bluuebunny/crossref_metadata_2025 huggingface dataset:
Finding Closure: Regulated Irf6 Expression is Required for Proper Wound Healing In Vivo
Authors: Reeb Tanner , Canady Franklin J , Tesdahl Brennan , Biggs Leah C , Dunnwald Martine
Published: November 2023

Chronic wounds affect 6.5 million people in the US, yet little is known about the genetic and molecular mechanisms regulating wound healing. Wound closure requires the concerted action of cellular proliferation, differentiation, and migration, all processes Interferon Regulatory Factor 6 (IRF6) has been shown to regulate in keratinocytes. Embryos deficient for Irf6 demonstrated impaired ex‐vivo embryonic wound closure, and Irf6‐deficient keratinocytes displayed decreased cellular migration resulting in delayed closure in an in vitro scratch assay. Furthermore, individuals with IRF6 mutations exhibited increased likelihood of wound healing complications following surgical repair. Therefore, we hypothesize that altered expression of Irf6 will lead to impaired wound healing in adult mice. Since Irf6−/− mice are perinatal lethal, we utilized two Irf6 mutant lines: an Irf6+/− line with decreased Irf6 expression, and a mouse line with ectopic expression of the Irf6 gene under the control of a keratin 14 promoter (K14‐Irf6). K14‐Irf6 leads to overexpression of Irf6 in the basal layer of the epidermis. The adult mice were wounded with a 6 mm biopsy punch, and the wounds were assessed at either 4 or 7 days post‐wounding. Following morphometric analysis, our results indicate that at day 4, Irf6+/− mice displayed an increase in epidermal area, epidermal volume, and wound volume compared to the wild type and K14‐Irf6 mice. By day 7 the K14‐Irf6 displayed a larger wound area and wound volume as well as impaired epidermal closure compared to wild type and Irf6+/− mice, and Irf6+/− displayed a larger percentage of epidermis in the wound compared to wild type and K14‐Irf6. To determine if changes in epidermal volume could be partially attributed to changes in keratinocyte proliferation, immunohistochemistry for PCNA was performed. Our results show an increase in proliferation in the basal keratinocytes of the migratory tongue in Irf6+/− mice compared with the two other groups (P = 0.06). We also performed Masson's Trichrome staining to determine levels of granulation tissue organization. While very little collagen deposition was present 4 days post‐wounding across the three groups, we observed a trend toward decreased collagen staining and granulation tissue organization in Irf6+/− samples compared with wild type and K14‐Irf6 samples. Together, these results demonstrate that the perturbation of the levels of Irf6 (either positively or negatively) leads to defects throughout the entire period of wound healing. The morphometric analysis data suggests that underexpression of Irf6 being more detrimental in early stages of wound healing (day 4) and overexpression being more detrimental in late stages of wound healing (day 7). Ultimately, these data will provide molecular mechanisms for the increased wound healing complications observed in patients with IRF6 mutations.

Support or Funding Information

R01 AR067739 (NIH)
Collagen analysis of the Achilles tendon injured and treated with F1 protein. A study using transmission electron, polarized light microscopy and western blot.
Authors: Cury Diego Pulzatto , Watanabe Ii‐Sei
Published: November 2023

Introduction

It is estimated that $30 billion are spent on musculoskeletal injuries in the United States each year and tendon/ligament injuries represent ~45% of these cases. Incidence of rupture of the Achilles tendon in North America is 5.5 to 9.9/100,000 habitants. After surgery, the patients return to their activity on approximately 85 days, but may range up to 270 days.

Aim

The effect of F1 protein extracted from Hevea brasiliensis in Achilles tendon repair in rats subjected to tenotomy.

Methods

Three months‐old male Sprague Dawley rats were divided into 3 experimental groups (90 total n): control (C), Kessler‐Tajima suture (K), Kessler‐Tajima plus F1 protein (KP). Catgut suture 5‐0 was used to surgery. Transmission electron microscopy was used to analyze collagen morphology and ultrastructural aspects; western blotting was used to quantify collagen type I and III and light microscopy under polarized light to demonstrates the collagen distribution. All groups were analyzed 2 and 4 weeks after surgery.

Results

Transmission electron microscopy analysis revealed ultrastructural characteristics, in which the presence of macrophages and mast cell were noted in K and KP groups in both analyzed times. Blood vessels among collagen fibrils were noted especially in the groups analyzed after 4 weeks of injury and surgery independent of the treatment. After 2 weeks of surgery in the treated (KP) and non‐treated (K) groups the tendons' weight was increased ~7 times and decreased to ~5.6 after 4 weeks in both groups. Tendon of K group (2 weeks after injury) presented a higher ratio of collagen type III than type I when compared to all other groups, however treated groups analyzed after 2 and 4 weeks presented more collagen type I than non‐treated groups, as well as a higher density of the fibrils collagen. Polarized light microscopy shows collagen fibers arrangement for all tissue. K and KP groups (2 weeks) presented more collagen type III near muscle, and type I near of calcaneus bone; both groups after 4 weeks presented the two types of collagen along the tissue with predominance of type I. C group had only collagen type I in this type of analyze.

Conclusion

F1 protein increases collagen type I and density of collagen fibrils when compared to non‐treated group, therefore apparently may improve tendon repair.

