bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological Examination of Ratite Orthopedic Disorders and Soft‐Tissue Visualization via Micro‐CT
Authors: Green Todd , De Miranda Michael , Larson Ashley , Bonitz Shayla , Gignac Paul , Kley Nathan , Kanatous Shane
Published: November 2023

Many hard‐tissue (e.g., bone, dentine, enamel) pathologies directly impact the development and function of soft‐tissue structures. Such pathologies can be unique to captive individuals and tend to be more common among large‐bodied captives such as crocodilians, elephants, and flightless birds. Emus (Dromaius novaehollandiae) are large‐bodied flightless birds that are uniquely efficient at both anaerobic sprinting and aerobic, sustained running. Native to Australia, they are commonly farmed in North America for oil and meat, but growth of the emu industry has been hampered by poorly understood orthopedic disease states (i.e., splayed‐leg disorder). Few studies have examined captive emu skeletal muscle, and none have documented the enzymatic profile of ratite cardiac tissue. We sought to bridge this gap by providing a quantified understanding of physical degradation in this large farm‐raised species. Surprisingly, we found the metabolic profile of captive emu cardiac tissue to be most similar to that of active, flying birds. Additionally, metabolic baselines for primary locomotor muscles between splayed and non‐splayed limbs indicate enhanced metabolic enzyme activities and myoglobin levels, resulting in a distinctly more “athletic” phenotype than control limbs. This compensatory increase in aerobic capacity demonstrates how soft tissues can mitigate functional issues caused by pathologies and suggests that muscle atrophy does not contribute to splayed‐leg disorder. Likely, other factors are to blame (e.g., nutrition). With the advent of more detailed imaging techniques, such as contrast‐enhanced micro‐CT imaging, we hope to test for signal of these factors and better understand the anatomy and physiology of health and disease states in ratite birds. Through documentation of the interplay between hard and soft structures, including their physiologies, we are now beginning to apply new tools to an ancient clade of poorly‐understood birds.

Support or Funding Information

Funding was provided to Dr. Shane Kanatous from the Colorado State University Department of Biology (muscle physiology study), and to Dr. Paul Gignac and Nathan Kley through National Science Foundation grants no. 1450850, 1457180, 0749750 and 1450842 (micro‐CT studies).
Iroquois 1 (Irx1) Marks the Stratum Intermedium and Periodontal Complex Cells
Authors: Romero‐Bustillos Miguel , Yu Wenjie , Eliason Steve , Amendt Brad A.
Published: November 2023

Iroquois 1 (Irx1) is a homobox transcription factor that was identified in craniofacial tissues through RNA‐sequencing and bioinformatics analyses. Irx1 is expressed in specific dental tissues during development and adult stages. Irx1 is present in the outer enamel epithelium and the stratum intermedium at prenatal stages. The expression of Irx1 is maintained until tooth eruption and at this stage it appears to play a role in root formation. Heterozygous Irx1 knockout mice show Irx1 expression in the Hertwig Epithelial Root Sheath and in the outer enamel epithelium at post‐natal stage p12 in molars. Epithelial rests of Malassez and isolated cells in the inter‐radicular cementum are positive for Irx1 expression. The presence of Irx1 is nonexistent in late stages on the root surface. Differences in Irx1 levels can be found in cultured cementoblast and periodontal ligament cells. Cementoblasts have a higher level of Irx1 than periodontal ligament cells. These findings were confirmed by real‐time PCR and protein levels. Under mineralization conditions cementoblasts show a reduction in the level of Irx1 expression. The presence of Irx1 during tooth development is indicative of the implication of this novel transcription factor in tooth formation. Moreover, the positive expression of Irx1 in cementoblasts under non‐mineralization conditions and its reduction of expression under‐mineralization conditions may suggest it regulates genes involved in root formation and possibly the origin progenitor cells of the cementum.

Support or Funding Information

Miguel Romero‐Bustillos would like to acknowledge the support received from the NIH under the R90 DE024296‐03 grant.
The Depth of Commonly Grafted Costal Cartilages at Different Locations along the Cartilages: Considerations for Autografts
Authors: Lynch Hannah L , Ganoe Maria R , Bender‐Heine Adam N , Holmes J Scott , Rickards Allen A , Armeni Mark A , Lambert H Wayne , Zdilla Matthew J
Published: November 2023

Costal cartilage grafting is a commonly used procedure in reconstructive surgery. The decision regarding which cartilage to select is often a question of the volume of material necessary to shape the graft. However, there is little information regarding the depth (superficial‐to‐deep) of commonly grafted costal cartilages. Therefore, this cadaveric study sought to determine the average depth of the 5th, 6th, 7th, and 8th costal cartilages at the medial, lateral, and intermediate locations along the cartilages. Results revealed the thickest cartilage is found at the medial portion of the 6th cartilage while the thinnest cartilage is found at the medial aspect of the 8th cartilage. The average thickness of each cartilage near its respective rib did not differ much between cartilage levels; however, the thickness of cartilage closest to the sternum differed widely. Moreover, any location along the 6th costal cartilage is, on average, thicker than any part of the 5th, 7th, and 8th cartilages.

