bluuebunny/crossref_metadata_2025 huggingface dataset:
Key science networking sites and how they work (9.3)
Authors: Mills David
Published: June 2021

Social networking sites offer professionals opportunities to identify and establish collaborations; review publication lists, explore the latest research trends, and use library and document‐sharing tools. They also afford the opportunity for recruiting students, showcasing your publications, and publicizing your research and accomplishments. The session will identify and distinguish between two types of social networking sites for engineers and scientists. The first are intended to help researchers manage their personal library of research papers, follow research trends, offer resources for collaborative networks, share and learn new protocols, and comment on the latest research trends. The second searches multiple databases to deliver the latest articles in a researcher's field, post e‐copies of their publications, share research in progress, and above all publicize your research. You can use embedded metrics to assess your impact, track how often your articles are downloaded and cited, identify researchers that are checking you out and how your publications are cited and where. The session will address implications of and potential loss of intellectual property and weigh the pros and cons. Participants will review case study examples and analyze them for best practices and errors to avoid, followed by group discussion on ways that participants can use social media to advance their own careers.
Regulation of PSCA expression by m‐Numb in the stomach (904.2)
Authors: Takahashi Tetsufumi , Suzuki Hidekazu , Matsunaga Kayoko , Imai Takao , Shibata Shinsuke , Tabuchi Yoshiaki , Okano Hideyuki , Nakamura Masahiko , Tsuchimoto Kanji
Published: June 2021

Prostate stem cell antigen (PSCA) is a GPI‐anchored cell surface protein expressed in the normal epithelium of several tissues including the isthmus region of the gastric mucosa. Recently, a genome‐wide association study revealed several SNPs in the PSCA‐gene to be associated with gastric cancer and duodenal ulcer risk. However, the underlying regulatory mechanisms of PSCA are still unclear.

Methods: Msi1‐KO mice with lowered expression of m‐Numb in the stomach and wild‐type ICR mice were used for in vivo studies. In vitro studies were carried out with the mouse gastric epithelial cell line MGE507. Overexpression and suppression of m‐Numb in MGE507 cells was achieved by transfection with lentiviral particles carrying an m‐Numb expression vector and shRNA expression vector, respectively. Gene expression was analysed with qRT‐PCR, and western blotting was used to measure protein levels.

Results: In Msi1‐KO mice, a significantly reduced expression‐ and protein level of PSCA was found when comparing the gastric mucosa to wild‐type ICR mice. Expectedly, the protein level of m‐Numb was also verified as lower in the KO‐line. Silencing m‐Numb expression in MGE507 cells resulted in a reduction in protein level of PSCA, and overexpression of m‐Numb led to an increased PSCA‐level.

Conclusion: PSCA gene expression and protein level was regulated by m‐Numb in the mouse stomach and in mouse gastric epithelial cells.
Mechanism and regulation of biotin uptake by mouse and human pancreatic beta cells/islets: physiological and molecular aspects (909.2)
Authors: Ghosal Abhisek , Sekar Thillai , Said Hamid
Published: June 2021

Biotin is essential for normal cellular function of all pancreatic cells. Little, however, is known about how pancreatic beta cells obtain biotin and the molecular identity of the system involved and its regulation. We addressed these issues using mouse‐derived beta‐TC‐6 cells as well as freshly isolated mouse and human primary pancreatic islets. Our results showed biotin uptake by beta‐TC‐6 cells is carrier‐mediated, Na‐dependent, cis‐inhibited by structural analogs and by pantothenic acid, and is saturable (apparent Km of 17 µM). Similarly, freshly isolated mouse and human primary pancreatic islets took in biotin via a carrier‐mediated Na‐dependent mechanism. These studies suggest the involvement of the Na‐dependent multivitamin transporter, SMVT; this was confirmed in studies using gene specific knockdown (shRNA) approach. Uptake of biotin was also adaptively‐regulated by extracellular vitamin levels through a transcriptional mechanism (s). Maintaining (96 hr) pancreatic beta cells in hyper‐glycemic condition (26 mM) did not affect biotin uptake. Exposer to specific cytokines (IL‐1β+TNF‐+IFN‐), whose level increases in diabetes, led to a significant inhibition in biotin uptake. The results show for the first time that biotin uptake by pancreatic beta cells is via a cytokine sensitive carrier‐mediated process that involves the SMVT system.

Grant Funding Source: Supported by DVA and NIH (DK58057, AA‐18071 and DK56061
Regulation of miR101/miR‐199a‐3p targeting cyclooxygenase‐2 by the epithelial sodium channel during embryo implantation (911.5)
Authors: Sun Xiao , Guo Jing‐Hui , Chen Hui , Ruan Ye‐Chun , Chan Hsiao‐Chang
Published: June 2021

Decidualization is a maternal prerequisite for embryo implantation. However, it remains unclear how signals from the embryo at the endometrial surface are converted into chemical signals, leading to decidualization in the stromal cells. Our previous study has demonstrated that the epithelial sodium channel (ENaC) plays a critical role in embryo implantation by regulating the decidulization‐inducing molecules cyclooxygenase‐2 (COX‐2)/PGE2. Recently, we also found that ENaC activation in human endometrial cells could triggers Ca2+ release, phosphorylation of cAMP response element‐binding protein (CREB), down‐regulation of mir101/mir199a‐3p with up‐regulation of COX‐2. In line with in vitro data, knocking down CREB in mice uterine resulted in mir101 and mir199a‐3p up‐regulation, COX‐2 down‐regulation and implantation failure. Taken together, these results indicate that ENaC‐dependent activation of CREB may suppress mir101/mir199a‐3p, resulting in up‐regulation of COX‐2 necessary for embryo implantation. The findings of the study suggest that ENaC may act as an important epigenetic regulator for mir101/mir199a‐3p targeting COX‐2 during embryo implantation.
Structural characterization of the anterolateral capsule of the knee ő a gross anatomic, histological, and magnetic resonance imaging study of the anterolateral ligament (914.6)
Authors: Caterine Scott , Litchfield Robert , Johnson Marjorie , Sandig Martin , Getgood Alan
Published: June 2021