Support or Funding Information

FAPESP #2015/24087‐3
The History Dependence of the EMG‐Force Relationship during Ankle Dorsiflexion
Authors: Paquin James , Power Geoffrey A
Published: November 2023

The steady‐state isometric contraction following a shortening or lengthening muscle action shows distinctly different force and activation levels as compared with a purely isometric contraction at the same muscle length. This study investigated how prior active lengthening (residual force enhancement; RFE) and shortening (force depression; FD) influences the isometric electromyography (EMG)‐Force relationship at submaximal and maximal levels as compared with purely isometric (ISO) contractions. By utilizing a series of force clamps and activation clamps at 20%, 40%, 60%, 80% and 100% of maximal isometric voluntary contraction (MVC) and maximal isometric integrated EMG (iEMG), respectively, the relative differences in neuromuscular efficiency (e.g., force/iEMG) between ISO, RFE, and FD were investigated. 17 healthy young male subjects were seated on a cybex dynamometer and performed voluntary contractions of the ankle dorsiflexors. Subjects first performed an MVC to determine relative intensities, then performed randomized active lengthening and shortening contractions at 25 deg/s over a 25 deg excursion ending at 115 degs respectively. These contractions were then compared with the purely isometric contraction at 115 degs. Surface EMG was used to record from tibialis anterior and soleus. In the RFE state when compared to the ISO state, there was significantly higher torque production during 100% MVC (~7%, P<0.05) with no significant change in agonist iEMG. Submaximally, less agonist iEMG was required to achieve the same amount of force at 60% MVC (P<0.05), and approaching significance for 40% and 20% MVC (P=0.07 and P=0.06 respectively), indicative of increased neuromuscular efficiency. In the FD state compared to the ISO state it was found that there was significantly less force produced during MVC (~15%, P<0.0001) with no significant change in agonist iEMG, but an increase in antagonist iEMG (P<0.05). In the submaximal FD state there was more agonist iEMG required to achieve the same levels of force at 80%, 60%, 40%, and 20% MVC (P<0.05), accompanied by significant increases in antagonist iEMG at 60% and 40% MVC (P<0.05). From these results, it can be concluded that the history dependent states have independent EMG‐Force relationships that exhibit significantly different levels of neuromuscular efficiency than the isometric EMG‐Force relationship.

Support or Funding Information

NSERC ‐ Natural Science and Engineering Research Council of Canada
The ERG1 K+ Channel Protein Is More Abundant in the Denervated Skeletal Muscle of Rodents
Authors: Cobb Brittan A , Anderson Luke B. , Latour Sawyer M , Mosole Simone , Davie Judith K. , Cheatwood Joesph , Carraro Ugo , Zampieri Sandra , Pond Amber L.
Published: November 2023

Skeletal muscle atrophy is the loss of muscle mass and strength that occurs with normal aging, neural and muscle injuries, starvation and diseases such as diabetes, neuropathies, muscular dystrophy, cancer and sepsis cachexia, etc. The atrophy of skeletal muscle can be incapacitating and is related to increased disability, morbidity and mortality in people. We have shown that the ERG1a alternative splice variant of the ERG1 K+ channel is up‐regulated in the skeletal muscle of mice experiencing atrophy as a consequence of cancer cachexia and disuse (i.e., hind limb unweighting, a condition in which muscle action potentials are reduced). In fact, the ERG1 protein is up‐regulated after 4 days of hind limb unweighting, which is prior to a significant loss in muscle cross sectional area, strongly suggesting ERG1 is involved in the onset of muscle loss. Further, we have shown that the ERG1a channel contributes to protein breakdown in muscle by increasing both ubiquitin proteasome proteolysis (UPP) and intracellular calcium concentration with a subsequent increase in calpain activity. Because skeletal muscle atrophy occurs rapidly in denervated skeletal muscle, we hypothesized that ERG1 would also increase in denervated muscles. To test this hypothesis, we performed sciatic nerve transection on mice and rats and collected gastrocnemius and soleus muscles at 0, 4, 7 and 14 days after surgery. Decreased muscle weight and myofiber cross sectional area demonstrate that the muscles were successfully denervated and underwent atrophy. Immunoblot with anti‐ERG1 antibodies demonstrates that indeed the ERG1 protein increases post denervation in mice. Further, immunohistochemistry demonstrates that ERG1 protein also increases in denervated soleus of rats. Interestingly, confocal microscopy analysis of these tissues shows that the ERG1 protein is localized in the sarcolemma and t‐tubules of denervated rat skeletal muscle as it is in heart. The t‐tubule location places it in the vicinity of the calcium release apparatus where it can affect calcium release. We conclude that ERG1 protein is upregulated in the t‐tubules and sarcolemma of denervated muscle and, because it is up‐regulated in other atrophy models, ERG1 is a consistent marker of skeletal muscle atrophy.