Support or Funding Information

WV Research Challenge Fund [HEPC.dsr.14.13] and

WVU Initiation to Research Opportunities (INTRO) Summer Research Program
The Anterior Contour of the Seventh Costal Cartilage: Considerations for Grafting to the Nasal Dorsum and Columella
Authors: Russell Michelle L , Bender‐Heine Adam N , Holmes J Scott , Rickards Allen A , Armeni Mark A , Lambert H Wayne , Zdilla Matthew J
Published: November 2023

When viewed from the anterior, the seventh costal cartilage tends to have an “L” shape that lends itself to a single‐block graft to reconstruct the dorsum and columella of the nose. However, it has been observed that the 7th costal cartilage can be bowed anteriorly. Because the anterior contour of the 7th costal cartilage has never been examined, this cadaveric study analyzed the anterior contour of the 7th costal cartilage as viewed from the superior via geometric morphometric techniques to determine the average contour and most likely contour variance. On average, the anterior contour of the seventh cartilage had moderate anterior bowing; however, the contour ranged from severely bowed to nearly flat. When present, the anterior bowing was most pronounced a distance two‐thirds the length of the cartilage away from the sternum. Although a single‐block “L” shaped cartilage graft may be possible in individuals with relatively flat anterior contours, in patients with significant bowing of the 7th costal cartilage one could cut the cartilage into two straight segments, one segment from the medial 2/3 and the other segment from the lateral 1/3 of the cartilage for separate dorsal and columellar grafts.

Support or Funding Information

WV Research Challenge Fund [HEPC.dsr.14.13] and

WVU Initiation to Research Opportunities (INTRO) Summer Research Program
Low Intensity Vibration to Improve Postural Stability, Reduce Chronic Low Back Pain and Improve Function
Authors: McCrae Shaun A , Einstein George P , Tulp Orien L
Published: November 2023

Low back and extremity pain has risen to one of the leading causes for physician visits and will impact approximately 80% of Americans during their lifetime. The cost of this epidemic will be approximately $8 Billion per year. The impact of this health issue is without prejudice and will not only impact the lives of approximately 31 million Americans but it will also impact the lives of countless family and friends as well as taking time and energy away from other patients who may have more pressing health issues. The idea of putting forth effort, at a level that could allow the individual the ability to significantly strengthen their core in such a way that it could provide the benefit needed to reduce axial and radicular pain is a difficult one at best. The thought that the individuals range of motion (ROM) would be pain free enough so as to allow the individual to put forth maximum effort and increase their muscle endurance is an unlikely idea, especially to the degree needed to effectively compress the core organs and displace them up towards the floor of the diaphragm and down toward the ceiling of the pelvis, thereby creating a larger distance between the diaphragm and the pelvis which could result in an unloading of the discs of the lumbar spine. This treatment may create an environment that will draw the discs out of the spinal canal and create a larger foraminal opening, thereby enabling the nerve roots at that level to exit the spinal canal with less potential stressors both of which would likely reduce the excitation of the nerve tissue at each specific level within the lumbar spine both bilaterally and within the spinal canal, resulting in a reduction in the expression of axial and radicular pain in the patient suffering from chronic low back and extremity pain.

Support or Funding Information

Supported by Institutional Resources of USAT College of Medicine and Einstein Medical Institute
Modulation of Chondrocyte Differentiation by the Hyaline Extracellular Matrix.
Authors: Domowicz Miriam S , Henry Judith G. , Glory Velazquez M. , Vaval Diandra S. , Schwartz Nancy B.
Published: November 2023

