bluuebunny/crossref_metadata_2025 huggingface dataset:
Sex Differences in the Severe‐ and Extreme‐Intensity Power‐Duration Relationships
Authors: Hurla Logan M. , Alexander Andrew M. , Didier Kaylin D. , Hammer Shane M. , Barstow Thomas J.
Published: May 2022

We have previously shown that, similar to the severe‐intensity power duration relationship (i.e., W’sev), there exists a separate, smaller power‐duration relationship for the extreme‐intensity domain (W’­ext) in men. We have also shown that there may be differences in contributions of central and peripheral fatigue between men and women during and immediately following extreme‐intensity exercise. The current study tested the hypotheses that 1) the W’extwould be significantly less than W’sev in both sexes, but not different between men and women, 2) absolute Maximum Voluntary Contractions (MVC) would be reduced to greater extent at end‐exercise following severe‐ and extreme‐intensity exercise in men compared to women but not when scaled relative to baseline, and 3) peripheral fatigue would be greater at task failure (Tlim) in men compared to women following severe‐intensity exercise, with no sex difference following extreme‐intensity exercise. Following familiarization, 7 men and 7 women completed three severe‐ (Tlim: 2‐4 min, S3; 5‐7 min, S2; 8‐12 min, S1) and three extreme‐intensity (70, 80, 90% MVC) isometric knee‐extension bouts to task failure. MVC and potentiated twitch force (Qpot) were measured and compared at baseline, task failure, and 150 s of recovery. W’ext was significantly smaller compared to W’sev in men (2.4 ± 1.2 kJ vs 3.9 ± 1.3 kJ; p = 0.03) and in women (1.6 ± 0.8 kJ vs 2.9 ± 1.7 kJ; p = 0.05), however there were no sex differences in either W’extor W’­sev. There were no sex differences in MVC at task failure following severe‐intensity exercise. However, MVC was lower in women following extreme‐intensity exercise compared to men, although there were no sex differences in MVC relative to baseline. Further, there were no differences in MVC by the end of recovery compared to baseline in men or women following extreme‐intensity exercise.

Qpot was not different at task failure following severe‐ and extreme‐intensity exercise between men and women. However, men showed lower Qpot relative to baseline, suggesting greater peripheral fatigue development, with the exception of exercise at 90% MVC. These data provide evidence that important sex differences exist in the contributions of peripheral fatigue to task failure and in recovery from exercise which depend on exercise intensity. Therefore, practitioners need to consider exercise intensity and the time course of recovery when prescribing exercise to men and women.
Aberrant Silencing of Ribosomal DNA Initiates a Global Stress Response that Promotes H3K9‐mediated Adaptation
Authors: Larkin Ajay J. , Ragunathan Kaushik
Published: May 2022

The covalent modification of histones establishes distinct phenotypic or epigenetic states without altering the underlying genetic code. This process allows cells to respond to acute changes in cellular homeostasis resulting from alterations in intracellular, environmental, or metabolic conditions. The consequences of post‐translational modifications of histones are multifaceted, leading to the emergence of chemotherapy resistance in cancers, drug resistance in fungal pathogens and temperature‐dependent initiation of flowering in plants. One modification, histone H3 lysine 9 methylation (H3K9me), is associated with heterochromatin formation, and under conditions of acute stress, heterochromatin relocates to silence novel genes that enhance cellular fitness. It is still a mystery how cells activate this adaptive pathway and mark specific genes with H3K9me to successfully adapt to adversity. We find that, in S. pombe cells, adaptive H3K9me is preceded by activation of a global stress response and downregulation of ribosome biogenesis machinery. Our results indicate that aberrant silencing of the ribosomal DNA repeats triggers adaptation via H3K9me and allows cells to respond to complex stresses by developing novel heritable phenotypes. My completed work provides me unprecedented temporal resolution into H3K9me adaptation and sets the foundation for investigation of more complex questions involving the H3K9me adaptive process.
Biochemical Characterization of the Radical SAM Methylase Involved in Tetrahydromethanopterin Biosynthesis in Methanogenic Archaea
Authors: McKinney Justin , Tunckanat Taylan , Clohan Katherine , Duin Evert , Allen Kylie
Published: May 2022

Tetrahydromethanopterin (H4MPT) is a tetrahydrofolate (H4F) analog that serves as a one‐carbon carrier cofactor in the methanogenesis pathway in methanogenic microorganisms. One major structural feature that distinguishes H4MPT from H4F and other pterin‐containing biomolecules is the presence of methyl groups at the C7 and C9 positions. Although the biosynthesis of H4MPT has been relatively well‐defined, the enzyme responsible for the methylation reactions at the unreactive carbon centers has remained unclear. Our previous work identified the radical S‐adenosyl‐L‐methionine (SAM) methylase, MjMptM, from Methanocaldococcus jannaschii that likely catalyzes the addition of both methyl groups during H4MPT biosynthesis. Our current data indicate that MjMptM does not utilize SAM as the methyl group donor and instead uses methylenetetrahydrofolate for this purpose, thus making MjMptM the founding member of “Class D” radical SAM methylases. Here, we describe our recent progress towards the biochemical and spectroscopic characterization of MjMptM. We demonstrated that the enzyme catalyzes two methylation reactions to produce a dimethylated folate species using methylenetetrahydrofolate as the C1 source for the in vitro methylation reactions and dihydrofolate as the in vitrosubstrate. Site‐directed mutagenesis and UV‐Vis spectroscopic analysis indicate that MjMptM harbors at least two [4Fe‐4S] clusters which reside in the two canonical CX3CX2C radical SAM motifs in the N‐terminus. This result was further confirmed by EPR spectroscopy, which shows the binding of at least two [4Fe‐4S] clusters with unique signals. Only one of the [4Fe‐4S] clusters catalyzes the reductive cleavage of SAM to produce 5’deoxyadenosine, the key first step in radical SAM enzyme catalysis, and thus the most N‐terminal auxiliary cluster is likely involved in electron transfer. Current work is focused on elucidating the details of the novel methylation reaction mechanism.
Identification of protein targets for NADPH oxidase 5 mediated oxidative modification
Authors: Sweeny Elizabeth A.
Published: May 2022