Support or Funding Information

This research was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number NIH NIAMS 1R03AR053706‐01A2 to ALP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
RECQ1 interacts with rDNA and promotes pre‐rRNA transcription
Authors: Parvathaneni Swetha , Lu Xing , Sharma Sudha
Published: November 2023

Ribosomal DNA (rDNA) is organized in tandem repeats and presents a significant barrier to replication fork progression. RECQ1 helicase is a major player in maintaining replication fork progression under stress. Nascent rRNAs generated during RNA Polymerase (Pol) I‐mediated rDNA transcription have a tendency to re‐associate with template rDNA and form rRNA:rDNA hybrids that can inhibit rRNA transcription, impede replication fork and facilitate rDNA recombination. Here, we show that RECQ1 is enriched in the nucleolus, the factory of ribosome biogenesis. RECQ1‐depletion in HeLa and U2OS cells significantly reduces pre‐rRNA levels. Genetic rescue experiments indicate a helicase activity‐dependent function of RECQ1 in rRNA biogenesis. RECQ1 binds to the rDNA promoter, enhances RNA pol I recruitment to rDNA promoter and enhances rDNA promoter activity. Depletion of RECQ1 causes ribosomal stress, dysregulated mRNA translation and reduced global translation. Consistent with this disruption of ribosome biogenesis, RECQ1‐depleted cells display impaired growth and proliferation. Collectively, these results identify rDNA as a novel target of RECQ1 helicase to maintain rRNA transcription and ribogenesis.

Support or Funding Information

This work was funded by the NIGMS/NIH grant SC1GM093999 to Sudha Sharma. We also acknowledge support from the NIMHD/NIH award number G12MD007597 and NIA/NIH award number 1R25 AG047843‐02. We thank Drs. Ritu Chaudhary, Xiaoling Li and Ashish Lal (NCI) for their help and support.
Characterization of the Interaction between Mps3 and Cdc5 in Saccharomyces cerevisiae
Authors: Antoniacci Lisa , Breymeier Corinne
Published: November 2023

Mps3 is a nuclear envelope protein identified in Saccharomyces cerevisiae and is involved in several chromosome functions such as sister chromatid cohesion, DNA damage repair, and telomere clustering. Mps3 is hypothesized to be involved in many of these processes through its association with several different chromatin associated proteins (Elg1, Htz1, Ctf18, Eco1). Recently our lab identified a physical interaction between Mps3 and the polo‐like kinase Cdc5. Cdc5 functions in regulation of many essential cell pathways including several that Mps3 also functions. Here we characterize the physical, genetic, and functional interaction of Mps3 and Cdc5.

Support or Funding Information

Schierling Endowed Chair Marywood University
Development of Novel Nickase Enzymes for Targeted Gene Modification in Higher Eukaryotes
Authors: Klipp Alexa , Antoine Michael , Kaplan Emily , Robins Lori , Turner Betsy , Hillers Kenneth
Published: November 2023

In the developing field of genomic engineering, several strategies for targeted, specific genetic modification have recently been developed. Many current methods, such as CRISPR, utilize double strand breaks (DSBs) in DNA to initiate an innate repair process called homology directed repair (HDR). DSBs can also trigger another repair pathway, non‐homologous end joining (NHEJ). However, NHEJ has a lower fidelity and can lead to harmful mutations or translocations. Single strand breaks (SSBs) in DNA stimulate HDR while avoiding the NHEJ pathway. Nickases are enzymes that are capable of inducing SSBs in DNA via a single cut, or nick, in one strand of DNA. Therefore, nickases are ideal for developing targeted and specific genetic modifications.

LAGLIDLAG homing endonuclease (LHEs) are monomeric enzymes with long recognition sequences; these properties make then good candidates for conversion to nickases. In collaboration with Dr. Lori Robins (University of Washington Bothell), we are carrying out a structure‐guided mutagenesis of various LHEs with the goal of converting them into functional nickases. Two LHEs, LtrWI and SmaMI, have been chosen to mutate into functional nickases. Site‐directed mutagenesis of these LHE genes is aimed at altering single amino acids that are believed to catalyze DNA hydrolysis in one of the active sites. By inactivating one of the active sites in the monomer and allowing the other active site to remain intact, this produces a semi‐functional enzyme capable of nicking DNA at specific sequences.

Through PCR‐directed mutagenesis of specific active‐site lysine residues, we have successfully produced LtrWI and SmaMI mutants with predominately nicking activity. Current efforts seek to extend these observations to additional LHEs, through identification and mutagenesis of homologous lysine residues in the cognate active sites.

Support or Funding Information

Thank you to the Cal Poly Student based fee and Chevron Applied Biotechnology Research Program for funding; Ellen Calcagno for administrative assistance and Michael Black for technical assistance; members of the Hillers/Turner lab for helpful discussion and support; Conor Beck for diagrammatic interpretation of data.
Differential Requirements for Nonhomologous End‐joining (NHEJ) Pathway Genes in DNA Repair and Telomere Stability
Authors: Rodriguez Nestor Daniel , Lewis Kevin
Published: November 2023

Deoxyribonucleic acid (DNA) is constantly being damaged due to environmental chemicals, free radicals, and radiation. These damaging agents cause varying effects from phosphate, sugar, or base modification to double‐stranded breaks (DSBs) in the DNA. If DSBs are left untreated in a cell, the consequences can be grave, potentially causing loss of function, mutations, and cell death. In order to repair DSBs, two independent pathways are used, homologous recombination (HR) and nonhomologous end‐joining (NHEJ). NHEJ is the preferred pathway in humans but secondary in the yeast Saccharomyces cerevisiae. The NHEJ pathway involves three major complexes in yeast cells: the Ku complex, Mrx complex, and DNA ligase IV complex. Each complex has a specific function in repairing DSBs.