The development of the skeleton involves a highly regulated, spatially organized series of events that require precise temporal control of a multitude of morphogens, growth factors, transcription factors, and mechanical‐signaling pathways. In the growth plate, the size of the limb element is regulated by the rate of chondrocyte proliferation and maturation to hypertrophy, accompanied by substantial changes in the extracellular matrix (ECM) composition. Chondroitin sulfate proteoglycans (CSPGs), in particular aggrecan, are critical for proper limb growth‐plate development in vertebrates, since several animal models have shown that defects in aggrecan composition and/or content lead to accelerated chondrocyte maturation and consequent chondrodysplasia. To explore how CSPGs in the extracellular matrix (ECM) influence chondrocyte differentiation we established a tissue‐culture system from primary chick chondrocytes, and altered their extracellular matrix by enzymatic digestion (chondroitinase or hyaluronidase treatment) or biosynthetic modification (sodium chlorate or 4‐nitrophenyl β‐D‐xylopyranoside treatment). We showed by qPCR that treatment with chondroitinase, hyaluronidase or chlorate, increased levels of collagen 10A1 (COL10A1) mRNA, a marker of chondrocyte hypertrophy, and enhanced matrix production as indicated by increases in SOX9, collagen 2A1 and aggrecan mRNA levels. Increased hypertrophy was confirmed by changes in cellular morphology and by mRNA in situ hybridization studies. In contrast, treatment with 4‐nitrophenyl β‐D‐xylopyranoside, which reduces CS chains in aggrecan but increases levels of free CS released, does not influence chondrocyte differentiation. Furthermore, levels of FGFR3 mRNA were increased in chondroitinase, hyaluronidase, and chlorate‐treated chondrocytes but that was not accompanied by changes in DNA content, i.e. cell proliferation. Thus, these results are consistent with observations in animal models, in which loss of aggrecan from the matrix induces acceleration of chondrocyte differentiation, allowing this culture system to be used to dissect matrix requirements and signaling pathways that lead to the increased rate of differentiation upon matrix aggrecan loss.

Support or Funding Information

Supported by U.S. Public Health Service Grants HD107332 and GM066522.
3D Imaging using MRI of a Non‐mineralized Complex Organism: The Jawless Sea Lamprey (Petromyzon marinus) as a Model
Authors: Wood Bradley M , Jia Guang , Carmichael Owen , McKlveen Kevin , Homberger Dominique G.
Published: November 2023

The three‐dimensional visualization of complex organisms lacking mineralized tissues has been challenging because (1) histology maintains cohesiveness of the tissues, but hinders a 3D conceptualization; (2) microdissection provides a three‐dimensional view, but is destructive; and (3) x‐ray CT imaging provides three‐dimensionality, but requires contrast staining that results in significant shrinkage due to the absence of mineralized tissue. MRI, however, is a non‐destructive method that provides soft‐tissue contrast without shrinkage. Jawless vertebrates, such as lampreys, are good models for organisms without mineralized tissues, yet are characterized by a complex connective tissue skeleton that provides a framework for their segmentally arranged musculature (myomeres). Furthermore, as the largest lamprey species, adult Sea Lampreys (ca. 75 cm long and 5 cm in diameter) are amenable to standard MRI scanning. We acquired high resolution MRI scans of a Sea Lamprey specimen by using an 8‐channel wrist coil and 3‐Tesla MRI scanner. Both 2D (horizontal and sagittal) and 3D scans were acquired as T1 and T2 weighted without moving the specimen in order to contrast tissues with high fat content (T1) and high water content (T2). For a field of view of 101 cm2, a slice thickness of 1.2 mm, and an acquisition matrix of 288 × 288, the voxel size was 0.451 mm × 0.271 mm × 1.2 mm. 3D scans take more time, but minimize gaps between slices. The MRI data were visualized by using the imaging software Avizo®. In our MRI data, certain connective tissue structures, such as myosepta, were not identifiable in their entirety in either T1 or T2 scans, but parts of them could be identified in one or the other. Therefore, segmentation (marking as a separate object) of a myoseptum was performed in T1 and the available information was augmented by toggling to T2, or vice versa. Using this approach, we were able to show how the sheet‐like myosepta curve and twist dorso‐ventrally and cranio‐caudally within the trunk. In combination with microdissection, the configuration of the visualized myosepta provides a framework for diagramming the length and orientation of the myomeric muscle fibers. The visualization‐of the connective tissue and muscle structures as separate objects is necessary for 3D animation as a step towards modeling lamprey locomotion.

Support or Funding Information

LSU Foundation to D.G. Homberger
Fibrosis and Scarring of the Brachial Plexus as Displayed by MRI/MRA/MRV
Authors: Collins James D , Saxton Ernestina H , Gelabert Hugh A , Carnes Alfred E
Published: November 2023

Bilateral rounding of the shoulders (laxity) associated with kyphosis of the thoracic spine causes costoclavicular venous compression and brachial plexopathy. This form of thoracic outlet syndrome is usually not amenable to surgical treatment in older patients, particularly in severe kyphosis of the thoracic spine. The objective of this presentation is to display the sites of landmark anatomy compressing the brachial plexus that decreases venous return to the nerves causing a shortage of oxygen and glucose needed for cellular metabolism causing fibrosis and scarring of the soft tissues marginating the brachial plexus. Magnetic resonance imaging (MRI) is the only modality that displays fibrosis and scarring of fascial plane anatomy obstructing venous and lymphatic return, not possible with Ultrasound and/or Computerized Axial Tomography (CAT).