NADPH oxidase 5 (NOX5) is a transmembrane signaling enzyme that produces superoxide in response to calcium binding. NOX5 has been identified as a key player for a variety of physiological and pathological conditions, yet a comprehensive understanding of its role in the cell has been lacking. Here we present a workflow to identify NOX5 protein partners as well as proteins which become oxidatively modified in a NOX5 dependent manner. These protein candidates can then be assessed for their role in mediating NOX5 dependent signaling in the cell which is involved in cellular differentiation, angiogenesis, and human disease. Investigating these results will allow us to understand how NOX5 carries out its physiological roles as well as provide indications for how dysregulation can lead to the initiation and progression of disease.
The Renin‐Angiotensin System after Pig Kidney Transplantation in Baboons
Authors: Navar L. G. , Hansen‐Estruch Christophe , Bikhet Mohamed H. , Javed Mariyam , Katsurada Akemi , Satou Ryousuke , Shao Weijian , Ayares David , Cooper David K. C. , Judd Eric
Published: May 2022

Resolution of many technical and immune‐related issues have allowed long‐term survival of baboons with life‐supporting pig kidney grafts. However, episodes of hypovolemia and hypotension during the post‐transplant period occur via an unexplained mechanism. It has been reported that pig renin has low reactivity on human angiotensinogen (AGT). Because the renin‐angiotensin system (RAS) is vitally important in the regulation of extracellular fluid volume and blood pressure, we evaluated the activity of the RAS in baboon’s post‐pig kidney xenotransplantation. Kidneys from genetically‐modified pigs were transplanted into 5 baboons (6 to 10 kg) that were immunosuppressed by blockade of the CD40/CD154 costimulation pathway, after excision of the native kidneys. Plasma renin, AGT (only baboon AGT could be measured), and angiotensin II levels were measured by ELISAs in (i) healthy genetically‐modified pigs (n=3), healthy non‐immunosuppressed baboons (n=6), and (iii) immunosuppressed baboons with pig kidney grafts. Samples were collected for up to 120 days post‐transplantation. Plasma renin levels were significantly greater in healthy pigs (586±139 (SE) pg/ml) than in intact baboons (430±28 pg/ml). The plasma renin levels in baboons with pig kidney grafts were similar to values in intact baboons (367±18 pg/ml), indicating the continuing production of renin by the pig kidney grafts in absence of baboon kidneys. Plasma AGT (which reflected only baboon AGT) levels were augmented in the transplanted baboons (21±0.8 µg/ml) compared to the pre‐transplant concentrations (9.9±0.8). Plasma angiotensin II levels were 16±1.3 pg/ml in intact baboons and trended lower from pre‐transplant levels in the transplanted baboons (10±1.6 pg/ml). Lower plasma angiotensin II concentrations in baboons with pig kidney grafts, even in the presence of increasing plasma AGT levels, may be a consequence of reduced, but not absent, reactivity of pig renin to cleave baboon AGT. The reduced angiotensin II levels in the transplanted baboons suggest an impaired ability to elicit a robust response to hypovolemic and hypotensive episodes.
The Role of a Regulatory Protein in the Differing Biofilms of Wild Isolates
Authors: Pepaj Franchesca , Wacker Sarah
Published: May 2022

Biofilms are complex communities of microorganisms that attach to surfaces and are surrounded by a self‐produced extracellular matrix that cannot be penetrated by many antibiotics. The bacterial strain Bacillus subtilis is a model organism for studying biofilm formation. Wild isolates of B. subtilis often form more robust biofilms than the laboratory strain that is typically studied. Our goal is to identify genetic variations in wild isolates that alter the strength of their biofilm. We focused on the gene that codes for the biofilm‐inducing protein SinI that acts through binding and inhibiting the biofilm repressor SinR. We found some wild isolates have a coding variation in the gene for SinI that may impact binding to SinR. We hypothesize that this genetic variation is partially responsible for the altered phenotype seen in the wild isolates. A deletion mutation of SinI was generated to determine whether a biofilm can form without it. As expected, a SinI mutant was unable to form a biofilm. To test the importance of the genetic variation found in SinI, we cloned plasmids containing the gene for SinI with the wild isolate variation. These plasmids were used to add SinI back into the deletion strain. This project provides insight into how natural variation arises among wild isolates to create differing biofilm phenotypes.
Med13 Plays a Dual Role in Promoting P‐Body Assembly
Authors: Hanley Sara E. , Cooper Katrina F.
Published: May 2022