The Ku complex is a heterodimer found in both humans and yeast (Ku70 and Ku80 for humans, Yku70 and Yku80 in yeast), which is responsible for first binding to the broken ends of the DNA and protecting the ends from degradation. A mutation in the Ku70/Ku80 complex genes causes severe combined immunodeficiency (SCID) in humans and animals. The Mrx complex is composed of Mre11, Rad50, and Xrs2 in yeast and binds to the DNA ends to form an end‐bridging complex. The last complex is the DNA ligase IV complex that is composed of Dnl4 and Lif1. This complex performs the last step in NHEJ whose function is to ligate the two DNA ends together.

Many past studies have studied NHEJ repair using plasmid end‐joining assays. In this method, cells are transformed with a plasmid containing a DSB that cannot be repaired by HR and the number of transformants with plasmids recircularized by NHEJ is determined. Previous reports observed that repair in yku, mrx and dnl4 mutants is reduced by 10–30 fold and suggested that the three major complexes are approximately equally important in the pathway. Using high efficiency transformation methods and early stationary phase cell cultures, we have been able to detect strong differences in the requirements for each complex. These results and additional experiments suggest that cell cycle phase distribution within cell cultures affects repair complex requirements.
Unstable Telomere Cap Structures in Saccharomyces cerevisiae yku70 Mutants Cause Altered Cell Cycle Phase Distributions
Authors: Posey Tressie , Lewis L Kevin
Published: November 2023

Eukaryotic cells keep their DNA compartmentalized within nuclei, but this does not guard the DNA from damage. There are a number of environmental and intracellular factors such as ionizing radiation, e.g., X‐rays and gamma rays, UV radiation, nucleases, DNA modifying agents, and free radicals that can damage the DNA. One of the most detrimental lesions that can occur in the DNA is a double‐strand break (DSB), where both strands of the double helix are fractured. This damage is the most difficult to repair and holds a number of consequences for the cell's viability. In humans, the main repair pathway for DSBs is NHEJ, or the Nonhomologous End‐Joining pathway. This repair pathway primarily uses three protein complexes to directly rejoin the two broken ends. In Saccharomyces cerevisiae (budding yeast) these three complexes are Mrx (Mre11, Rad50, and Xrs2), DNA ligase IV (Dnl4 and Lif1), and the Ku complex (Yku70 and Yku80). The Ku complex binds to the broken ends first, protecting them from further degradation. Mrx then bridges the gap between the broken ends and DNA ligase IV covalently links the ends together.

In this project, yeast yku70 cells, which exhibit a growth defect when grown at 37° C, were analyzed microscopically after growth at multiple temperatures (20° C, 30° C, and 37° C). Normally, yeast cells spend approximately the same proportion of time in G1, S, and G 2/M during log phase growth, but yku70 cells exhibit an increased number of G2/M cells. This phenomenon was not seen in other NHEJ mutants such as dnl4 cells. Further experiments involving the quantitation of cells with different nuclear morphologies and testing the role of DNA damage checkpoints will be presented.
DNA double‐strand break repair deficiency is associated with changes in cell cycling and cell morphology in Saccharomyces cerevisiae
Authors: Weis Monica , Lewis L Kevin
Published: November 2023

Chromosomal DNA experiences a variety of different types of damage often and naturally. Considered potentially the most lethal of DNA lesions, double‐strand breaks (DSBs) involve a complete severance of both strands of DNA at some point in the chromosome. If not repaired, DSBs can lead to an increase in DNA mutations, chromosome loss events and ultimately loss in cell viability. In eukaryotes, DNA DSBs are repaired almost exclusively by two mechanisms, nonhomologous end‐joining (NHEJ) and homologous recombination (HR). NHEJ is the simpler of the two pathways, involving the direct rejoining of broken chromosome ends, while HR involves the use of a region of a homologous chromosome as a template for repair. RAD52 group proteins (Rad51, Rad52, Rad54, etc.) play an important role in the HR pathway. In Saccharomyces cerevisiae homologous recombination is considered the primary pathway for repair. Checkpoint proteins also play an important role in DSB repair by making sure that damage is repaired before a cell completes the cell cycle. If there is significant damage that requires attention, such as DSBs induced by chemicals or radiation, growth arrest is induced and the cells accumulate in G2 phase.

Yeast rad52 mutants cannot repair DSBs by HR and have elevated levels of unrepaired DNA lesions. In the current project, we have observed that rad52 cell cultures have high levels of enlarged large‐budded cells, and that log phase cells spend 64 minutes of each 128 minute cell cycle in G2 phase. Similar results were seen with rad51, rad54, rad55, rad57, and rad59 mutants, but not rdh54 cells. Inactivation of 7 critical DNA damage checkpoint genes (RAD9, RAD17, RAD24, CHK1, DDC1, MRC1, and MEC3) in rad52 cells, creating double mutants, abolished the high large‐budded cell phenotype. It is likely that rad52 cells have elevated levels of unrepaired DNA lesions leading to constitutively activated damage‐responsive cell cycle checkpoints. Additional experiments involving analysis of changes in nuclear and cellular morphology will be presented.
Exploring the Role of ATP Hydrolysis in the RecA Protein via Stalled DNA Intermediates
Authors: Sowin Laura , Cox Michael M
Published: November 2023

A central participant in the recombinational DNA‐repair system is the recombinase protein, RecA, which acts as a DNA pairing enzyme that is responsible for promoting an exchange of homologous DNA strands. Physical studies have revealed three DNA binding sites buried within the interior of the RecA filament. This current understanding of RecA is incomplete as it fails to explain how a four‐strand DNA exchange occurs between two duplex DNA fragments.

