research projects were carried out. The laboratory has become a favorite among the 600 middle and high school students who have visited our facilities. Current science education strategies should be applied in Exercise Science education.

Supported by a grant from the NSF‐DUE‐CCLI (0511219).
An Extended Analysis of the Histological Variants of Tau‐Negative FTLD
Authors: Josephs Keith Anthony , Stroh Alex , Amador‐Ortiz Catalina , Graff‐Radford Neill , Dickson Dennis W
Published: June 2021

Objective: To determine if subcortical, brainstem and cerebellar lesion pattern characteristics of the different types of tau‐negative frontotemporal lobar degeneration (FTLD) would be similar to neocortical and hippocampal patterns [Type 1 has neuronal cytoplasmic inclusions (NCI) and dystrophic neurites (DN), type 2 mainly DN, and type 3 mainly NCI (Mackenzie et al., 2006)].

Design/Methods: We analyzed 40 cases of tau‐negative FTLD from Mayo Clinic, Florida. For each case, 12 standardized sections covering 16 different brain regions or nuclei were sampled including neocortex, hippocampus, subcortical grey, brainstem and cerebellum. TDP‐43 immunohistochemistry was performed and abnormal immunoreactivity semiquantitated.

Results: Thirty‐six cases were classified into one of the three types. For subcortical grey and brainstem regions, we did not see predominance of DN in type 2, and the density of NCI and DN were similar in type 3. Type 2 cases had significantly more NCI in basal ganglia compared to the other types (p<0.05). We found a trend that fine DN in hippocampal CA1 region was more frequent in type 1 compared to type 2 and 3 (p=0.07). A novel finding was that NCI were Pick body‐like in type 2 but granular‐like in type 3.

Conclusion: This study extends the patterns of pathology previously described in the three variants of tau‐negative FTLD and shows how subcortical areas are affected by TDP‐43.
TDP‐43 Neuronal Cytoplasmic Inclusions in the Amygdala of Patients with Advanced Alzheimer Disease
Authors: Hu William T , Parisi Joseph E , Josephs Keith A , Dickson Dennis W
Published: June 2021

TAR DNA binding protein‐43 (TDP‐43)‐immunoreactive neuronal cytoplasmic inclusions (NCIs), characteristically associated with frontotemporal lobar degenerations (FTLD‐U and FTLD‐MND), have been found in the hippocampal dentate granule cells of about 20% of cases with advanced Alzheimer disease (AD). The extent and distribution of TDP‐43 pathology in brain regions other than hippocampus in AD is not known, especially in AD cases without hippocampal NCIs.

Sections of hippocampus, amygdala, neocortex, and medulla from patients with clinical dementia and pathologic features of AD were analyzed for the presence of TDP‐43‐immunoreactive NCIs.

22 cases of advanced AD (Braak Stage V‐VI) without hippocampal NCIs and 23 cases of advanced AD with hippocampal NCIs were included. 6 hippocampal‐negative cases (27%) had TDP‐43 positive NCIs in the amygdala. All 23 cases of hippocampal‐positive cases had numerous TDP‐43 NCIs in the amygdala, with decreasing density of NCIs in the other brain regions.

In summary, TDP‐43 immunoreactive NCIs are found predominantly in the medial temporal structures in advanced AD, and the amygdala appears to be the area most vulnerable.
Acoustic pathways revealed: simulated sound transmission and reception in Cuvier's beaked whale (Ziphius cavirostris) using finite element modelling
Authors: Cranford Ted W. , Krysl Petr , Hildebrand John A.
Published: June 2021