In response to stress, cells rapidly adjust gene expression, translation, and protein levels to mitigate damage and initiate repair pathways. The Cdk8 Kinase Module (CKM) is a highly conserved, detachable unit of the Mediator complex that plays a vital role in regulating transcription and communicating stress signals from the nucleus to other organelles. Previously, our laboratory has shown that the scaffold protein within the CKM, Med13, is exported from the nucleus and degraded via a selective autophagy pathway following nitrogen starvation. In periods of starvation, cells attenuate translation and biosynthetic pathways to reserve nutrient pools. To rapidly adapt to starvation, cytosolic ribonucleoprotein (RNP) granules such as processing bodies (P‐bodies) tightly regulate mRNA accessibility and translation. Here we show that Med13 plays a dual role in promoting P‐body assembly. In physiological conditions, Med13 functions as a positive transcriptional regulator of P‐body formation as the deletion of MED13 significantly decreases the quantity of P‐bodies. In addition, during stress Med13 co‐localizes with the P‐body marker, Edc3, suggesting that Med13 may also play a scaffolding role within these membrane‐less organelles. Taken together, these data demonstrate the duality of Med13 as a transcriptional regulator of P‐bodies in unstressed conditions and a scaffolding component during stress. The nutrient sensing kinase, Ksp1, plays an important role in mediating P‐body localization of Med13 and other mRNA binding proteins following nitrogen starvation. These data demonstrate a novel cytosolic role for Med13 and expand the known proteome of P‐bodies.
Inhibition of prelimbic cortical inputs to the patch compartment of striatum reduces habitual methamphetamine self‐administration
Authors: Horner Kristen Ashley , Bavadekar Vaishnavi , Dama Ashitha , Jain Maahi , Muchandi Hyma , Taylor Ashton , Ahn Jamin , Christy David J.
Published: May 2022

An imbalance in activity between that patch and matrix compartments of striatum is thought to result in inflexible and repetitive behaviors, such as psychostimulant‐induced stereotypy. Habitual drug abuse is also inflexible, suggesting that an imbalance in activity between the patch and matrix s may underlie addiction. In addition, the patch system receives information regarding reward from regions such as the prelimbic cortex, which likely also contributes to the development of habitual drug use. However, whether enhanced activation of the patch compartment is related to habitual drug use is not known. The current study examined the role of the patch compartment in the development of habitual methamphetamine (METH) use by inhibiting inputs from the prelimbic cortex, which projects specifically to the neurons of the patch compartment and carries information related to reward, emotion and motivation. Rats were bilaterally infused in the prelimbic cortex with the inhibitory designer receptor exclusively activated by designer drugs (DREADD) pAAV‐CaMKIIa‐hM4D(Gi)‐mCherry‐AAV5, or vehicle and allowed to recover for 6 weeks. The rats were then implanted with jugular catheters and trained to self‐administer METH using a random interval paradigm known to generate habitual behaviors. Following the acquisition of METH self‐administration, rats were then trained to associate METH with a negative stimulus, by pairing METH with lithium chloride or saline, as a control. Confirmation of lithium chloride‐induced aversion to METH was determined using conditioned place aversion. After the development of aversion to METH, animals were reintroduced to the operant chambers and given the opportunity to self‐administer METH. Our data indicates that inhibition of prelimbic inputs to the patch compartment using inhibitory DREADDs reduced METH self‐administration upon reintroduction to the operant chamber in animals that had developed an aversion to METH, indicating that habitual drug use may have been attenuated in these animals. Furthermore, these animals showed almost equal levels of activation in the patch and matrix compartments, while animals that continued to self‐administer METH showed an enhanced activation of the patch compartment relative to the matrix compartment. These data suggest that activation of the patch compartment by prelimbic cortex may be necessary for the development of habitual METH self‐administration.
Absence of Gli1 Decreases PDGFRβ Expression Following Ductal Ligation Injury
Authors: Altrieth Amber , O'Keefe Kevin , Feminella Sage , Gellatly Victoria , Nelson Deirdre , Larsen Melinda
Published: May 2022

Fibrosis results from excess extracellular matrix deposition, which alters normal tissue architecture and impedes function. Salivary gland fibrosis can be caused by irradiation therapy, as a treatment for head and neck cancer, as well as by the autoimmune disease Sjögren’s Syndrome. The mechanisms that contribute to fibrosis are complex and poorly understood; however, defining the cell types and signaling pathways involved could reveal potential therapeutic targets. Stromal cell subpopulations have been shown to contribute to fibrosis. Specifically, Gli1 is a transcription factor expressed by mesenchymal stem cell‐like cells, which can contribute directly to fibrosis by transdifferentiating into myofibroblasts following injury. In the salivary gland, it is unknown which stromal cells expand following fibrotic injury or if the Gli1+ cells contribute to fibrotic injury. We hypothesize that Gli1+ cells contribute to salivary gland fibrosis by undergoing a transdifferentiation into myofibroblasts. To induce salivary gland fibrosis, we used a ductal ligation model, where a ligation is placed on the main ducts feeding from the submandibular and sublingual glands to the mouth and performed immunohistochemistry. Following ligation injury, there is a progressive fibrotic response where both collagen deposition and actively remodeled collagen increase. There is also an expansion of the PDGFRα+ stroma and F4/80+ macrophage populations. Using single‐cell RNA‐sequencing data, we found that a majority of Gli1+ cells at embryonic day 16 also express PDGFRα, suggesting that Gli1+ cells are a subset of both the PDGFRα and PDGFRβ stromal cell subpopulations, as in other organs. After performing a ductal ligation surgery on glands of mice genetically null for Gli1, we found that trichrome and remodeled collagen area trend downwards and there is a significant decrease in PDGFRβ area. We used a lineage tracing strategy to track Gli1‐expressing cells in salivary glands of Gli1CreERT2;(CAG)ROSA26TdTomato mice. In response to injury, there was an expansion of Gli1+ cells with a subset of these cells beginning to express vimentin and PDGFRβ. As there was a significant decrease in PDGFRβ area following ligation injury in the absence of Gli1 function, Gli1 signaling may stimulate PDGFRβ expression. Our results suggest that although Gli1 signaling and Gli1+ cells do not significantly contribute to injury‐induced fibrosis in the salivary gland; PDGFRα+ and PDGRFβ+ stromal cells may be potential therapeutic targets.
Characterizing the transcriptional signature regulated by the AHR ligands TCDD and kynurenine in colon cancer cells
Authors: Perez‐Castro Lizbeth , Conacci‐Sorrell Maralice
Published: May 2022