The bacterial RecA homologue is unique in that it couples ATP‐hydrolysis with DNA strand exchange. ATP hydrolysis occurrs uniformly throughout the RecA filament, with only a small fraction of ATP used for filament disassembly. The mechanism by which the RecA filament utilizes the rest of ATP hydrolysis to perform DNA strand exchange reactions is not fully understood. My aim is to explore the hypothesis that the majority of ATP utilized by the reaction is hydrolyzed at binding sites located longitudinally along both the interior and exterior of the filament, providing the energy needed to rotate one segment of DNA around another.

This will be explored by performing RecA mediated strand‐exchange reactions with a variety of linear, double stranded DNA substrates containing heterologous regions. When the RecA protein is run into a heterologous barrier, a stalled DNA‐protein intermediate molecule is the product. Formation of these intermediates has been obtained. Imaging with Electron Microscopy shows topological changes that offer fundamental insight into the universal mechanics of RecA‐mediated strand exchanges. Furthermore, evidence shows that introduction of the protein Uup modifies the structure of the intermediates, providing insight into Uup's function.
Effects of DNA Damage on Natural Transformation in Bacillus subtilis
Authors: Korry Benjamin Joseph , Belenky Peter A
Published: November 2023

Horizontal gene transfer is an important mechanism that promotes rapid evolution in microbial communities. Understanding how environmental conditions impact horizontal gene transfer in microorganisms can identify factors that lead to the transfer of virulence and antibiotic resistance genes. Natural transformation is a form of horizontal gene transfer that allows bacteria to take up and incorporate extracellular double stranded DNA. Competence machinery utilized for the import of DNA is typically induced by cellular state or external stimuli. Following import, DNA is recombined into the genome in a RecA‐dependent fashion. Cellular stress has been shown to modulate natural transformation in various bacterial species by targeting either the competence or recombination machinery. Here we investigate the links between genotoxic stress and changes in the rate of natural transformation in naturally competent B. subtilis. We find that genotoxic agents have divergent impacts likely related to their effects on DNA replication and processing in bacteria. Traditional high‐throughput methods are applied to analyze transcriptional responses induced by genotoxic agents in order to link known mechanisms of natural transformation to phenotypic outcomes.

Support or Funding Information

“Monitoring the Impact of Climate Change Stressors on Horizontal Gene Transfer in Marine Microbial Populations” 2016. Rhode Island Research Alliance Collaborative Research Grant, Rhode Island Science & Technology Advisory Council
Stress Response and Natural Transformation in Vibrio cholerae
Authors: Rowan Aislinn D , Belenky Peter A
Published: November 2023

Antimicrobial resistance is a major threat facing modern medicine, which is accelerated by various forms of horizontal gene transfer (HGT). A contributing problem is that antibiotics themselves, especially those inducing DNA damage and the SOS response, can actually increase the rates of transfer of resistance genes through conjugation and transduction. Evidence from other species indicates that DNA‐damaging stress may also have effects on natural transformation, a form of HGT in which extracellular DNA is promiscuously taken up and recombined into the genome and which may contribute to the spread of resistance genes. We are studying the effects of DNA‐damaging stress in Vibrio cholerae, an important human pathogen capable of both natural transformation and the SOS response. Here, we demonstrate that genotoxic agents can alter the rates of natural transformation in V. cholerae and explore the mechanisms by which these changes occur.

Support or Funding Information

“Monitoring the Impact of Climate Change Stressors on Horizontal Gene Transfer in Marine Microbial Populations.” 2016. Rhode Island Research Alliance Collaborative Research Grant, Rhode Island Science and Technology Advisory Council (STAC).

Brown University Presidential Fellowship
Down‐Regulation of Ubiquitin‐Specific Protease 24 Regulates DNA Repair by Homologous Recombination in Lung Cancer Cells
Authors: Wang Shao‐An , Wang Yi‐Chang , Hung Jan‐Jong
Published: November 2023

Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double‐strand breaks. Ubiquitin‐specific peptidases (USPs) belong to a large family of cysteine proteases and are deubiquitinating enzymes that specifically recognize and remove ubiquitin from proteins. USP disorders have been reported to be involved in many human diseases, such as neurodegenerative disorders, inflammation, and cancer progression. In this study, we want to elucidate the role of a novel deubiquitinating enzyme, ubiquitin‐specific peptidase 24 (USP24) in DNA damage repair. Our recent study showed that downregulation of USP24 increases cell cycle progression by enhancing the G1‐S transition. The molecular mechanism involves a decrease in the USP24 level, which reduces the expression of E2F4 and thus increases the G1/S transition. In this study, we found that USP24 knockdown enabled the cells to overcome CPT‐induced cell death was also reversed when E2F4 overexpression was performed after USP24 knockdown. Furthermore, RAD51 involved in DNA damage repair was also dramatically up‐regulated after USP24 knockdown and USP24 knockdown A549 cells formed more colonies than scramble knockdown group after DNA damaging agent CPT treatment. E2F4 overexpression can inhibit USP24 knockdown induced RAD51 promoter activity. Loss of transcription repressive effect of E2F4, RAD51 was up‐regulated and increased CPT resistance through promoting homologous recombination. Based on these results, E2F4 is one of the critical factors in USP24 knockdown induced genes upregulation, and E2F4 overexpression can partially reverse USP24 knockdown induced proliferation and CPT resistance. Taken together, we have proofed that USP24 plays important roles in DNA damage repair and drug resistance.