The longer venous obstruction, transient ischemia or permanent obstruction ischemia if unrelieved, progressively affect the nerve fibers in increasing numbers and to an increasing degree. Pathology develops with edematous swelling and vascular congestion. If the pressure is unrelieved and continues to increase, the nerve(s) suffer a first degree or a conduction block injury. Compression ischemia with degeneration and fibrosis develop. In absence of relief, the endoneurial tubes and funniculi atrophy and with increasing ischemia, fibrosis becomes marked.

This presentation displays costoclavicular compression of the brachial plexus with fibrosis and scarring in two patients. One patient post resection of the scalene triangle complicated by laceration of the second division of right subclavian artery and the second patient with bilateral scalenectomy with incomplete first rib resection.

Monitored bilateral MRI, MRA and MRV of the brachial plexus is the only modality of choice to display costoclavicular compression and fibrosis and scarring of the brachial plexus.
Impact of Histone Mutations on Mismatch Repair and Cancer
Authors: Fang Jun
Published: November 2023

Mutations in histone 3 (H3), particularly H3K36M/I and H3G34V/R have been identified as novel drivers for several types of human cancer. Because H3K36me3 is essential for DNA mismatch repair (MMR) in vivo by recruiting mismatch recognition protein MutSa to replicating chromatin, cancers with H3K36M/I mutations are expected to have a defective MMR system, which leads to genome instability and cancer development. However, how H3G34V/R mutations confer tumorigenesis is unknown. Given that both Arg and Val contain a bigger side chain in comparison to Gly, we hypothesize that these big side chains at position 33 of H3 impairs H3K36 interactions with MutSa and/or SETD2, the major methyltransferase responsible for H3K36 trimethylation. We show here both in vitro and in vivo evidence that G34V/R mutations block H3K36 trimethylation and its interaction with MutSa. The crystal structures of the SETD2 catalytic domain bound to H3K36 peptide with SAH (S‐adenosylhomocysteine) reveal that the SETD2 catalytic domain adopts an open conformation, with the K36 peptide snuggly positioned in a newly formed substrate channel, which is essential for SETD2‐H3K36 interaction. It is obvious that substitution of G34 with V34 or R34 obstructs the catalytic domain and the SETD2‐H3K36 interaction. We therefore conclude that like H3K36M/I mutations, H3G34V/R mutations promote mutagenesis and tumorigenesis by blocking MMR in vivo

Impact of H3G34D/R/V on H3K36 methylation in vivo by Mas‐spec analysis. A. H3K36 methylation ratio in G1‐S boundary cell lines. B. H3K36 methylation ratio in unsynchronized cell lines. Unsynchronized cells have less H3K36me3 than G1‐S boundary cells.

A H3K36 methylation ratio in G1‐S boundary cell lines

H3.3‐WT

H3.1‐WT

non
45.8%
93.8%
57.6%
82.9%
50.0%

mono‐
44.0%
1.7%
11.6%
16.3%
8.7%

di‐
9.4%
3.7%
16.2%
0.8%
14.2%

tri‐
0.8%
0.9%
14.6%
0.0%
27.1%

B H3K36 methylation ratio in unsynchronized cell lines

H3.3‐WT

H3.1‐WT

non
56.2%
52.3%
37.3%
91.8%
30.8%

mono‐
27.9%
18.7%
18.9%
7.4%
18.0%

di‐
14.7%
26.2%
34.5%
0.8%
43.3%

tri‐
1.2%
2.8%
9.3%
0.0%
7.9%
Identifying the contribution of sumoylation and ubiquitination to yeast Replication Factor A function through the examination of “lysine‐less” RFA mutants
Authors: Senger Barbara L , Haring Stuart J
Published: November 2023

Replication Protein A (RPA) is a heterotrimeric protein complex present in all eukaryotes that is essential for DNA replication, repair/recombination, and cell cycle regulation. The major biochemical function of RPA is binding to single stranded DNA (ssDNA) intermediates, especially those formed during the processes above. In response to DNA damage, the N‐terminus (NT) of the 32 kDa subunit, Rpa2, becomes hyper‐phosphorylated by protein kinases that are integral to the DNA damage response (DDR).

Recently, phosphorylation of RPA has been demonstrated to be influenced by other upstream post‐translational modifications, including ubiquitination and sumoylation. In response to DNA damage, sumoylation occurs on the Rpa1 C‐terminus, which recruits in proteins that allow for ubiquitination. The covalent attachment of ubiquitin (and SUMO) has been proposed to affect RPA function through signaling for degradation via the proteasome, changes in protein interactions, and/or changes in downstream post‐translational modification of RPA. Sumoylation and ubiquitination of RPA occur on multiple lysine residues present in all three subunits. It is possible that there is a post‐translational modification cascade required to ultimately influence RPA function.