AHR was first identified as the receptor that binds to the pollutant TCDD, which causes tumorigenesis. Since its identification, AHR has been credited to play roles in multiple aspects of carcinogenesis such as increased cellular proliferation, immune evasion, and metastasis. A wide variety of tumors express increased AHR in comparison to the tissue of origin, thus suggesting a positive correlation with tumorigenesis. While most of the research has been focused on the role of activated AHR, a topic that has not received enough attention is whether activation of AHR by different agonists leads to different transcriptional outputs. In this study, we investigated the immediate transcriptional output of colon cancer cell lines exposed to TCDD and Kyn in an AHR‐dependent manner. We found that the expression of CYP1A1, ALDH1A3, ABCG2, ADGFR1, SCIN, and ITPR1 were commonly activated by TCDD, and Kyn, suggesting that these are canonical targets of AHR in colon cancer cells. The activation of expression of CYP1A1, ALDH1A3 and ABCG2 suggests that xenobiotic response pathways are induced upon AHR activation, independent of its ligands. TCDD, which can induce carcinogenesis in various rodent models, induced a larger group of 40 genes, including several potential oncogenes, regulators of cell proliferation, cytoskeletal organization, and signaling. Additionally, we further investigated the effects of SCINin colonic cancer cells. SCINdisrupted proliferation of colon cancer cells when silenced. Gain of function of this gene in colon has not been studied. Our study indicates that this gene can be critical for attachment and activation of the Wnt pathway. Genes activated by AHR are crucial for multiple aspects of carcinogenesis and suggests that targeting AHR by using AHR antagonists could have therapeutic value by limiting cell growth.
Cellular and Mitochondrial Damage by the Antiviral agent Tenofovir in Renal Epithelial Cells
Authors: Valentovic Monica A. , Brown Kathleen C. , Murphy Rachel , Cremeans Brad , McGuffey Rachel , Cox Ashley
Published: May 2022

Tenofovir disproxil fumarate (TDF) is a highly effective HIV antiviral drug approved for treating Human Immunodeficiency Virus and Hepatitis B. It is one of the first line drugs used to treat HIV and is efficacious in both novel and treatment‐experienced patients. The prodrug is administered orally and deesterified to the active drug TFV. TFV is associated with nephrotoxicity. However, the precise mechanisms for cytotoxicity remain unknown. Our hypothesis is that TFV causes mitochondrial dysfunction including diminished ATP formation and oxidative stress. HK‐2 cells were seeded and grown to confluency for 48h followed by 24‐72h exposure to 0‐28.8uM TFV. The vehicle was phosphate buffered saline (PBS). Cell viability was assessed using the MTT assay. Mitochondrial dysfunction was examined by measuring mitochondrial oxygen consumption, ATP levels, cytochrome c leakage, and by assessing the Cell Energy Phenotype using an Agilent Seahorse system. Oxidative Stress was evaluated by studying protein carbonylation and 4‐hydroxynonenal (4‐HNE) adducts by Western blot. All groups were a minimum of 3 independent studies of different cell passages. Analysis of statistical differences were performed using a One or ANOVA with a 95% confidence interval, p<0.05. TFV reduced HK‐2 cell viability at 24‐72h as shown by the MTT Assay. TFV reduced ATP levels and increased cytochrome c leakage at 72 h compared to PBS‐control. Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) were decreased following 24‐72h exposure to 28.8uM TFV. TFV induced oxidative stress as protein carbonylation and 4‐HNE adduct formation were elevated relative to PBS controls. Protein expression of LC3BI and LC3BII were compared between vehicle and TFV treated groups. TFV mediated oxidative stress, induced mitochondrial damage as indicated by decreased ATP, OCR, and ECAR, and cytochrome c leakage.
Computational models elucidate dynamics in the intergenerational inheritance of mtDNA mutations
Authors: Franco Melissa , Cote‐L’Heureux Auden , Khrapko Mark , Woods Dori , Tilly Jonathan , Khrapko Konstantin
Published: May 2022