Finite Element Modeling (FEM) techniques are used to study the effects of mechanical perturbation in complex structures. Simulated sound sources placed inside and outside of an adult male Cuvier's beaked whale (Ziphius cavirostris) reveal pathways for acoustic propagation into and out of the head. Sound sources located at the left and right phonic lips produce beams that converge just outside the head. This result supports the notion that dual sonar sources can interfere constructively to form a sonar beam in front of the animal. The most intriguing FEM results concern pathways by which sounds reach the hearing apparatus. A 40 kHz planar wave that approaches from in front of the animal may be transmitted through the lower jaw by flexural wave coupling. The simulations also reveal a previously undescribed “gular pathway” for sound reception. Propagated sound pressure waves enter the head from below and between the lower jaws, continuing toward the bony ear complexes through the internal mandibular fat bodies. This new pathway has implications for the evolution of underwater hearing in odontocetes. The techniques developed for this study can be used to study acoustic perturbation in a wide variety of marine organisms.
Congenital and acquired atrophy of the shoulder girdle muscles in thoracic outlet syndrome (TOS) patients displayed by MRI/MRA/MRV: Sprengel's deformity and poliomyelitis
Authors: Collins James D. , Saxton Ernestina H. , Miller Theodore Q. , Ahn Samuel S. , Carnes Alfred
Published: June 2021

Bilateral MRI/MRA/MRV displays abnormalities of the osseous structures and soft tissues of the cervicothoracic spine that disrupt fascial planes of the brachial plexus and compress the lymphatics, veins and arteries that supply the nerves. Congenital osseous abnormalities include cervical ribs, synchondrosis, Klippel‐Feil and Sprengel's deformity, among many others. Acquired abnormalities, including inflammatory, traumatic, neoplastic and infectious, also cause symptoms of thoracic outlet syndrome. Monitored multiplanar MRI/MRA/MRV displays sites of compression of the brachial plexus. Images were acquired on a 1.5 Tesla GE Signa unit, 44 cm field of view and saline water bags to enhance signal to noise ratio. This presentation displays an adult Sprengel's deformity and the sequelae of childhood polio resulting in costoclavicular compression of the bicuspid valves within the draining veins within the neck, supraclavicular fossae, and of the subclavian and axillary arteries with binding nerve roots. In both Sprengel's and polio the patients had significant atrophy of the shoulder muscles with costoclavicular compression. The patient with Sprengel's deformity had acute sloping ribs with tension on the sternocleidomastoid and scalene muscles. The omovertebral bar was not fused to the scapula. The patient with polio also ruptured his carotid artery lifting a heavy object and required stent placement.
Identification of a cDNA clone to the chicken extracellular matrix protein hLAMP‐1
Authors: Sinning Allan R.
Published: June 2021

The cardiac extracellular matrix protein hLAMP‐1 is part of a multi‐component complex that initiates the transformation of endothelium into mesenchyme within the atrioventricular canal and proximal outflow tract. Previously a 1100 bp cDNA named KMS2 identified as hLAMP‐1 was isolated from a cDNA library made from the quail cell line QCE‐6 (Genbank accession # AY313452). This report describes the isolation of a cDNA sequence in the chick against hLAMP‐1. Initially primers devised from KMS2 were used in reverse transcription PCR of stage 15‐16 chick embryos. The PCR fragments were then cloned for sequencing using a TOPO TA cloning kit. The obtained sequences showed a 98% homology to KMS2 and were submitted to Genbank (accession # EF424417). To extend the original chicken cDNA, 3 prime and 5 prime RACE was conducted on Stage 13–14 and 16–17 embryos and the results compared to KMS2. These experiments extended the chicken sequence to that of KMS2. In addition a 711 bp splice variant was seen in the stage 16–17 embryos and may represent a functional difference between these stages. Taken together these results confirm that these clones represent a portion of hLAMP‐1 and should provided the basis for isolating a full‐length clone and determining the function of this molecule during development.
The role of the notochord in dorsal aortae fusion
Authors: Garriock Robert John , Mikawa Takashi
Published: June 2021