For over 140 years, a structure located on the lateral side of the knee has been recognized but poorly understood by surgeons. Most recently termed the Anterolateral Ligament (ALL), it has been described to consistently originate on the lateral femoral condyle and insert onto the tibia posterior to Gerdy’s tubercle. This study aims to characterize the anatomical properties of the ALL utilizing magnetic resonance imaging, cadaveric dissection, and histological/immunohistochemical sectioning of fresh‐frozen human cadaveric knees. Preliminary results show that in each of the 7 cadaveric specimens undergoing 3T MRI analysis, the ALL can be identified as a distinct, densely arranged structure. The ALL was then clearly visualized in all 9 cadaveric dissections, showing variable origin on the lateral femoral condyle and consistent insertion on the tibia. H&E histological sectioning of 2 cadaveric specimens also indicates that the ALL is ligamentous in nature, revealing a dense, regular network of collagenous tissue. In 1 specimen, immunohistochemistry specific to neurofilament proteins found in axons show peripheral nervous innervation of the ALL, indicating ligamentous tissue. These results show that the ALL can be confidently identified in a multitude of different modalities, providing clear evidence that the ALL is a distinct ligament of the lateral knee.
Tear analysis of the anterior cruciate ligament using a novel staining method (914.8)
Authors: Gillespie Michael , Jorgensen Adam , Wilkes Daniel , Wisco Jonathan
Published: June 2021

The purpose of this project is to create data driven, three‐dimensional fiber maps of various grade‐III sprained human anterior cruciate ligaments. In order to increase visibility of these fibers, we have created a stain made from powdered sugar and blue dye that reveals their fine architecture. Application of the stain to human cadaveric ACL and calcaneal tendon has revealed otherwise undistinguishable fiber boundaries. We performed the following six steps on each of five un‐embalmed human cadaveric knees. 1) MRI imaging, 2) Induced valgus force to simulate common ACL tear, 3) MRI re‐imaging, 4) Dissection to reveal ACL, 5) Stain ACL, 6) MicroScribe both torn halves. With our newly developed staining technique, the intricate surface architecture of torn human cadaveric ACL was exposed to a degree never before possible. This allowed for more complete and accurate MicroScribe 3D digitizing and the creation of maps that exhibit the intricate details of how and where ACLs typically tear. A more thorough understanding of ACL tear microstructure will offer insight into why current repair techniques are ineffective and how they can be improved. The overarching goal is the development of an effective repair method to replace the current method of reconstruction. This would eliminate the many post‐operative complications that accompany ACL reconstruction.

Grant Funding Source: Supported by Mentoring Research Grant, College of Life Sciences, Brigham Young University
Lymphoid formation of mucous membrane of a human rectum (916.5)
Authors: Maul Ya , Aubakirov A. , Suleimenova F.
Published: June 2021

Mucous membrane of a rectum constantly is subject to a significant antigene influence taking into account the reservoir function of the organ. However, problems of interrelation of lymphoid tissue associated with mucous membrane of the organ with the epithelium integument and with other tissue structures were not studied enough.

Aim: to study histomorphological interrelation of lymphoid tissue localised in the mucous membrane of a rectum with epithelium cover of the organ and adjusting tissue structures.

Material and methods of investigation: Human rectum from the period of birth till the eighty seven years. Morphometric, histomorphological, statistic methods have been used.

Results: In the process of research we revealed some interesting facts: A great number of postcapillary venules with the migrating lymphocytes through their walls.

In the anorectal zone of the anal section a lymphoid tissue is represented as a continuous “ring” consisting of the lymphoid nodes and diffuse lymphoid tissue.

In the mucous membrane of the rectum we revealed periarterial macrophagal‐lymphocyte clutches.The clutches had the form of ovoid or ellipse.

Stratified flat non‐cornified epithelium similar to the esophageal epithelium is revealed in the area of anal columns and sinuses. In this section of the rectum there is not a lymphoid tissue or it is shown as single nodes.

Conclusions: In the mucous membrane of the rectum the presens of well‐ developed lymphoid apparatus was proved morphologically that allows to consider the rectum as the peripheric organ of immunogenesis Besides, we suggest a continuous “ring” of the lymphoid tissue localized in the anorectal zone of the anal section to classify as the analogue of “Fabritsious bad” of a man.
Characterization of calcium phosphate composite two solution bone cements (922.20)
Authors: Chari Jonathan , Rodriguez Lucas , Aghyarian Shant , Rodrigues Danieli
Published: June 2021

Introduction: Acrylic bone cements are used in a variety of orthopedic applications including vertebral decompression and implant augmentation. However, they are bioinert with no ability to integrate with bone. We hypothesize that correction of this drawback can be achieved through the addition of the calcium phosphate (CaP), Brushite, providing a porous scaffold for bone interdigitation while retaining desirable mechanical, rheological, and handling properties.