Support or Funding Information

MOST‐103‐2320‐B‐006‐035‐MY3
Regulation of Interleukin 33 by Transforming Growth Factor‐β in Epithelia Cells, Fibroblasts and Keratinocytes
Authors: Woelfel Jennifer M , DenHaese Jason P , Gervasi Megan E , Ruvio Stacy
Published: November 2023

Interleukin 33 (IL‐33), a member of the IL‐1 family of cytokines, plays a key role in inflammatory responses functioning as an alarmin, alerting the immune system to tissue damage. IL‐33 signaling molecules following damage, IL‐33 coordinates recruitment and activation of immune cells leading to the release of mediators such as VEGF, which drives angiogenesis and skin remodeling.I L‐33 is expressed in epithelial cells, stromal cells, and endothelial cells. High levels of IL‐33 as has been implicated in diseases such as asthma, inflammatory skin and rheumatological diseases. Preliminary studies have shown that the pluripotent cytokine Transforming Growth Factor‐β (TGF‐β) represses expression of IL‐33 in mouse and human mammary epithelial cells, human kidney and lung epithelial cells and human primary fibroblast in vitro. TGF‐β is a cytokine involved in a number of cellular responses including cell differentiation, growth inhibition, extracellular matrix sti mulation, immunosuppression, and immunomodulation. Our current studies revealed that repression of IL‐33 was dependent on the TGF‐β canonical pathway of signaling. Continuing studies will utilize mast cells to determine the biological implications of repression of IL‐33 by TGF‐β.

Support or Funding Information

Work supported by the Department of Biology and Mathematics, D'Youville College.
Regulation of renal proximal tubule function by CREB‐regulated transcriptional coactivators and salt‐inducible kinase
Authors: Taub Mary , KIm Dongwook , Cutuli Facundo
Published: November 2023

The kidney plays a central role in blood pressure regulation, via its ability to control sodium reabsorption via locally produced natriuretic, and anti‐natriuretic factors. Previously, natriuretic and anti‐natriuretic factors, including dopamine and norepinephrine, have been observed to alter sodium reabsorption by changing the phosphorylation state of Na,K‐ATPase in the renal proximal tubule (RPT). Protein Kinase A and calcium‐mediated signaling pathways are involved. These same signaling pathways control transcription of the Na,K‐ATPase β subunit gene atp1b1 in RPT cells. In this report, evidence is presented that (1) recently discovered cAMP‐regulated transcriptional coactivators (CRTCs), and salt‐inducible kinase 1 (SIK1) contribute to the transcriptional regulation of atp1b1 in RPT cells and (2) renal effectors, including norepinephrine, dopamine, prostaglandins, and sodium, play a role. Evidence for the role of CRTCs includes the loss of transcriptional regulation of atp1b1 by a dominant‐negative CRTC, and a CREB mutant with an altered CRTC binding site. In a number of experimental systems, SIK1 phosphorylates CRTCs, which are then sequestered in the cytoplasm, preventing their nuclear effects. Consistent with such a role of SIK1 in primary RPT cells, atp1b1 transcription increased in the presence of a dominant‐negative SIK1. Regulation by dopamine, norepinephrine, and monensin was also disrupted by a dominant‐negative SIK1. These latter observations can be explained if these renal effectors phosphorylate and inactivate SIK1. Our results support the hypothesis that Na,K‐ATPase in the RPT is regulated at the transcriptional level via SIK1 and CRTCs, in addition to the previously reported control of the phosphorylation of Na,K‐ATPase.

Support or Funding Information

NHLBI
Engineering a Tissue‐Specific Cell‐Based Therapeutic Delivery System
Authors: Pandey Anjali , Keenan Jessica L , Doiron Kailyn L , Siggers Trevor
Published: November 2023

Delivery of protein‐based therapeutics to specific tissues is a major hindrance to the treatment of many diseases, including solid tumors. Cell‐based therapeutic delivery vehicles, which are naturally capable of sensing and responding to their environment, are a promising approach to improving the targeting of therapeutics to pathological sites. To improve the specificity of cell‐based delivery vehicles, we are developing a cell‐based delivery system that expresses a therapeutic protein upon sensing multiple signals indicative of the disease state. As a proof of concept for the system, we are working to target solid tumors using macrophages; macrophages are naturally recruited to solid tumors, making them an ideal delivery vehicle. However, they also localize to non‐pathological sites including the lungs, liver, and spleen, requiring additional engineering to restrict therapeutic delivery to pathological sites. This system could be used to deliver anti‐cancer therapeutics, such as interleukin‐2, specifically to tumors, thereby reducing deleterious off‐target effects associated with systemic administration. To accomplish this goal, we are coupling endogenous signals characteristic of the tumor environment to transgene expression. Therapeutic protein expression will be triggered by simultaneous activation of two native transcription factors (TFs), HIF‐2 and PPARγ:RXRα (PPARγ), which are activated by hypoxia and immunosuppressive signals indicative of solid tumors, respectively. When both TFs are activated, they will facilitate the recruitment of a novel engineered scaffold protein and transcriptional activator (miniCBP) to a DNA response element (DRE), triggering expression of the therapeutic protein. To identify an appropriate DRE, we will use protein‐binding microarrays (PBMs) to screen tens of thousands of DNA sequences. We will then test the system in vitro in HEK293 cells and primary human monocytes. We anticipate that this work will advance the specificity of cell‐based delivery vehicles and represents a potentially transformative approach for delivering potent protein‐therapeutics directly to tumors, increasing efficacy and reducing toxicity.
Analysis of Differentially‐Expressed Genes in a Gamma‐Secretase Conditional Knockout Mouse
Authors: Biefeld Chelsea , Harvey Amanda , Zhu Julia , Yu Gang , Dries Daniel
Published: November 2023