Mitochondria, widely studied for their crucial role in cell bioenergetics, carry independent genomes that are inherited in offspring maternally. Understanding of the dynamics of the mitochondrial genome (mtDNA) is critical to further study of a large host of diseases (such as MELAS and LHON) that are caused by mtDNA mutations. One of the key steps in the inheritance of mtDNA is the germline bottleneck in the development and proliferation of primordial germ cells, during which process the mtDNA copy number of these cells fluctuates greatly. This leads to key alterations in and increased variance of mutation frequencies and allows complex patterns of selection at the organellar and cellular level. Furthermore, there can be varying degrees or patterns of selection that are present throughout the bottleneck process. Gaining an understanding of how these pressures interact with other factors such as genetic drift and Muller’s ratchet can better elucidate the inheritance of mtDNA mutations. To date, however, there is little consensus on how this process occurs and though numerous models have been developed to explain trends in experimental data, a thorough analysis of the different components and parameters of these models and how they relate to biological systems is lacking. Drawing from extensive intergenerational datasets of pathogenic mutations in mice and humans, we develop models based on trends that appear in the transmission of mtDNA between mothers and their offspring. The combination of diverse datasets comprising high fidelity single molecule‐sequence and NGS data allows sufficient resolution to support detailed, multivariate analysis. We derive novel metrics for describing these data and create biologically informed simulations that can flexibly account for constant, variable, and multiphasic modes of selection. We apply these models to datasets generated from simulating bottlenecked cell proliferation, using previously proposed and newly derived bottleneck frameworks and population dynamics. The resulting mutational profiles allow deeper understanding of the mechanisms underlying the biological data and provide a foundation for further development of computational approaches to studying germline mtDNA. Our simulations thus represent a substantial step forward in the understanding of the complex inheritance of mitochondrial mutations.
Anti‐cancer Effects of Dual‐action Inhibitors of Nek2 and EGFR Kinases: Investigation of Synergy
Authors: Bhuiyan Ashif I. , Reghuvaran Santha Asha , Das Tuhin , Hernandez Dianne , Musayev Rafael , Ghoshal Sarbani , Talele Tanaji T. , Pathak Sanjai K.
Published: May 2022

While lung cancer is the leading cause of mortality among all cancer types for both men and women, breast cancer dominates cancer diagnosis among women patients. Never in mitosis gene A (NIMA)‐related kinase 2 (Nek2) is a member of NEK family, a Ser/Thr kinase, that is overabundant in both cancer types. Studies from our laboratory have shown that abnormal expression of Nek2 kinase can promote aggressive tumor behavior and metastasis. Nek2 overexpression also drives drug resistance. These tumors also overexpress another key oncogene, epidermal growth factor receptor (EGFR), that has shown to induce cell proliferation, angiogenesis, and tumorigenesis. Hence, concomitant inhibition of Nek2 and EGFR kinases can potentially be a promising therapeutic strategy for both lung and breast cancers. Our goal is to develop anticancer agents that targets inhibition of both Nek2 and EGFR activities. Our hypothesis is such dual‐action inhibitory agents will provide superior efficacy due to a more predictable PK/PD profile in vivo. In this work, we first test our hypothesis that dual inhibition of Nek2 and EGFR kinases by small molecules will indeed yield net signaling output that results in synergistic cancer cell death in A549 (lung) and MDA‐MB‐231 (breast) cells. Using a novel drug‐like pharmacophore identified in our laboratory, we have also developed small molecule inhibitors that inhibits both Nek2 and EGFR activities. We anticipate that these findings will render new opportunities toward development of targeted small molecule therapeutics for both lung and breast cancers.
Structures, Mechanism, and Functional Relevance of Filament Formation by SgrAI
Authors: Ghadirian Niloofar , Shan Zelin , Lyumkis Dmitry , Horton Nancy
Published: May 2022

SgrAI is a type II Restriction endonuclease (REases) enzyme from the bacterium Streptomyces griseus. It has been proposed that SgrAI protects the host DNA from off‐target cleavage activity, through a new regulation mechanism via filamentation. Binding of SgrAI to invading phage DNA activates the enzyme leading to DNA sequence specificity expansion from 3 recognition sites to 17 and increased DNA cleavage rate. Like other types of II REases, SgrAI cleaves DNA in a Mg2+ dependent manner. But unlike other REases that form homodimers and cleave near their 4‐6bp recognition sequence, SgrAI forms a filament when in its active form, and a dimer when found in its inactive form. In its active form, SgrAI exhibits an increased rate of DNA cleavage 200 to 1000‐fold faster. The mechanistic underlying these events remain unclear.

We have been investigating the details of SgrAI’s structure and cleavage activity on its secondary site DNA sequences to complete our understanding of SgrAI’s kinetic pathway and filament formation. In our recent discoveries, we described two new structures of the SgrAI enzyme, one is the Cryo‐EM structure of the filamentous form of SgrAI bound to intact primary site DNA, and the other one is a high‐resolution x‐ray crystal structure of apo SgrAI. These structures help us to understand important conformational changes that could be contributed to the catalytic mechanism of this enzyme.
Developing a diceCT brain atlas: Comparison of diceCT neuroanatomy and brain histology in a rat model
Authors: George Jacob , Green Todd , Vazquez‐Sanroman Dolores , Gignac Paul
Published: May 2022

Significant advancements and refinement of neurological imaging modalities can enable new understanding in nervous system organization. One recent example, diffusible iodine‐based contrast‐enhanced computed tomography (diceCT), is an iodine staining and X‐ray μCT‐imaging technique that allows for the differentiation of myelinated from unmyelinated nervous tissues at finer scales of spatial resolution than standard three‐dimensional (3D) brain‐imaging tools allow. DiceCT is versatile, capable of imaging specimens across several orders of magnitude in size. It enables large and complex structures such as human brains to be studied alongside those of less complex nervous systems (e.g., fish, frogs, rodents, birds). One key feature is that the staining agent can be removed, allowing the subsequent application of acetate, fluorescent, and immunofluorescent histology following diceCT imaging. Nevertheless, the relative novelty of diceCT as a neuroanatomical visualization tool also demands ground‐truthing by determining its practical reliably for distinguishing precise boundaries between brain structures. For example, some adjacent, tissue‐level neuroanatomical features are poorly differentiated due to staining similarity or inherently obscure boundaries (such as the thalamus, which houses many small white‐matter tracts). To address this, we compare 3D orientation‐matched image stacks of diceCT juvenile Sprague Dawley rat brains to the Paxinos and Watson atlas, a gold‐standard rat‐brain reference. Visual identification of brain structures was attempted for all brain regions. Cardinal boundaries for each structure's dorsal, ventral, medial, and lateral edges were scored as distinguishable or non‐distinguishable. Right/left staining asymmetries were also scored. We used this outline to compare and contrast the neuroanatomical structures identified by diceCT with those described by Paxinos and Watson. Preliminary results indicate that 42% of all scored structures were visible via most (e.g., 3–4) of their cardinal boundaries, whereas 25% of atlas structures showed no distinguishable boundaries using diceCT. Overall, diceCT rat‐brain sections align well with standard histological sections, and visibility of neuroanatomical features is similar to tissue‐based, 3D magnetic resonance imaging atlases of comparable resolution.
Mechanisms of mitochondrial promoter recognition in humans and other mammalian species
Authors: Zamudio‐Ochoa Angelica , Morozov Yaroslav , Sarfallah Azadeh , Anikin Michael , Temiakov Dmitry
Published: May 2022