To examine the importance of sumoylation and ubiquitination on yeast RPA (called Replication Factor A; RFA) function, mutant forms of Rfa1 and Rfa2 were generated in which ALL of the lysine residues in each subunit were mutated to arginines: rfa1‐K‐>R (39 residues mutated), rfa2‐K‐>R (15 residues mutated), and the rfa1‐K‐>R rfa2‐K‐>R double mutant (54 residues mutated). These mutants were examined with respect to growth, the DNA damage response, and checkpoint initiation/maintenance/exit. We have demonstrated that all “lysine‐less” mutants are viable and have varying degrees of DNA damage sensitivity. Despite mutation of the targets for sumoylation and ubiquitination, we observe phosphorylation of Rfa2, suggesting that sumoylation/ubiquitination and phosphorylation of Rfa2 may be independent of one another. Additionally, mutations in RFA that lead to DNA damage sensitivities have been demonstrated by us and others to have a major defect in checkpoint regulation. Therefore, we are in the process of determining if lack of sumoylation/ubiquitination affects checkpoint function of RFA, particularly its role in exit from a checkpoint.

Support or Funding Information

This work was supported by award NSF‐CAREER‐1253723 from the National Science Foundation to SJH.
“Purification and Characterization of Deinococcus radiodurans Polynucleotide Phosphorylase a phosphate stimulated Mn2+‐dependent nuclease”
Authors: Spence Madison N. , Uranga Lee , Lusetti Shelley
Published: November 2023

DNA double strand breaks, if unrepaired, pose a threat to the cell because they can cause a wide variety of genetic alteration and apoptosis. Therefore, DNA double‐strand break repair pathways are critical for the survival of all organisms. Deinococcus radiodurans is one of the most radiation resistant organisms known exhibiting a D10 dose of 10,000 gy. Its survival is attributed to robust DNA repair mechanisms. Previous work with DNA repair proteins has identified a potential interacting partner, Polynucleotide Phosphorylase (PNPase). PNPase was first characterized for its polymerase and phosphorylase activities on RNA, where it is involved in regulating RNA turnover. In its synthetic mode, PNPase is a metal‐dependent template independent single stranded RNA polymerase that adds nucleotide diphosphates (NDPs) onto an existing RNA chain; as well as a Mg2+‐PO4 ‐dependent RNA phosphorylase. In vitro studies have shown that PNPase can also work on single stranded DNA substrates when manganese is used as a metal cofactor. Our lab is interested in how PNPase activity can be influenced by other DNA repair proteins and vice versa. The purified D. radiodurans PNPase displays the expected DNA degradation activity with phosphate and its NDP‐dependent polymerase activity. However, we have also observed that the enzyme can degrade DNA in the absence of added phosphate suggesting nuclease activity; and that the addition of phosphate stimulates this Mn2+ ‐dependent nuclease activity.

Support or Funding Information

R01GM104375
Rabbit Polyclonal Antibodies to Detect C7orf49 Proteins Toward Understanding Double Strand Break Repair
Authors: Englund Bailey
Published: November 2023

DNA double‐strand breaks are a highly cytotoxic form of DNA damage. Non‐homologous end joining (NHEJ) is an important mechanism that repairs double‐strand breaks in DNA. Accurate function of NHEJ protects genome integrity; however, malfunctions within the mechanism can lead to genomic changes resulting in carcinogenesis. The basic requirements for the NHEJ process have been determined, but there are still questions about the step‐wise assembly of the system. Our preliminary data indicates chromosome 7 open reading frame 49 (C7orf49) produces short open reading frame (sORF)‐encoded proteins that may participate in non‐homologous end joining (NHEJ). C7orf49 encodes three alternatively spliced protein isoforms, which will be referred to as spliced forms 1, 2 and 3 (SF‐1, SF‐2 and SF‐3, respectively). Transcriptome analysis and ribosomal profiling indicate that the C7orf49 locus is both transcribed and translated in human cells; but it is presently unclear: 1) whether endogenously expressed C7orf49‐encoded proteins are stable or degraded, 2) how abundant C7orf49‐encoded proteins are, 3) what isoforms are expressed, and 4) how DNA damage affects the abundance of C7orf49‐encoded proteins. The goal of this project is to determine whether our anti‐C7orf49 antibodies, rabbit polyclonal antibodies, can be used to detect C7orf49‐encoded proteins in human cell lysates. Antibodies were tested via Western blot under variable conditions with both wild type and C7orf49‐deficient HEK293 cells. C7orf49 antisera recognized SF‐1 and SF‐2 as recombinant purified proteins or when ectopically expressed in HEK293 cells. Compared to pre‐immune sera, immune sera recognized bands in wild type cell extracts, which may be encoded by C7orf49. Future experiments will investigate which isoforms are expressed, and what affects DSB‐inducing drugs have on C7orf49‐encoded protein abundance, to help determine how these proteins contribute to NHEJ.