Methods: Composite two solution cement samples were prepared by Brushite addition yielding a pre‐mixed cement with up to 75% of the polymer replaced. Viscosity was assessed using frequency sweeps on non‐setting cements with a DHR‐3 Rheometer. Compression tests followed the ASTM F451 standard using a Bionix MTS 370. To study injectability, a pneumatic gun was used to extrude 1mL of cement through the system and the time measured.

Results: CaP addition was shown to be feasible with no deleterious effect on cement mechanical properties. Rheology showed that the variation in viscosities between samples is not significant (p<0.05) and pseudoplasticity was sustained. Injectability of the cement formulations varied considerably, while some showed “plugging” after the cartridge was emptied.

Significance: The versatility of the pre‐mixed two solution bone cement allows for the incorporation of CaP fillers thereby enhancing interdigitation with bone.

Grant Funding Source: Supported by The University of Texas System, UT Transform Award (Dr. Danieli Rodrigues)
Creating 3D virtual models from CT and MRI for anatomy education (923.2)
Authors: Chen Jian , Pavlov Anna , Smith Andrew , Sinning Allan , Conway Marianne , Cui Dongmei
Published: June 2021

CT and MRI are gaining more attention in contemporary anatomy education due to the ability to create detailed 3D constructions, made possible by thinner sections. 3D virtual models are particularly helpful for understanding the spatial orientation of anatomical structures in the mind’s eye before performing a dissection. We use AMIRA® software to create 3D structures from CT and MR images. AMIRA allows for the development of 3D reconstructions by the following steps: (1) select image data into working pool; (2) outline and tag segmentations from sections (axial, coronal, or sagittal); (3) connect segmentations from all images ensuring the same orientation; (4) generate the surface; and (5) modify the 3D structure presentation. 3D structures from thin MRI and CT sections give more detail to students. AMIRA® has many advantages in 3D image creation, including a simple and straightforward creation process, friendly and convenient user interface, clear demonstration of detailed structures due to a high voxel, ability to see not only the outside but the inside of structures with the navigation tool, and quickly generating surfaces from MRI in only one step. High quality 3D reconstructed images can be used as a strong tool for students and residents outside of the anatomy lab or the operating room due to the ability to create lifelike, space‐oriented structures from real CT and MRI images.
A half‐sized écorché by Talrich (19th century) description of an anatomical model in plaster on a posterior view (923.3)
Authors: Le Floch‐Prigent Patrice , Gillot Jean‐Baptiste
Published: June 2021

During the 19th century, the anatomical models in plaster were frequent but were still in balance between the expressionism of the previous centuries and the cold reality of the anatomical dissections. This half‐sized, posterior view of a model of Jules Talrich who was an official molder in the “Faculté de Médecine de Paris” in this times (1856) was exactly between this two extremes with a tendancy to a complete respect of the representation of the anatomical structures. The écorché was in a prone position near the anatomical position of reference with the exception of an abduction of 15° degree of each lower limb and a left upper limb with the hand laid on the occiput, the arm in elevation and the elbow flexed at 90°. The different layers of the represented muscles were described in each main region. The representation of the muscles were noticeable different between the two lower limbs. The anatomical veracity of the model was very good and the artistic rendering was limited. A metallic plate embedded in the plaster permitted to authentify its author as Talrich. The model was 87cm high, 56cm wide and 11cm thick. It was posteriorly hollowed on the opposite side and not colored.
ITCH E3 ubiquitin ligase positively regulates TGF‐β signaling to EMT via Smad7 ubiquitination (930.6)
Authors: Woo Chang‐Hoon , Lim Jae Hyang
Published: June 2021

Transforming growth factor‐beta (TGF‐β) has been shown to regulate pleiotropic cellular responses including cell growth, differentiation, migration, apoptosis, extracellular matrix production, and many other biological processes. Although non‐Smad signaling pathways are increasingly being reported to play many roles in TGF‐β‐mediated biological processes, Smads, especially recetor‐regulated Smads (R‐Smads), still play a central role for mediating TGF‐β signaling to endothelial mesenchemal transition (EMT) processes. Biological activity of R‐Smads is thus tightly regulated in multiple points. Inhibitory‐Smad (I‐Smad) Smad7 act as critical endogenous negative feedback regulator for Smad‐signaling pathways by inhibiting R‐Smad phosphorylation and also by inducing activated type I TGF‐β receptor degradation. Emerging roles of Smad7 in health and disease have been increasingly reported, the molecular mechanisms that regulate Smad7, however, are not well understood yet. In this study, we have shown that E3 ubiquitin ligase ITCH acts as a positive regulator of TGF‐β signaling and subsequent EMT‐related gene expression. Interestingly, ITCH‐mediated positive regulation of TGF‐β signaling was dependent on Smad7 ubiquitination and subsequent degradation. Further study revealed that ITCH acts as an E3 ubiquitin ligase for Smad7 polyubiquitination, thereby acting as an important regulator for Smad7 activity and positively regulating TGF‐β signaling and TGF‐β‐mediated biological processes. Our findings thus uncover new regulatory mechanism controlling Smad7 by ITCH.
Structural and chemical perspectives of RNA polymerase II transcriptional fidelity (939.5)
Authors: Wang Dong
Published: June 2021

Maintaining a high transcriptional fidelity is essential to life. A long‐standing key question has been how RNA polymerase II (Pol II) recognizes DNA template and substrate and achieves such a high transcriptional fidelity. Here we use a combined structural, computational, and chemical biology approaches to gain novel insights of the molecular basis and chemical interactions in controlling Pol II transcriptional fidelity. We focused on the three key checkpoint steps in controlling transcriptional fidelity: specific nucleotide selection and incorporation, differentiation of RNA transcript extension of a matched over mismatched 3’‐RNA terminus, and preferential removal of misincorporated nucleotides from the 3’‐RNA end (proofreading). Our recent work on the systematic evaluations of individual chemical interactions and the roles of sugar integrity in controlling Pol II transcriptional fidelity will be discussed.