The paired dorsal aortae are the first prominent vessels to develop in the amniote embryo. The dorsal aortae are kept separate on either side of the midline. This bilaterality of dorsal aortae is induced in part by secretion of BMP antagonists (chordin and noggin) from the notochord that inhibit blood vessel formation and migration into the midline region despite the presence of positive vascular regulators VEGF and Shh. During subsequent development, the paired dorsal aortae fuse at the midline to form a single dorsal aorta. It is not known what causes the paired dorsal aortae to fuse. Here, we show that at the time of dorsal aorta fusion the notochord expresses significantly less BMP‐antagonist while expressing similar levels of VEGF and Shh. This correlative observation is supported by in vivo experimentation where implants of older notochord (not expressing BMP antagonist) exert a positive influence on vascular development. These observations suggest that dorsal aortae fusion is largely regulated by a reduction in BMP‐antagonism at the midline.
Communication between integrin receptors facilitates epicardial cell adhesion and matrix organization
Authors: Dettman Robert Warren
Published: June 2021

During heart development, formation of the superficial epicardium requires α4β1 integrin, a receptor for fibronectin (FN) and vascular cell adhesion molecule (VCAM‐ 1). Here we investigated the role of RGD binding integrins during chick epicardial development. We detected transcripts for α5, α8, αv, β1, β3, and β5 integrins in the proepicardial organ (PE). α5β1 was organized into focal complexes and fibrillar adhesions. αvβ3 integrin was localized in focal complexes and α8β1 was organized along actin filaments. Migration of EMCs in vitro was reduced by RGD‐containing peptides. We used adenoviruses expressing an antisense to chick α4 (AdGFPα4AS), full‐length human (Adhα4V5) or C‐terminal deleted α4 (Adha4ΔCV5) to test if α4 regulates expression or function of RGD‐binding integrins. Expression and localization of α5, α8, and αv integrins was unchanged after AdGFPα4AS infection. However, these cells were less able to adhere to vitronectin and FN120. α5 was expressed in EMCs infected with all three viruses; however in Adhα4ΔCV5 infected cells α5 was diminished in fibrillar adhesions and new FN matrix assembly was abnormal. We propose that cooperation between α4β1 and RGD integrins in the PE and epicardium is important for EMC adhesion and subepicardial matrix formation.
The Subscapular Artery in the Axillary Region: Tracking its Anatomical Distribution for Clinical Application
Authors: Garry Norah E , Marquez Samuel
Published: June 2021

The subscapular arterial vessel, the largest branch of the axillary artery, is a key player in the blood supply of the pectoral girdle and associated upper limb musculature. Its multiple branches ensure the functional integrity of the region. Identification of its pattern and distribution is clinically essential to avoid insult during invasive surgical procedures. Forty‐six subscapular trunks from twenty‐three donor bodies were examined from the first year gross anatomy course for SUNY Downstate College of Medicine. In this study we recorded subscapular anatomical patterns, noting variation of arterial branching. 82.6% of our sample exhibited the template pattern reported in anatomy textbooks. The vessel originates from the posterior aspect of the third part of the axillary artery, following the inferior margin of the subscapularis muscle for a few centimeters before dividing into two terminal branches, the circumflex scapular and thoracodorsal arteries. Variations in the overall arterial pattern of the axillary artery appear to be taken up by the subscapular vessel, for example, the posterior circumflex humeral artery comes off the subscapular or the thoracodorsal vessels in three individuals. Another common variation we encountered was the origination of the lateral thoracic artery from the thoracodorsal instead of the second part of the axillary artery. Knowledge of the frequency and distribution of the subscapular vessel will aid in successful transplantation procedures used in reconstructive surgeries.
A slow RecA protein, and its suppression in vivo
Authors: Hao Li , Cox Michael M.
Published: June 2021