Support or Funding Information

Support received from Leslyn Hanakahi and Matthew Summerlin at University of Illinois Chicago ‐ Rockford
DNA Repeat Expansion and Mismatch Repair: A Recurrent Problem
Authors: Fuselier Kayla T B , Grabczyk Ed
Published: November 2023

At least 22 human neurodegenerative diseases are caused by DNA repeat expansion. Repeat expansion can occur at any developmental stage or during the life of an affected individual. Somatic repeat expansion is particularly interesting because it has the potential to affect disease age‐of‐onset, severity, and progression. Thus, understanding the factors that cause this somatic expansion is crucial and makes it a prime therapeutic target for these disorders. Previous studies have found that a major driver for repeat expansion is DNA mismatch repair (MMR). Though MMR is important for genomic stability, it has been conversely associated with trinucleotide repeat instability.

Our lab has focused on the GAA·TTC trinucleotide repeats that are associated with Friedreich ataxia (FRDA), a progressive neurodegenerative disorder that currently has no treatment. Our objective is to refine our understanding of the role MMR proteins play in the repeat expansion that causes FRDA. We have previously shown that the expansion rate is associated with transcription within the repeat and requires the MMR recognition complex MutSβ and the subsequent action of a MutL endonuclease complex. In our human cell model, we have established that the necessary endonuclease complex is MutLγ, a heterodimer of MLH1 and MLH3. Our research indicates a pivotal role for MLH3 in GAA·TTC expansion, specifically a single isoform, MLH3 isoform 1 (MLH3 iso1). Humans have two splice variants for MLH3; MLH3iso1 includes exon 7, an endonucleolytic domain, and MLH3iso2 that excludes exon 7. All known MutL complexes require MLH1, making it a poor therapeutic target. In contrast, MLH3 is a constituent of only MutLγ, a minor form expressed as a small percentage of total MutL complexes. Therefore, MLH3 isoforms are a much more attractive target to halt GAA·TTC expansion rather than pursuing other DNA mismatch repair enzymes.

Support or Funding Information

Friedreich Ataxia Research Alliance (FARA)
Activation of Transcription‐Coupled 5′‐RNA Capping by TFIIH
Authors: Noe‐Gonzalez Melvin , Conaway Joan , Conaway Ronald
Published: November 2023

Transcription is coupled in time and space to various mRNA processing events including 5′ capping, splicing, and polyadenylation. Capping of the 5′ end of pre‐mRNA, the first detectable mRNA processing event, is thought to occur by the time the transcript is only 25–30 nucleotides long and correlates with hyperphosphorylation of the C‐terminal domain (CTD) of transcribing Pol II. Notably, previous studies have shown that capping is over 100‐fold more efficient when coupled to Pol II transcription.

Here we exploit a highly purified, reconstituted Pol II transcription system to investigate the mechanism of co‐transcriptional capping. We present direct evidence that TFIIH functions as a potent activator of co‐transcriptional capping in this system. The TFIIH‐associated CAK protein kinase most likely contributes to TFIIH action in transcription‐coupled capping in light of evidence (i) that CAK inhibitors, H8 and THZ1, inhibit TFIIH function and (ii) that recombinant CAK can at least partially replace TFIIH. Finally, we use DNA:RNA scaffolds to show that capping of transcripts in artificial Pol II elongation complexes also requires TFIIH and its CAK kinase in the absence of other general transcription factors.

Taken together, these data support the model that TFIIH‐dependent phosphorylation, most likely of the Pol II CTD, plays an integral role in transcription‐coupled RNA capping.
The Heme‐Binding PAS Domain Mediates Dimerization in the CO‐Sensing Transcription Factor BxRcoM‐1
Authors: Pinhancos Rebeca C. , Bowman Hannah E. , Dent Matthew R. , Young Byron H. , Berndsen Christopher E. , Burstyn Judith N
Published: November 2023