gamma‐secretase is an intramembrane protease involved in Alzheimer's disease and neurodevelopment. Here, a conditional knockout mouse was made in which a component of the gamma‐secretase complex (Nicastrin) was deleted from oligodendrocytes. These mice displayed schizophrenia‐ and compulsive‐like phenotypes. In order to identify genes and pathways that elicit these phenotypes, RNA‐Seq was used to perform transcriptome analysis. A bioinformatics pipeline was created to process this data and search for differentially expressed genes within the conditional knockout mice. Gene ontology programs were used to evaluate the genes and identify biological processes and functions that may play a role in the behavioral aspects of these mice.

Support or Funding Information

We would like to thank the Howard Hughes Medical Institute, the National Institutes of Health (Grant R01 AG029547), and the International Healthspan Institute for providing funding.
Determining the effect environmental conditions have on the activity of the 3′–5′ exoribonuclease Rrp6
Authors: McAdoo Brooke
Published: November 2023

Eukaryotic cells contain quality‐control systems that monitor RNA biogenesis. These systems feature ribonucleases that prevent the accumulation of nonfunctional RNAs as well as regulate normal mRNAs. Rrp6 is a 3′–5′ exoribonuclease that plays a critical role in the 3′‐end formation and degradation pathways in the nucleus. In Sacchromyces cerevisiae Rrp6 physically and functionally interacts with the exosome, a highly conserved RNA‐processing complex. Rrp6 possesses activities that are both dependent and independent on its association with the exosome. It has been well documented that the deletion of RRP6 (rrp6‐Δ) leads to increased levels of both coding as well as non‐coding RNAs. However few experiments have addressed how changes in nutrient source, temperature, and under hypoxic conditions affect the activity of Rrp6, specifically its ability to regulate coding and non‐coding RNAs. To address this question we evaluated the level of several Rrp6 regulated RNAs in wild‐type and rrp6‐Δ cells grown in media containing either galactose or glucose, grown at 30 degrees and 37 degrees and in the presence or absence of oxygen. We calculated the growth rate of the yeast under these conditions and used quantitative real‐time polymerase chain reaction (qRT‐PCR) and ΔΔ Ct analysis to determine changes in RNA expression. The goal of these experiments is to better understand the activity of Rrp6p, which is critical regulator of RNA levels in all eukaryotes.

Support or Funding Information

Supported by internal funding by St. John Fisher College.
Resolving specificity over a large footprint distinguishes androgen and glucocorticoid receptor DNA binding
Authors: Zhang Liyang , Martini Gabriella D. , Rube H. Tomas , Bussemaker Harmen J. , Pufall Miles A.
Published: November 2023

The glucocorticoid (GR) and androgen receptors (AR) share an identical DNA binding interface, bind a similar core motif, yet occupy largely distinct genomic loci to regulate different gene programs. Their interplay is particularly important in the context of some castration resistant prostate cancers (CRPC), where GR can functionally substitute AR to drive the growth of tumor in the absence of androgen signaling. Some differences in the genomic localization of AR and GR can be explained by selective association with binding partners, however it is not clear how they discriminate the remaining sites. Examination of the crystal structures suggested that specificity might extend over a larger footprint (≥20bp) which had not been accessible to experimental and computational techniques previously. We therefore developed a refined version of SELEX‐seq coupled to an iterative algorithm, SelexGLM, to model the free energy of binding over a ~30bp binding site. The position specific affinity matrices (PSAMs) for these two factors reveled difference both within and outside the 15bp core motif, with AR being less tolerant to changes and thus more specific. Part of this specificity was derived from readout not just of specific base pairs, but also the shape of DNA. AR also exhibited preference for A‐tracts flanking the core sequence that narrow minor groove width. In contrast, GR specificity was largely restricted to the core sequence, with preference for a wider minor groove within that motif. The enhanced specificity of AR appears to be intrinsic to the protein, and thermodynamically driven. Isothermal titration calorimetry revealed that AR DNA‐binding is more enthalpically driven than GR binding, which is more entropically driven. This demonstrates, on a comprehensive scale, that, much like direct readout, DNA shape driven binding is enthalpically driven, and that entropically driven binding is associated with decreased specificity. Thus, this work provides a biophysical rationale for how GR can functionally substitute for AR in some castration resistant prostate cancers: in the absence of AR, overexpression of the less selective GR is able to bind AR sites. Further, this work demonstrates that combining high‐throughput and classical biophysical approaches provides the deep understanding of specificity that is necessary to dissect the regulation of fine‐tuned systems.