Recognition of mammalian mitochondrial promoters requires the concerted action of mitochondrial RNA polymerase (mtRNAP) and transcription initiation factors TFAM and TFB2M. In this work, we found that transcript slippage results in heterogeneity of the human mitochondrial transcripts in vivo and in vitro. This allowed us to correctly interpret the RNAseq data, identify the bona fide transcription start sites (TSS), and assign mitochondrial promoters for >50% of mammalian species and some other vertebrates. The divergent structure of the mammalian promoters reveals previously unappreciated aspects of mtDNA evolution. The correct assignment of TSS also enabled us to establish the precise register of the DNA in the initiation complex and permitted investigation of the sequence‐specific protein‐DNA interactions. We determined the molecular basis of promoter recognition by mtRNAP and TFB2M, which cooperatively recognize bases near TSS in a species‐specific manner. Our findings reveal a role of mitochondrial transcription machinery in mitonuclear coevolution and speciation.
Detection of the Pin1‐ligand interactions using 19F NMR spectroscopy
Authors: Chen Xiao‐Ru , Igumenova Tatyana I.
Published: May 2022

Pin1 is a peptidyl‐prolyl cis‐trans isomerase with specificity towards the pSer/pThr‐Pro protein motifs. Through its cis‐trans isomerization activity, Pin1 regulates the stability of oncogenic and tumor suppressor proteins. The involvement of Pin1 in cancer development makes it an attractive target for therapeutic intervention. To that end, a wide variety of small‐molecule inhibitors were developed to specifically target Pin1. A major challenge that remains in the Pin1 field is the high percentage of false‐positive readouts. Hence, there is a need to establish a robust and facile binding assay to directly validate Pin1 as the inhibitor target. We present a 19F Nuclear Magnetic Resonance (NMR)‐based approach that directly reports on the binding of inhibitors and substrates to Pin1, and enables identification of which Pin1 domain, WW or PPIase, harbors the binding site. The method relies on the introduction of 19F labels into the strategic positions of Pin1. Upon fluorination, Pin1 remains well‐folded and catalytically active. The ligand binding process is monitored by the changes in the position and linewidth of the Pin1 19F peaks in the NMR spectra. Moreover, quantitative information about the affinity of the Pin1‐ligand interactions is readily available from the chemical shift‐based binding curves. This approach is demonstrated for several small‐molecule inhibitors and the bivalent peptide substrates of Pin1.
Distribution and Frequency of the Variant rs10738445 of BNC2 Gene Associated to Idiopathic Scoliosis in Puerto Rico
Authors: García Tubéns Nicole M. , Ramírez Aponte Edwin G. , Martínez Cruzado Juan C.
Published: May 2022

Idiopathic Scoliosis is a deformity of the spine, due to the growth of the person, shaped like an “C” or “S”; at a minimum Cobb angle of 10°. Some studies show that Idiopathic Scoliosis may be a condition due to multifactorial effects. However, recent studies have found a susceptibility allele rs10738445 increases the expression of the BNC2 gene especially in homozygous for the haplotype CC. The BNC2 protein is present in the myoblasts and uterus, spinal cord, bone, and cartilage tissue. Thus, suggesting that there is a relationship between the increase in the gene and the etiology of Idiopathic Scoliosis. Other findings suggest a functional role for BNC2 in the development and progression of spinal deformity in patients with Idiopathic Scoliosis. The SNP rs10738445 of the BNC2 gene appeared in the genomic study 1000 Genomes Project, and Puerto Rico showed a genotypic frequency of 0.53 for the haplotype CC. Using rt‐PCR, 622 samples of the general population of Puerto Rico were genotyped for the SNP rs10738445. In this study, we obtained that the genotype CC of rs10738445 has a genotypic frequency of 0.059 in Puerto Rico, with a higher prevalence in the western area. The differences in genotypic frequencies could be the result of the bias in the sampling of the Puerto Ricans that participated in the 1000 Genomes Project. We are planning to genotype patients diagnosed with Idiopathic Scoliosis and other spinal deformities for this variant in Puerto Rico.
Highly conserved binding region of ACE2 as a receptor for SARS‐CoV‐2 between humans and Hippopotamus
Authors: Hayashi Takuma
Published: May 2022

Background

Several cases of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection transmitted from human owners to their dogs have recently been reported. The first ever case of SARS‐CoV‐2 transmission from a human owner to a domestic cat was confirmed on March 27, 2020. Mother and daughter of Hippopotamus amphibius (Hippo) at the Antwerp Zoo in Belgium have been reported infected with SARS‐CoV‐2. SARS‐CoV‐2 is believed to have been infected by the hippo caretaker.