The gases NO, O2, CO and H2S are small‐molecule effectors involved in signaling pathways. Heme serves as an effector‐binding site in many proteins that sense these molecules, including gas‐sensing transcription factors, and the effector‐binding signal is transduced through varying allosteric mechanisms. Bacterial transcription factors are excellent systems in which to study gas‐mediated allostery due to their simplicity relative to the multi‐protein complexes involved in eukaryotic transcription. RcoM‐1 is a heme‐containing, CO‐responsive transcription factor that appears to regulate aerobic CO oxidation in the non‐pathogenic bacterium Burkholderia xenovorans. The RcoM‐1 heme plays a key role in CO‐sensing. Under reducing conditions, the Fe(III) heme undergoes a redox‐mediated ligand switch in which a neutral donor replaces an axial cysteine(thiolate) ligand, Cys94. The neutral donor ligand Met104 was identified through a spectroscopic study of three Met‐to‐Leu variants. RcoM‐1 variants in which Met104 is replaced with a non‐coordinating Leu residue exhibited electronic absorption and magnetic circular dichroism spectra that, under reducing conditions, are indicative of a five‐coordinate, high‐spin (S = 2) Fe(II) heme center. CO replaces Met104, binds to the Fe(II) heme, and presumably modulates DNA‐binding activity. The structure of RcoM‐1 is still unknown; however, sequence homology suggests that RcoM‐1 is a unique, single‐component signaling protein that contains both a PAS‐like heme‐binding domain and a LytTR‐like DNA‐binding domain. We probed the oligomeric status and topology of this novel single‐component transcription factor. Size exclusion chromatography (SEC), SAXS, and analytical ultracentrifugation data (MW/MS = 2.25 ± 0.01) are consistent with a dimeric structure for RcoM‐1. Further SEC data show that an RcoM‐1 truncate containing only the heme domain is dimeric and that variants lacking the His74 ligand, which bear no heme, are also dimeric. Taken together, these data suggest that RcoM‐1 dimerizes via its heme domain, but dimerization does not require heme. A stable, dimeric RcoM‐1 homology model that dimerizes via the heme domain was built that fits well within the molecular envelope predicted by SAXS.

Support or Funding Information

The National Science Foundation supported these studies under CHE‐1213739 (to J.N.B.)
Transcriptome Analysis of Conditional Knockout Mice
Authors: Harvey Amanda L , Biefeld Chelsea , Sztroin Kelcie , Zhu Julia , Yu Gang , Dries Daniel R.
Published: November 2023

Gamma‐secretase is an intramembrane protein that cleaves type I transmembrane proteins. It is known to be involved in Alzheimer's disease and neurodevelopment. Previous studies have investigated the role of gamma‐secretase role in neurons. However, it has seldom been considered in glial cells, the non‐neuronal cells of the brain. Here, a conditional knockout mouse was made in which Nicastrin, a substrate receptor for gamma‐secretase, was rendered nonfunctional in 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 searching for differentially expressed genes in the experimental mice. Gene ontology programs were used to evaluate the genes and identify biological processes that may play a role in the behavioral aspects of these mice. Disease pathways identified in the analysis include Alzheimer's disease, myelination, schizophrenia, and oligodendrocyte function. Cellular pathways include extracellular communication and immune responses. Together this analysis presents a picture of the cellular pathways involved in the non‐cognitive aspects of Alzheimer's disease as it relates to the non‐neuronal white matter of the brain.

Support or Funding Information

Acknowledgments 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.
Using Cryo‐electron Microscopy to Discover Box C/D s(no)RNP Structure
Authors: Baserga Susan J , Yip W.S. Vincent , Shigematsu Hideki , Taylor David W.
Published: November 2023

Ribosomal RNA (rRNA) modifications are essential for ribosome function in all cellular organisms. Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze 2′‐O‐methylation, one type of rRNA modification found in Eukarya and Archaea. Negatively stained electron microscopy (EM) models of archaeal box C/D sRNPs from our laboratory have demonstrated the dimeric sRNP (di‐sRNP) architecture, which has been corroborated by nuclear magnetic resonance (NMR) studies. Because of the limitations of the structural techniques, the orientation of the box C/D sRNAs in the di‐sRNP has remained unclear. Here, we have used cryo‐EM to elucidate the orientation of the sRNAs in a M. jannaschii box C/D di‐sRNP. The cryo‐EM reconstruction suggests a parallel orientation of the two sRNAs. This is further confirmed by results from biochemical and structural analyses of sRNPs assembled with mutant sRNAs that indicate a potential interaction between the sRNA stem ends. Our results suggest that the parallel arrangement of the sRNAs juxtaposes their stem ends into close proximity to allow for a stabilizing interaction that helps maintain the di‐sRNP architecture.