Grant Funding Source: NIH GM085136, NIH GM102362
Regulation of acetyl‐CoA homeostasis and global histone acetylation (942.1)
Authors: Galdieri Luciano , Zhang Man , Vancura Ales
Published: June 2021

Acetyl‐CoA is a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Concentration of acetyl‐CoA affects histone acetylation and links intermediary metabolism and transcriptional regulation. We have identified acetyl‐CoA carboxylase, encoded in yeast by the ACC1 gene, as the key regulator of acetyl‐CoA homeostasis. Acc1p catalyzes the carboxylation of acetyl‐CoA to malonyl‐CoA, the first and rate‐limiting reaction in the de novo synthesis of fatty acids. Attenuated expression of ACC1 results in increased cellular level of acetyl‐CoA, increased histone acetylation and altered transcriptional regulation. The glucose sensor Snf1p, the yeast ortholog of the mammalian AMP‐activated protein kinase (AMPK), phosphorylates and inactivates Acc1p. Inactivation of Snf1p results in an increased conversion of acetyl‐CoA to malonyl‐CoA, reduced pool of cellular acetyl‐CoA, and globally decreased histone acetylation. Collectively, our results demonstrate that signaling pathways that affect acetyl‐CoA homeostasis regulate global histone acetylation and transcriptional regulation. Our current experiments test the hypothesis that global histone acetylation functions as a signal reflecting acetyl‐CoA homeostasis and metabolic state of the cell.

Grant Funding Source: Supported by NIH
Tryptophan fluorescence quenching kinetic studies of steps of open complex formation by E. coli RNA polymerase (945.1)
Authors: Xia Xue , Murray Jan , Record Thomas , Yang Zao
Published: June 2021

To initiate transcription, E. coli RNA polymerase (RNAP) binds promoter DNA, bends the start site region into the active site cleft, opens it using binding free energy, and places the start site base of the template strand in the active site. Structural studies of the open complex show that bending is accompanied by insertion of the ‐11 base into a pocket on the specificity (σ70) subunit. We are using stopped flow fluorescence measurements of tryptophan (W) quenching to investigate when in the mechanism of open complex formation this base‐flipping step occurs, and to characterize the kinetics of this step for different promoters for comparison with other kinetic assays for formation of closed and open complexes. For the λPR promoter at 42°C, tryptophan quenching kinetics fit two exponentials, as expected for the rapid‐equilibrium formation of an ensemble of closed complexes, including the base flipped species, followed by the slower irreversible opening step; rates of these steps are comparable to those determined by other assays. The kinetics are complicated by slower decays which appear to be photobleaching.

Grant Funding Source: NIH GM103061
QscR regulates expression of the formate dehydrogenase genes in Methylobacterium extorquens AM1 (946.9)
Authors: Valdez Rachael , Skovran Elizabeth
Published: June 2021

Methylobacterium extorquens is a model organism for understanding methylotrophic growth. It is of interest as a platform for production of value added chemicals, such as biofuel and bioplastics, from methanol which is inexpensive and readily available. During methanol growth, methanol is first oxidized to formate by methanol dehydrogenase and the formaldehyde activating enzyme. The cell then faces an important choice. Formate can be oxidized to CO2 producing cellular reducing power, or be converted to methylene‐H4F which is assimilated into biomass. Four formate dehydrogenases are known to exist in M. extorquens, though how these enzymes are regulated is unknown. QscR plays an important role during methylotrophic growth by activating expression of the Serine Cycle genes, which constitute the main carbon assimilation pathway during methylotrophic growth. QscR directly senses formyl‐H4F, an intermediate produced after formate but before the Serine Cycle. Microarray data suggest that QscR may also regulate the expression of the formate dehydrogenases genes. To assess in vivo regulation of formate dehydrogenase expression, fluorescent transcriptional reporter fusions were created. QscR was also purified and promoter binding was assessed. At least two of four formate dehydrogenase promoters were shifted by QscR. We also show that QscR derepresses its own transcription in the presence of formate suggesting that a feedback loop exists, which results in derepression of qscR expression when formate builds up and repression of qscR expression as levels of formate decrease and formyl‐H4F increases. This co‐metabolite sensing by QscR would allow the cell to coordinate carbon assimilation and dissimilation. A deeper understanding of the regulation at the formate branch point will be essential in engineering M. extorquens to balance energy and reducing power generation with carbon assimilation into value added chemicals.

Grant Funding Source: Supported by: San Jose State University Research, Scholarship and Creative Activity Grant
Assessing the orientations of lipoprotein Pal in the outer membrane of Escherichia coli (948.4)
Authors: MacPherson Victoria , Shaw Juliana , Pichichero Michael , Michel Lea
Published: June 2021

Pal is a well‐characterized peptidoglycan‐associated lipoprotein found in the periplasm of Escherichia coli (E. coli). Although the biological function of Pal is not understood, the protein has been shown to interact with Tol B, Lpp, and several other periplasmic proteins. We recently discovered that Pal’s homologue, P6 of nontypable Haemophilus Influenzae (NTHi), is inserted into the outer membrane in two orientations—one facing in toward the periplasm and one facing out toward the extracellular space. One other lipoprotein, Lpp of E. coli, was also shown to exhibit two orientations in the outer membrane. In an effort to collect data in support of our hypothesis that lipoprotein dual orientation is a more widespread phenomenon, we utilized confocal microscopy and flow cytometry to assess the localization and orientation(s) of Pal in E. coli. Our results demonstrate that Pal is indeed present on the surface of E. coli, suggesting that Pal is also a dual oriented lipoprotein.