Objective

These reports have influenced us to perform a comparative analysis among angiotensin‐converting enzyme 2 (ACE2) homologous proteins for verifying the conservation of specific protein regions. One of the most conserved peptides is represented by the peptide “353‐KGDFR‐357 (H.sapiens ACE2 residue numbering), which is located on the surface of the ACE2 molecule and participates in the binding of SARS‐CoV‐2 spike receptor binding domain (RBD).

Methods/Results

ACE2 works as a receptor for the SARS‐CoV‐2 spike glycoprotein between humans, dogs, cats, tigers, Hippo, and other animals, except for snakes. The three‐dimensional structure of the KGDFR hosting protein region involved in direct interactions with SARS‐CoV‐2 spike RBD of the Hippo ACE2 appears to form a loop structurally related to the human ACE2 corresponding protein loop, despite of the reduced available protein length (805 residues of the Hippo ACE2 available sequence vs 805 residues of the human ACE2) (Figure). The multiple sequence alignments of the ACE2 proteins shows high homology and complete conservation of the five amino acid residues: 353‐KGDFR‐357 with humans, dogs, cats, tigers, Hippo, and other animals, except for snakes.

Conclusion

Where the information revealed from our examinations can support precision vaccine design and the discovery of antiviral therapeutics, which will accelerate the development of medical countermeasures, the World Health Organization recently reported on the possible risks of reciprocal infections regarding SARS‐CoV‐2 transmission from animals to humans.
Highest Resolution MicroCT Scan of the Human Brainstem Reveals Putative Anatomical Basis for Infrequency of Medial Medullary Syndrome
Authors: Feldman Kaylea M. , O'Keefe Yasmin , Gignac Paul M. , O'Brien Haley D.
Published: May 2022

Posterior circulation or brainstem strokes account for approximately 25% of acute strokes, affecting arterial territories in the medial or lateral divisions of the midbrain, pons, or medulla. Out of all brainstem regions, medial medullary strokes are the rarest, making up fewer than 1% of reported posterior circulation cases. Medial Medullary Syndrome (MMS) is a clinical triad of contralateral hemiplegia, loss of fine touch / position sense, and hypoglossal palsy that may result from ischemia or hemorrhage of small perforating branches of the anterior spinal, vertebral, or basilar arteries. The relative infrequency of MMS is surprising given the prevalence of stroke in other perforator artery territories as these vessels are more susceptible to small vessel disease. Indeed, small perforator‐type vessels from other parent arteries contribute to more than 50% of all strokes in a population (e.g. lacunar branches of the middle cerebral artery and paramedian pontine branches of the basilar artery). This disparity in prevalence remains unexplained in part because clinical anatomy has been historically limited by an unfavorable ratio of fine blood vessel size to coarse MRI and CT scan resolution. Here, we capture and digitally dissect the highest‐ever‐resolution diffusible iodine‐based contrast‐enhanced CT (diceCT) scanned images of a cadaveric brainstem to map MMS arteries in never‐before‐captured detail. A cadaveric brain was fixed in formalin and stained using a 3% concentration of Lugol’s iodine (I2KI) for 12.5 weeks (refreshed every 2 weeks), before microCT scanning at 57 microns (a scale twice as fine as any previous scans). The resulting image stack was segmented in Avizo 2020.1. Results show that within the MMS anterior spinal artery territory, there are numerous, small sulcal artery branches perforating the medulla within the anterior median fissure. These branches proceed in parallel through the anteroposterior depth of the medulla as expected; however, we also identify a network of intraparenchymal, rostrocaudal anastomoses between these sulcal perforating branches. This network of intraparenchymal sulcal artery anastomoses has never been described, and may provide a significant collateral supply of oxygenated blood flow throughout the medial medulla. By ramifying deeper tissues, this anastomosis can help explain the infrequency of MMS.
GAA Deficiency Disrupts Distal Airway Cells in Pompe Disease
Authors: El Haddad Léa , Lai Elias , Murthy Preetish , Tata Purushothama R. , ElMallah Mai K.
Published: May 2022

Pompe disease is an autosomal recessive glycogen storage disease caused by mutations in alpha‐glucosidase (GAA) ‐ an enzyme responsible of hydrolyzing lysosomal glycogen. GAA deficiency results in systemic lysosomal glycogen accumulation and cellular disruption. Skeletal muscle, motor neuron pathology and airway smooth muscle cells are known to contribute to respiratory insufficiency in Pompe disease, but the role of distal airway cells including alveolar type 1 and type 2 cells (AT1 and AT2, respectively) has not been evaluated. AT1 cells depend on lysosomes for cellular homeostasis to maintain a thin barrier for gas exchange whereas AT2 cells depend on lysosomal structures for surfactant production. Using an established mouse model of Pompe disease, the Gaa−/− mouse, we compared lung histology using Periodic acid‐Schiff staining, quantification of immunohistochemistry staining and electron microscopy, and pulmonary mechanics using forced oscillometry FlexiVent system, between Gaa−/− and age‐matched wild‐type (WT) mice. We also perfomed single cell‐RNA seq (scRNA‐seq) on the distal airways of Gaa‐/‐ mice. Lysosomal glycogen accumulation and engorged lamellar bodies were observed in the AT2 cells in Gaa−/− but not WT mice. Furthermore, AT2 positive surfactant protein‐C (SPC) staining and lysosomal positive lysosomal associated membrane protein 1 (LAMP1) staining is more evident with increased colocalization of LAMP1 with the type 2 cell marker after quantification of each marker in Gaa−/− airway cells. The Gaa−/− mice exhibited a significant decrease in total and central airway resistance (Rrs and Rn, respectively), as well as a significant increase in lung compliance (Crs) versus WT mice. Moreover, in the dimensionless and volume‐independent shape constant k that describes the curvature of the upper portion of the deflation limb of the Pressure‐Volume curves, a statistically significant change was observed in Gaa−/− mice. This indicates a change in the intrinsic elastic properties of the respiratory system in Pompe disease. Finally, a robust transcriptomic dysregulation in AT1 and AT2 cells was detected scRNA‐seq in Gaa‐/‐mice relative to WT mice. We conclude that GAA enzyme deficiency leads to glycogen accumulation in the distal airway stem cells that may contribute to respiratory impairments in Pompe disease. Our findings will inform the clinical care of patients with Pompe disease and will provide essential information for the development of novel therapies in Pompe disease that will address this airway pathology.
Biochemical characterization of the bile acid synthetic enzyme 3β‐hydroxy‐Δ5‐C27 steroid dehydrogenase/isomerase type VII
Authors: Gardner Sarah , Marmorstein Ronen
Published: May 2022