Support or Funding Information

Acknowledgments: This research was supported by the National Institutes of Health (NIH) K99/R00 CA149088, the Roy J. Carver Charitable Trust 01–224, and the National Science Foundation CAREER grant 1552862 (MAP), and grant R01HG003008 (HJB).
Functional Characterization of Caprin2 in Mouse Eye Development and Its Associated Developmental Defect Peters Anomaly
Authors: Borders Nathaniel Cole
Published: November 2023

Using the iSyTE bioinformatics tool developed by our lab we identified a novel RNA binding protein and RNA granule component Caprin2 that is enriched at early mouse embryonic time points starting at embryonic time point (E) 10.5 onward based on microarray data. In situhybridization (ISH) and immunostaining experiments were performed on mouse embryonic lens tissue to validate these findings. This analysis demonstrated that Caprin2 mRNA and protein were localized to lens fiber cells, supporting the hypothesis that it has potential function in lens development. Recently, our lab demonstrated that a lens‐specific conditional knockout of Caprin2 (Caprin2cko/cko) caused compaction of the central region of lens fiber cells and Peters anomaly, a lenti‐corneal stalk. However, the Peters anomaly defect showed only 8% penetrance in Caprin2cko/cko mice. We hypothesized the low penetrance of this ocular defect resulted from the residual Caprin2 protein present at E12.5 in the mutant mice. Caprin2 germline (Caprin2‐−/−) deletion knockout mouse mutants were generated to test this hypothesis. Caprin2 protein was confirmed to be deleted constitutively through western blotting and immunostaining on Caprin2‐−/− lenses at birth and at E14.5 respectively. Phenotypic characterization has thus far been performed on the Caprin2‐−/− mice through embryonic histology and scanning electron microscopy. Histology of the mouse mutant demonstrated no significant morphological difference from wild type mice. Scanning electron microscopy also had no significant difference, though more samples remain to be analyzed. Further analyses will include immunostaining for important lens development markers including Pax6, Foxe3, p63, Spry1/2, and Shrm3.
Angiotensin II‐mediated Repression of Guanylyl Cyclase/Natriuretic Peptide Receptor‐A Gene Expression Involving CREB, HSF4a, and HDAC1/2 in Mesangial Cells
Authors: Arise Kiran K , Kumar Prerna , Pandya Krishna , Pandey Kailash N.
Published: November 2023

Atrial natriuretic peptide (ANP) is an endogenous cardiac hormone that binds to guanylyl‐cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and regulates sodium excretion, water balance, and steroidogenesis; all directed toward the reduction of blood pressure and blood volume. The present study was aimed at understanding the mechanisms involved in angiotensin II (Ang II)‐mediated repression of Npr1 (coding for GC‐A/NPRA) gene transcription. The mouse mesangial cells (MMCs) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and ITS (insulin, transferrin, and sodium selenite) and treated with Ang II for 24 h. The luciferase assay of the Npr1 promoter deletion constructs showed that the region approximately –1182/–916 bp upstream of the transcription start site contains the cis‐elements involved in ANG II‐mediated transcriptional repression of Npr1. Real‐time quantitative PCR and Western blot analyses revealed that Npr1 mRNA expression and NPRA protein levels were reduced by 60 – 65% in Ang II‐treated MMCs. Treatment with Ang II also significantly decreased ANP‐stimulated intracellular accumulation of cGMP as compared with ANP‐stimulated cells. Gel mobility shift assays confirmed that Ang II enhanced the binding of transcription factors cAMP‐response element‐binding protein (CREB) and heat shock factor 4a (HSF4a) to the Npr1 promoter. Increasing concentrations of Ang II also decreased the acetylation levels of histone H3 at lysine 9 and 14 and histone H4 at lysine 8. In contrast, there was significant increase in protein expression of prohypertrophic Class 1 histone deacetylase (HDAC) 1 and 2. Treatment of MMCs with Ang II increased the activity of HDAC in a dose‐dependent manner. The present findings provide a novel regulatory mechanism of Ang II‐mediated repression of Npr1 transcription and expression, which plays an important role in the control of hypertension and cardiovascular homeostasis.

Support or Funding Information

This work was supported by NIH grants (HL057531 and HL062147).
Further development of a CRISPR/Cas9 platform for 60% of Duchenne muscular dystrophy patients
Authors: Fernandez Karla
Published: November 2023

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder that affects approximately 1 in 3,500–5,000 males worldwide, and is caused by mutations in the DMD gene causing loss of dystrophin protein expression. This results in progressive muscle degeneration, leading to lethal conditions of cardiac and respiratory function. Since approximately 60% of the out of frame mutations in DMD occur between exons 45–55, the Spencer lab developed a platform utilizing CRISPR/Cas9 gene editing to restore the DMD reading frame by creating a large deletion of exons 45–55. To expand on this platform we are analyzing DNA homology between humans and larger animal models to design and test new guide RNAs (gRNAs) that would be specific across multiple species. Homology is being analyzed using BLAST to check for previously designed gRNAs that are conserved across dog, pig, and monkey genomes and to design new gRNAs. Secondly, we are analyzing muscle specific promoters. We hypothesize that putting Cas9 behind a muscle specific promoter would restrict Cas9 expression to solely muscle tissue and reduce potential off target effects. By analyzing gene expression and immunostaining of candidate promoters in muscle sections, we expect to determine a candidate promoter to drive Cas9 expression only in muscle tissue and eliminate non‐specificity. In conclusion, these studies allow us to identify gRNAs for large animal models as well as a muscle specific promoter for Cas9, to expand the development and safety of the CRISPR/Cas9 platform which has the potential to serve as a therapy for 60% of DMD patients.