Grant Funding Source: Supported by the Camille and Henry Dreyfus Special Grant Program in the Chemical Sciences.
Genetic variability and the quantitative proteome (95.3)
Authors: Aebersold Ruedi
Published: June 2021

The question how genetic variability is translated into phenotypes is fundamental in biology and medicine. Powerful genomic technologies now determine genetic variability at a genomic level and at unprecedented speed, accuracy and (low) cost. To date the effects of genomic variability on the expressed information of the cell has been mainly studied by transcript profiling. In this presentation we will discuss emerging computational and quantitative proteomic technologies to relate genotypic variation to the proteome. Proteomic data to support such correlations need to be quantitatively accurate, highly reproducible across multiple measurements and samples and generable at high throughput. Data with these qualities can now be generated by the targeted proteomic methods selected reaction monitoring (SRM) and, at higher throughput, by SWATH‐MS. We will discuss the principles of these mass spectrometric methods, discuss the computational challenged they pose for data analysis and demonstrate with selected applications their ability to determine the effect of genetic variability on the quantitative proteome.
The structure of a tail‐anchor membrane protein‐binding complex reveals the regulation of Get3 by Get4 (950.4)
Authors: Gristick Harry , Rao Meera , Chartron Justin , Rome Mike , Shan Shu‐ou , Clemons William
Published: June 2021

Tail‐anchored (TA) membrane proteins are targeted to the endoplasmic reticulum via the post‐translational GET pathway. A critical step in this essential pathway is the transfer of TA substrates from the co‐chaperone Sgt2 to the Get3 ATPase, mediated by the hetero‐tetrameric complex Get4/Get5 (Get4/5). However, a major outstanding question is how Get4 binds to Get3 and regulates its activity. Here we report a 5.4 Å crystal structure of an ATP‐bound Get3‐Get4/5 complex from Saccharomyces cerevisiae (Sc), the largest pathway components solved to date. Our structure reveals the role of nucleotide in complex formation and the presence of two functionally distinct binding interfaces. Mutational analysis confirms that one interface is required for the specificity of binding, while the other interface is involved in the regulation of Get3 ATPase activity. Additional functional assays demonstrate that Get4/5‐mediated regulation of ATP hydrolysis by Get3 is essential for efficient TA protein targeting. Our work also supports a model for Get3/TA complex formation in which two opposing Get3 dimers are bridged by a single Get4/5 hetero‐tetramer. Finally, we present a 2.8 Å crystal structure of an apo ScGet3‐Get4/5 complex that provides evidence for a rapid initial binding state mediated by electrostatics. This work illustrates how Get4/5 regulates Get3, priming it for TA loading, a critical step in this important pathway.

Grant Funding Source: NIH
The role of a ubiquitin processing protease in Dictyostelium development (952.4)
Authors: Plubell Deanna , Knotts Alice , Lindsey David
Published: June 2021

The ubiquitin processing protease, UbpA (a homolog of yeast Ubp14 and human IsoT/USP5), is required for the growth‐to‐development transition of Dictyostelium. When ubpA¯ cells starve, they accumulate ubiquitin‐containing species that likely include ubiquitin conjugates of proteins that must be degraded for the growth‐to‐development transition to occur. In previous work, we tentatively identified these proteins by S5a‐affinity chromatography and MuDPIT mass spec analysis. Among the putative ubiquitin conjugates was PsmC5. Here we report that a ubiquitination assay showed that his‐tagged PsmC5 is ubiquitinated in lysate prepared from starving cells. In other experiments, we have shown that UbpA is required for the growth‐stage expression of lmcA, which is involved in sensing nutrient levels and signaling cells to develop upon starvation. Overexpression of lmcA partially rescues the defect in ubpA¯ cells, suggesting that the ubpA¯ phenotype is at least partially due to the absence of LmcA. To identify interacting partners of LmcA and elucidate LmcA‐dependent pathways, we purified TAP‐tagged LmcA expressed in Dictyostelium. When the putative LmcA‐interacting proteins were further purified by S5a‐affinity chromatography, PsmC5 was recovered, suggesting that ubiquitinated PsmC5 interacts with LmcA. These results will lead to a better understanding of the mechanisms cells use to sense starvation stress and respond by making the transition from growth to development.
Molecular mechanisms of substrate degradation by the 26S proteasome revealed by CryoEM (952.6)
Authors: Matyskiela Mary , Lander Gabriel , Martin Andreas
Published: June 2021

The 26S proteasome is the major eukaryotic ATP‐dependent protease, responsible for regulating the proteome through degradation of ubiquitin‐tagged substrates. The proteasome regulatory particle, containing the heterohexameric AAA+ ATPase motor and the essential deubiquitinase Rpn11, recognizes substrates, removes their ubiquitin chains, and translocates them into the associated peptidase after unfolding. Despite recent insights into the architecture of the regulatory particle, little is known about the structural requirements and mechanisms for substrate processing. Here, we present the first structure of the 26S proteasome during substrate degradation and show that it switches from a pre‐engaged to a translocation‐competent conformation. This conformation is characterized by a rearranged ATPase ring with uniform subunit interfaces, a widened central channel coaxially aligned with the peptidase, and a spiral orientation of pore loops that suggests a rapid progression of ATP‐hydrolysis events around the ring. Importantly, Rpn11 moves from an occluded position to directly above the central pore, facilitating substrate deubiquitination concomitant with translocation.