Cholesterol is a key biological molecule that is a vital precursor of steroid molecule biosynthesis, including steroid hormones, bile acids and vitamin D3.These processes require 3β‐hydroxysteroid dehydrogenase (3β‐HSD) enzymes, which catalyze dehydrogenation of hydroxysteroids. This family of enzymes catalyzes a wide variety of reaction mechanisms, carrying out a combination of oxidation, isomerization and decarboxylase reactions that result in the formation of cholesterol, steroid hormones and bile acids. 3β‐hydroxy‐Δ5‐C27 steroid dehydrogenase/isomerase type VII (HSD3B7) is responsible for the NAD+ dependent conversion of 7α‐hydroxycholesterol (7‐OHC) to 7α‐hydroxy‐3‐oxo‐4‐cholestenoate (7‐HOC), the second step of the bile acid synthesis pathway, which is vital for nutrient absorption and excretion of excess lipids. This oxidation reaction is one of many steps that increases the solubility of cholesterol for either storage or secretion. HSD3B7 shows substantial functional differences from other family members leaving significant gaps in our understanding of its mode of catalysis and substrate specificity. To this end, I have characterized HSD3B7 using Michaelis‐Menten kinetics, thermal shift assays, and begun x‐ray crystallographic studies. These studies reveal that HSD3B7 displays activity towards multiple bile acid precursors with differing hydrocarbon tail oxidation, with Km values in the low micromolar range. HSD3B7 retains its catalytic efficiency towards these substrates, suggesting that its substrate binding pocket can withstand changes in polarity upon alterations to this hydrocarbon tail. Lastly, HSD3B7 can be crystallized after modification to its membrane associated region, which will result in one of the first structures of a 3β‐HSD enzyme. Together these studies provide key insight into the mechanism of substrate specificity of HSD3B7 and appears amenable for X‐ray crystal structure determination.
Protein Kinase A Catalytic subunit:Smoothened interface ‐ Role of a parallel holoenzyme in negative feedback in Hedgehog Signaling
Authors: Venkatakrishnan Varun , Arveseth Corvin , Myers Benjamin , Anand Ganesh S.
Published: May 2022

During signal transduction in the Hedgehog pathway, the G protein‐coupled receptor (GPCR) Smoothened (SMO) signals intracellularly via a novel mechanism involving sequestration of the Protein Kinase A (PKA) catalytic (C)‐subunit at the membrane. Activation of Hedgehog signaling leads to negative feedback regulation mediated through inhibition of PKA‐C by SMO at the ciliary membrane. While it is known that PKA‐C interacts with SMO at its C‐terminal proximal cytoplasmic tail domain (pCT), the dynamics of PKA‐C:SMO interactions is poorly understood. Here, we report a map of the PKA‐C:SMO interface by Amide Hydrogen Deuterium Exchange Mass spectrometry (HDXMS) over 1‐10 min timescales. SMO binding on PKA‐C resulted in global decreases in deuterium exchange (Dex). Peptides spanning the kinase activation loop (188‐198), P+1 loop (199‐211) and pseudo‐substrate recognition site between β5 and αD (129‐145) showed significant protection (>0.5 Da) when bound to SMO, identifying these regions as the SMO binding interface on PKA‐C. Interestingly, the footprint of SMO binding on PKA‐C was identical to that of the RIa regulatory subunit in PKA‐I holoenzymes. Furthermore, increased Dex (0.4 Da) was observed at the region spanning αC and β4 (83‐100), indicating that SMO binding destabilizes the active kinase conformation to allosterically inhibit PKA‐C. Correspondingly on SMO, PKA‐C binding elicited only low magnitudes of Dex protection (0.4 Da) in regions flanking the pseudo substrate site (573‐590, 622‐631). Increased Dex (0.4 Da) was observed at the sterol binding pocket (96‐112) in the extracellular Cysteine rich domain, indicating that PKA‐C binding may allosterically impact SMO activation by modulating its ability to bind sterols. A significant increase in Dex (0.9 Da) was observed at SMO helix 8 (537‐546) which is positioned parallel to the membrane, indicating that C binding promoted a disordered helix 8 conformation. We speculate that the pCT exists in a membrane‐bound or folded conformation in free SMO, but PKA‐C interaction switches the binding mode of pCT leading to a significantly Dex‐protected state. Thus, PKA‐C:SMO interactions reveal an unexpected intersection between cyclic nucleotide signaling and the Hedgehog pathway, defining a novel mechanism of PKA‐C regulation beyond the canonical PKA signalosome.