A conserved (K/R)X(K/R) motif is found at the subunit‐subunit interfaces in bacterial RecA protein filaments. In the E. coli RecA protein, this motif is made up of residues K248 and K250. Both of these residues have a role in ATP hydrolysis (in trans across the interface) and in the transmission of conformational information across the interface. A K250R mutation creates a RecA protein that promotes both ATP hydrolysis and DNA strand exchange at a six‐fold lower rate than the wild type protein. E. coli strains overexpressing this mutant RecA protein also grow much slower than wild type strains. Suppressor mutations appear quickly, and most suppressor mutations inactivate RecA protein (leading to high levels of UV sensitivity). One suppressor mutation did not inactivate the RecA protein, but instead produced a recA variant that allowed normal rates of growth under normal growth conditions, while partially restoring UV resistance. The characteristics of this suppressor mutation and the purified mutant protein containing it will be reported.
Roles of catalytic residues on the nucleotide recognition domain of T7 DNA polymerase
Authors: JIN ZHINAN , Johnson Kenneth A.
Published: June 2021

Previous kinetic analyses of active site residues of DNA polymerases have not addressed the roles of these residues in the conformational change preceding nucleotide incorporation. Here we assessed the roles of three T7 DNA polymerase active site residues, K522, H506, and R518, which form electrostatic interactions with α−, β–, γ‐phosphates of the incoming nucleotide, respectively. We quantified effects of mutations on the rate and equilibrium constants governing the nucleotide‐induced conformational change and the rate of catalysis. Stopped‐flow data were obtained using a conformationally sensitive fluorophore linked to the fingers domain to monitor conformational dynamics. These data were correlated with quench‐flow data that measured rates of incorporation (kpol) by global data fitting. Kinetic analyses of dNTP(αS) incorporation of mutant enzymes, K522A, K522H, H506A, H506K, R518A, and R518K, were completed. All the mutants showed a slower conformational change of the fingers domain than the wild type enzyme. After this conformational change, there is a rate‐limiting step with a rate comparable to kpol measured by quench‐flow experiments. The direction of fluorescence change implied that the closing of the fingers domain delivers incoming nucleotide to a misaligned state for these mutants. Surprisingly, only small thio elemental effects were observed: 4 for K522A, H506A, and 1.3 for K522H, H506K. Our data suggest that these active site residues play important roles in the fast conformational change and accurate alignment of the active site during correct nucleotide incorporation.
Simultaneous Mutation of Methylated Lysine Residues in Histone H3 Causes Enhanced Gene Silencing, Cell Cycle Defects, and Cell Lethality in Saccharomyces cerevisiae
Authors: Jin Yi , Rodriguez Amy M. , Stanton Julie D. , Kitazono Ana A. , Wyrick John J.
Published: June 2021

The methylation of specific lysine residues in histone H3 is integral to transcription regulation; however, little is known about how combinations of methylated lysine residues act in concert to regulate genome‐wide transcription. We have systematically mutated methylated histone lysine residues in yeast and found that the triple mutation of H3K4, H3K36, and H3K79 to arginine (H3 K4,36,79R) is lethal. The histone H3 K4,36,79R mutant causes a mitotic cell cycle delay and a progressive transcription defect that initiates in telomere regions and then spreads into the chromosome. This effect is mediated by the silent information regulator (SIR) silencing complex, as we observe increased binding of the SIR complex to genomic regions adjacent to yeast telomeres in the H3 K4,36,79R mutant and deletion of SIR2, SIR3, or SIR4 rescues the lethal phenotype. Curiously, a yeast strain in which the histone methyltransferase genes are simultaneously deleted is viable. Indeed, deletion of the histone methyltransferase genes can suppress the H3 K4,36,79R lethal phenotype. These and other data suggest that the cause of lethality may in part be due to the association of histone methyltransferase enzymes with a histone substrate that cannot be methylated. This work was supported by American Cancer Society grant RSG‐03‐181‐01‐GMC.
Structural characterization of histone‐binding PHD fingers
Authors: Kutateladze Tatiana G , Champagne Karen , Pena Pedro , Hom Robert , Musselman Catherine , Hung Tiffany , Gozani Or
Published: June 2021