Grant Funding Source: Supported by NIH grant R01‐GM094497‐01A1, ACS grant 121453‐PF‐11‐178‐01‐TBE
Membrane fluidity and lipoperoxidation of placental mitochondria in diabetic rats (953.2)
Authors: Figueroa‐García María del Consuelo , Cortés‐Rojo Christian , Saavedra‐Molina Alfredo , Mejía‐Zepeda Ricardo
Published: June 2021

Oxidative damage on biological molecules has been proposed as a major cause of alterations observed during diabetes. In this study, using the excimer forming monitor dipyrenylpropane (DPyP), we measured membrane fluidity of placental mitochondria in diabetic rats with 19 days of gestation and its respective normoglycemic controls. A significant increment (p < 0.001) of mitochondrial membrane fluidity was found in diabetic rats, along with MDA increment. Physicochemical parameters had changes, i.e. lower activation energies in gel (23.0 ± 5.0 vs. 18.2 ± 4.0 kJ/mol) and liquid‐crystalline (13.9 ± 1.3 vs. 12.0 ± 0.4 kJ/mol) phases in the diabetic group (p < 0.05); the transition temperature was lower in diabetic (37.8 ± 1.6 vs. 30 ± 2.0 °C), p < 0.05. There were several changes in fatty acid composition. These results provide further evidence that oxidative stress may play a role in the increment of mitochondrial membrane fluidity during pregnancy in diabetic rats despite the physical effects of lipoperoxides in membranes.

Acknowledgements: CONACYT postdoctoral Fellowship to MCFG; financial support from DGAPA‐PAPIIT UNAM to RMZ; financial support from CIC‐UMSNH (2.16 to ASM) and CONACYT (169093 to ASM).
Macromitophagy is a longevity assurance process that in chronologically aging yeast limited in calorie supply sustains functional mitochondria and maintains cellular lipid homeostasis (953.3)
Authors: Titorenko Vladimir , Beach Adam , Richard Vincent , Leonov Anna , Piano Amanda , Feldman Rachel
Published: June 2021

Macromitophagy controls mitochondrial quality and quantity. It involves the sequestration of dysfunctional or excessive mitochondria within double‐membrane autophagosomes, which then fuse with the vacuole/lysosome to deliver these mitochondria for degradation. To investigate a physiological role of macromitophagy in yeast, we examined how the atg32Δ‐dependent mutational block of this process influences the chronological lifespan of cells grown in a nutrient‐rich medium containing low concentration of glucose. Under these longevity‐extending conditions of caloric restriction (CR) yeast cells are not starving. We also assessed a role of macromitophagy in lifespan extension by lithocholic acid (LCA), a bile acid that prolongs yeast longevity under CR conditions. Our findings imply that macromitophagy is a longevity assurance process underlying the synergistic beneficial effects of CR and LCA on yeast lifespan. Our analysis of how the atg32Δ mutation influences mitochondrial morphology, composition and function revealed that macromitophagy is required to maintain a network of healthy mitochondria. Our comparative analysis of the membrane lipidomes of organelles purified from wild‐type and atg32Δ cells revealed that macromitophagy is required for maintaining cellular lipid homeostasis. We concluded that macromitophagy defines yeast longevity by modulating vital cellular processes inside and outside of mitochondria. Supported by NSERC of Canada.
Comparison of different techniques for monitoring of mammalian cell respiration (960.4)
Authors: Kondrashina Alina , Dmitriev Ruslan , Papkovsky Dmitri
Published: June 2021

Cell and tissue oxygenation in vivo are maintained within narrow physiological range and their deviations from the norm lead to pathological states such as ischemia, cancer and inflammation. The selection of the analytical method for O2 monitoring is dictated by the required experimental design, sensitivity, throughput and measurement task. Comparison of different cell respiration techniques is important for their standardisation and benchmarking. Using intracellular and extracellular phosphorescent oxygen probes, we measured respiration of MEF cells in hermetically sealed quartz cuvettes, 96 well plates sealed with mineral oil on top of the samples (partial seal), and local oxygenation of resting cells in an open plate. In the latter cell oxygenation levels were shown to be determined by their respiration activity and ambient O2, whereas in quartz cuvettes absolute oxygen consumption rates (OCR) were directly proportional to cell numbers. Measurements of oxygen concentrations were performed with adherent and suspension, resting and stimulated cells on both platforms in parallel employing intracellular oxygen sensitive probe. We found a sigmoidal relationship between OCR and oxygenation with a linear correlation in the range 20‐200 μM O2 and leveling off at both high and low cell numbers which indicates changes in mitochondrial oxidative phosphorylation.

Grant Funding Source: Supported by EU Grant Agreement number PITN‐GA‐2010‐264772 (ITN CHEBANA)
Cytokinesis requires supervillin‐mediated binding to myosin II and synergism with anillin (962.1)
Authors: Luna Elizabeth , Smith Tara , Fridy Peter , Li Yinyin , Basil Shruti , Arjun Sneha , Friesen Ryan , Leszyk John , Chait Brian , Rout Michael
Published: June 2021

Cytokinesis requires coordination of cortical myosin II activation with central spindle alignment. Supervillin and anillin are two actin‐ and myosin II‐associated vertebrate proteins implicated in maintenance of the cytokinetic furrow in early cytokinesis, The nature of their cross‐talk is unknown. Point mutations within an evolutionarily conserved sequence in supervillin, residues 99 ‐ 153, eliminate binding to the nonmuscle myosin II heavy chain (MHC), confer dominant‐negative inhibition of cytokinesis, and abrogate the ability of full‐length supervillin to rescue RNAi‐mediated depletion of supervillin. While an observed ~2‐fold increase of anillin in supervillin‐knockdown cells does not eliminate cytokinetic failure, knockdown of both proteins together yields more than additive increases in numbers of multinucleated cells, as compared with depletion of either protein alone. Localizations of supervillin, anillin, MHC (total myosin), and phosphorylated regulatory myosin II light chain (pMRLC, activated myosin) in dividing cells suggest that supervillin and anillin synergize to confine MHC to the furrow. Anillin alone is sufficient to maintain normal levels of pMRLC at the furrow in late anaphase, but supervillin is required for normal pMRLC localization in early anaphase and around the cytokinetic bridge during daughter cell separation. Protein affinity isolations of GFP‐tagged supervillin and anillin indicate that these proteins do not significantly associate with each other although myosin II, actin and other cytoskeletal proteins co‐purify with each bait protein. None of these proteins co‐isolated with GFP alone. Stark differences between specifically co‐isolating kinases, phosphatases, and other signaling proteins reinforce the conclusion that supervillin and anillin regulate cytokinesis through separate pathways.

Grant Funding Source: Supported by the National Institutes of Health and the University of Massachusetts Medical School.
Identification of key components involved in mtDNA inheritance during cell division (965.1)
Authors: Camacho Beatriz , Osman Christof , Walter Peter
Published: June 2021

Majority of cellular DNA is located in the cell nucleus, however the mitochondrion also possesses DNA. Mitochondrial DNA (mtDNA) is packaged into protein DNA complexes called nucleoids required for production of ATP. Mutations in mtDNA inheritance contribute to human diseases. Despite its importance, little is known about the mechanisms. Using live cell microscopy in yeast, it was established that mitochondrial nucleoids localize to the tips of mitochondrial tubules, which migrate into the daughter cell during cell division, ensuring mtDNA inheritance. Tip localization of mtDNA lead us to hypothesize that there is protein or complexes of proteins that anchor mtDNA to mitochondrial tips. To identify and characterize these molecular factors, a novel genetic approach was designed using Saccharomyces cerevisiae that relies on synthetic biology. mtDNA was artificially tethered to the inner mitochondrial membrane by expression of a synthetic protein. This synthetic protein consists of Yme2, a transmembrane protein of the inner mitochondrial membrane, and the LacI protein that binds LacO repeats, which were inserted into the mtDNA. The presence of this synthetic tether renders the endogenous protein complex that anchors mtDNA to the membrane dispensable. Using a genetic tool known as white and red markers, a genetic sectoring screen was performed to identify tethering components. White, sector and red colonies were obtained, but only red colonies indicated dependence on the artificial tether for mtDNA. Red colonies were isolated for further identification of the molecular factors that tether mtDNA to mitochondrial tips. This novel genetic approach will impact our understanding of mtDNA metabolism.
Mechanism of preventing doxorubicin‐induced mitochondrial toxicity with cardiolipin‐targeted peptide, SS‐31 (966.1)
Authors: Quach Bonnie , Birk Alex , Szeto Hazel
Published: June 2021

Doxorubicin (DOX) is an effective chemotherapeutic that intercalates DNA and inhibits topoisomerase II in cancer cells. However, clinical use of DOX is limited by its cardiac toxicity, resulting from its ability to bind to cardiolipin (CL) and inhibit mitochondrial function. SS‐31, a mitochondria‐targeted tetrapeptide was shown to prevent DOX‐induced toxicity in cardiac and skeletal muscle, and cultured myocytes. However, the mechanism of protection against DOX by SS‐31 is not well understood. Here, by taking advantage of the intrinsic fluorescence of DOX, we developed assays to measure DOX binding to free CL, CL‐containing liposomes, mitoplasts and mitochondria. We demonstrated that SS‐31 prevents DOX binding in different CL‐containing model systems. Furthermore, we showed that SS‐31 could inhibit DOX binding to endogenous CL and uptake in mitoplasts and mitochondria in a dose dependent manner. Therefore, SS‐31 protects against mitochondrial DOX toxicity by preventing its binding to CL. Targeting CL with SS‐31 has clinical implications in increasing the therapeutic window of DOX and potentially addressing other therapeutics with off‐target side effects associated with CL and mitochondrial toxicity.
Limitations of modified substrate recognition by Taq DNA polymerase mutants (967.2)
Authors: Hadley Emma , Chia Hannah , Leconte Aaron
Published: June 2021

DNA Polymerase, which is required for enzymatic DNA synthesis, does not recognize or incorporate 2’ modified substrates although they are useful in a number of biotechnology applications. Through directed evolution, mutant polymerases have been identified with the ability to incorporate 2’ modified substrates; however, they are incapable of full primer extension limiting their use. Here we compare the ability of 5 previously evolved enzymes to extend with 2’OH and 2’OMe modified nucleotides. Comparative studies reveal stark differences in activity amongst previously evolved mutants. In particular, we observe that while all of the enzymes are capable of incorporating a modified nucleotide onto an unmodified primer with moderate to high efficiency, there are significant differences between enzymes in the rate of incorporation of additional modified nucleotides onto the modified primers. Understanding the limitations of modified substrate incorporation, in conjunction with other mutant polymerase characterization currently being done, will help guide future endeavors to rationally design mutant polymerases that can overcome these extension limitations.
Comparative characterization of Taq DNA polymerase mutants capable of recognizing 2' modified nucleotides (967.3)
Authors: Leconte Aaron , Chia Hannah , Hadley Emma , Gochi Andrea , Valencia Alfredo
Published: June 2021