Post‐translational histone modifications and protein modules that bind these epigenetic marks are essential components of the chromatin remodeling and gene expression machinery. The PHD (plant homeodomain) finger is found in many chromatin‐associated complexes and transcription factors, however the biological function of this domain remains unclear. Recently, it was found that a subset of PHD fingers recognizes histone H3 tri‐methylated at lysine 4 (H3K4me3). In this study we aimed to characterize the determinants of the PHD finger's binding specificity toward modified and unmodified histone tails using X‐ray crystallography, NMR and fluorescence spectroscopic approaches. Here we report the crystal structures of ING1, ING4 and ING5 in complex with histone peptides, and identify residues important for the specific histone interactions. The histone binding sites were also mapped by resonance perturbation analysis and mutagenesis, and binding affinities were measured using tryptophan fluorescence. Together our results provide novel insights into the molecular mechanisms underlying the activity of the histone‐binding PHD fingers, and impart a greater understanding on how the histone code is read.

Supported by NIH (GM071424 and CA113472 to TGK).
The histone H3 variant CENP‐A localizes to sites of DNA repair induced by laser microirradiation of living cells
Authors: Zeitlin Sam G. , Baker Norman M , Wang Jean JY , Berns Michael W , Cleveland Don W
Published: June 2021

The conserved centromere protein A (CENP‐A) is most similar to histone H3, with a unique N‐terminal tail. CENP‐A is known to be essential for recruitment of other kinetochore proteins to mediate attachment of mitotic spindle microtubules to chromosomes. Recently, we reported that DNA damaging mechanisms influence CENP‐A binding to DNA in vitro. We now report that CENP‐A localizes to sites of DNA damage induced by a laser in interphase nuclei of living cells, along with known DNA repair markers such as 53BP1, phosphorylated H2AX, Nbs1 and Chk2. CENP‐A tagged with GFP rapidly localizes to sites of damage, while other tagged histones such as H3 and H2B do not.These observations suggest that CENP‐A may function in DNA repair as part of a non‐nucleosomal complex, in addition to its known function in kinetochore formation.

SGZ was supported by the California Institute of Regenerative Medicine
SRp20, hnRNP A1 and F regulate alternative splicing of insulin receptor via binding to the intronic regulatory elements
Authors: Talukdar Indrani , Sen Supriya , Webster Nicholas
Published: June 2021

Insulin receptor has two splice variants which are produced due to alternative splicing of 36 base pair long exon 11. Inclusion and exclusion of this exon in the matured RNA produces two isoforms, B and A respectively. Previously our lab has generated a minigene construct containing exon 10–12 and the surrounding introns of human IR. Deletion mutation of this construct identified a purine rich ISE near the 5′ and an ISS near the 3′ end of intron 10. For accurate identification of these elements linker scanning mutants were created throughout the ISE and ISS. These constructs were transfected into HepG2 cell lines. The ISE mutants suggest the presence of multiple element binding sites in this region. From ISS mutants, a 12 nt long silencer sequence has been identified, deletion or mutation of which caused a constitutive incorporation of exon 11. Sequence analysis of ISE revealed that it contains potential binding sites for hnRNP A1, F and H proteins. An in vitro RNA pull down assay taking portions of the ISE sequence followed by western blot analysis confirms the binding of these proteins to this region. Co transfection of the minigene with hnRNP A1 inhibits and hnRNP F activates the inclusion of exon 11 in HepG2 cells. Co transfection of the minigene with siRNA against hnRNPA1 and F increases and decreases the inclusion of exon 11 respectively. RNA pull down assay with the ISS revealed the binding of CUG‐binding protein 1 (CUGBP‐1) and SRp20. Co transfection of wild type minigene with CUGBP‐1 inhibits the inclusion of exon 11, whereas, mutation of the 12‐nt region of the minigene abolishes the effect of CUGBP‐1. Surprisingly, co‐transfection of the minigene with SRp20 increased the inclusion of exon 11, suggesting that SRp20 might bind to an enhancer element as well. This study identifies novel intronic regulatory elements and reveals the role of hnRNP A1, F and SRp20 as regulatory proteins for IR splicing.