In addition to encoding all known life, DNA is also an invaluable biotechnological tool. Chemical modifications to DNA, such as 2’ modifications, are expected to increase the biotechnological utility of DNA; however, these modified forms of DNA are limited by their inability to be effectively synthesized by DNA polymerase enzymes. Previous efforts have identified mutant Thermus aquaticus DNA polymerase I (Taq) enzymes capable of recognizing 2’ modified DNA nucleotides. While these enzymes recognize these modified nucleotides, they are not capable of synthesizing DNA containing more than six to eight modified nucleotides. Here, we describe comparative biochemical studies that seek to better understand these previously evolved enzymes through quantitative characterization. We describe the identification of specific amino acids and combinations of amino acids that contribute most to unnatural activity. Notably, we have also found that these enzymes are capable of using substrates beyond the originally selected substrates, introducing the possibility of new evolved enzyme‐modified substrate pairs. Collectively, through quantitative characterization of mutations, substrate specificity, extension efficiency and structure, we hope to elucidate key features of previously evolved DNA polymerases. These studies should inform future DNA polymerase engineering efforts, whether via rational design or directed evolution.
A protein crystallography and small molecule informatics‐based discovery engine for the development of selective chemical probes for in vivo investigations of protein kinases (968.3)
Authors: Grum‐Tokars Valerie , Roy Saktimayee , Minasov George , Anderson Wayne , Watterson D.
Published: June 2021

There is a critical need for selective serine‐threonine protein kinase (S/T PK) inhibitors appropriate for in vivo studies. We used a discovery engine based on high resolution co‐crystallography of PKs in complex with ligands and robust databases for small molecules with documented in vivo properties and potential for good ADMET property development (Chico et al., 2009a, 2009b). As a proof‐of‐concept, we targeted the active site of the stress activated p38α MAPK, a well‐studied target that has been intractable for PK specificity when targeting the active site. Further, we focused on the especially difficult challenge of p38α MAPK involvement in CNS pathology progression where it is activated by diverse stressors in both glia and neurons as part of a synaptic dysfunction cycle. Novel, highly selective, CNS penetrant, active site bound, p38α MAPK inhibitors were developed and validated by large scale kinome and pharmacological screens, cellular target engagement, and in vivo proof of efficacy (Watterson et al., 2013).

Grant Funding Source: NIH grants R01 NS056051, R01 AG031311, U01 AG043415
Screening of secondary metabolites biosynthesized with novel amino acid carrier protein system (970.3)
Authors: Matsuda Kenichi , Hasebe Fumihito , Tomita Takeo , Shiwa Yu , Yoshikawa Hirofumi , Shin‐ya Kazuo , Kuzuyama Tomohisa , Nishiyama Makoto
Published: June 2021

LysW has been identified as a carrier protein in lysine biosynthetic pathway of hyperthermophilic bacteria Thermus thermophilus that protect the unstable intermediate and mediate latter step reactions efficiently. Recently it was revealed that LysW function as a carrier protein not only in lysine biosynthesis but also in arginine biosynthesis in hyperthermophilic archaeon, Sulfolobus acidcaldarius, suggesting that LysW is widely possessed by many species. We found the gene encoding LysW homolog in genome of many Actinomycetes. These Actinomycetes possess lysine and arginine biosynthetic pathway same as general bacteria, suggesting that lysW homolog is involved in secondary metabolism. In this study, we aimed to investigate the diversity of the biosynthetic machinery employing the carrier protein, and to discover a variety of novel compounds biosynthesized with this system. PCR screening revealed that 50 Actinomycetes strains out of 848 possess lysW homologs. Draft genome sequence analysis of 32 strains possessing lysW homologs revealed that various PKS and NRPS genes are encoded in the vicinity of lysW homologs, suggesting that lysW homologs are involved in biosynthesis of various secondary metabolites. The vicinity region of lysW homolog was cloned with cosmid vector and introduced into S. lividans TK23. The recombinant S. lividans TK23 produced a novel compound.
Characterization of glycosyltransferases in the biosynthetic pathway of capsular polysaccharide A (970.4)
Authors: Sharma Sunita , Troutman Jerry
Published: June 2021

Capsular Polysaccharide A (CPSA) is a polymer of a tetrasaccharide subunit found on the surface of the symbiotic gut bacteria Bacteroides fragilis. It has been suggested that CPSA is vital for maintaining a normal balance between the various types of immune cells in a healthy mammalian host. If this balance is disturbed, various autoimmune disorders may develop in the human body. Our laboratory is applying an in vitro method to enzymatically synthesize CPSA, which is assembled on a bactoprenyl diphosphate scaffold one sugar at a time. The initiating sugar transferase, WcfS has been previously identified and characterized. Utilizing reverse phase HPLC, and fluorescent bactoprenyl monophosphate analogues we will describe the functional characterization of the remaining glycosyltransferases involved in CPSA biosynthesis, WcfN, WcfP and WcfQ.

Grant Funding Source: NIH