Support or Funding Information

Supported by NIH R01GM0115710
Functional Significance of Intermediate Cleavages in the 5′ETS of Schizosaccharomyces pombe pre‐rRNA
Authors: Van Loggerenberg Warren , Nellimarla Srinivas , Park James , Nazar Ross N.
Published: November 2023

The biosynthesis and incorporation of mature ribosomal RNAs (18S, 5.8S and 25S rRNA) into ribosomes is dependent on coordinated and interdependent processing events, acting as a “quality control mechanism”. Transcribed spacers are critical to processing and act as “biological springs” to enable the appropriate configuration of maturing termini. The pre‐rRNA 5′ external transcribed spacer (5′ ETS) of Schizosaccharomyces pombe contains structural elements critical for initiation and efficient pre‐rRNA processing. Examination of termini in the 5′ ETS region based on ligation‐mediated RT‐PCR revealed two major intermediate cleavage sites (A1 and A2) in addition to the A0 cleavage, which forms the mature 5′ end of the 18S rRNA. Mutational analyses at the A2 intermediate cleavage site indicate that this cleavage is not critical to rRNA maturation and can be eliminated. Mapping of the 5′ ETS region in cells with temperature sensitive nucleases, using RT‐PCR, refute formation of the mature 5′ end of 18S rRNA by RNase III‐like endonucleolytic cleavage across an extended hairpin. Mutational analysis around the 5′ ETS/18S rRNA junction indicate cleavage is not dependent on sequence and/or structure, but can dramatically affect rRNA yield. Additionally, mutations in U3 snoRNA or a complementary site in the 5′ 18S rRNA pseudoknot can significantly affect the 18S/25S rRNA ratio. These results raise the possibility that the 5′ end of the 18S rRNA is the result of ribozyme cleavage and that the U3 snoRNA acts as a chaperone to stabilize RNA structure.

Support or Funding Information

Supported by the Natural Sciences and Engineering Research Council of Canada.
BDF2 Transcript Sensitivity to RNase III‐Mediated Decay is Heavily Governed by its Transcript Localization and Vulnerability to Cleavage
Authors: Wang Charles , Roy Kevin , Chanfreau Guillaume
Published: November 2023

Bromodomain factors are emerging as key players in transcription regulation through chromatin remodeling and transcription initiation. Due to their significant impact on the eukaryotic transcriptome, they are often under the control of several regulatory mechanisms to ensure the appropriate modulation of gene expression. One such factor in yeast, Bromodomain Factor 2 (BDF2), has been established as a target of both RNase III‐mediated decay (RMD) and spliceosome‐mediated decay (SMD) for extensive surveillance. Although our previous work has shown that the RMD pathway for the BDF2 transcript is hyper‐activated by salt stress and other environmental conditions, the mechanism driving the enhanced cleavage of BDF2 RNA under these conditions remains obscure. Through the anchor‐away technique, we show that inhibiting the export of the BDF2 transcript out of the nucleus can recapitulate the stress‐induced RMD hyper‐activation. In addition, we also show in vitro and in vivo that the cleavage efficiency of the RNase III (Rnt1p) on BDF2 may further contribute to its vulnerability to RMD. Altogether, these results suggest that the activity of RMD is heavily governed by the localization of its target transcripts, and that the efficiency of cleavage may further contribute to the sensitivity of these transcripts to degradation.

Support or Funding Information

Cellular and Molecular Biology Training Grant
Structural and Functional Characterization of a Human Ribonuclease Implicated in Obesity
Authors: Abshire Elizabeth , Chasseur Jennifer , Bohn Jennifer , DelRizzo Paul , Goldstrohm Aaron , Trievel Raymond
Published: November 2023

Obesity affects a significant number of Americans and greatly increases the risk of developing cardiovascular disease, type‐II diabetes, fatty liver disease, and certain cancers. Understanding how obesity is regulated opens up new avenues of pharmacological interventions to treat obesity, concurrently reducing the risk of co‐morbidities. Mice lacking the broadly expressed enzyme Nocturnin (NOCT) are resistant to diet‐induced obesity and other studies have observed a role for this enzyme in regulating intestinal trafficking of dietary fats and promoting adipogenesis. We have characterized human NOCT and determined that it represses the activity of reporter RNAs in tethered function assays promotes decay of reporter RNAs in vivo. These observations are consistent with a proposed function for NOCT in RNA decay and suggest that there may be a novel pathway that regulates lipid metabolism by targeting mRNAs for degradation. Though the broader physiological effects of gene deletion of NOCT have been described, discovering its molecular function remains an important goal. We have utilized structural and approaches to determine the first crystal structure of the human homolog of NOCT to aid in elucidating its catalytic mechanism. Additionally, we have developed cell‐based assays that will be used to study NOCT function and analyze the effects of active site mutants. These studies describe the structure and activity of human NOCT and provide the foundation for characterizing this enzyme as a potential drug target for obesity and related co‐morbidities.

Support or Funding Information

National Institutes of Health Chemistry‐Biology Interface Training Program (NIH grant 5T32GM008597), American Heart Association Midwestern Affiliate Predoctoral Fellowship, Michigan Nutrition and Obesity Research Center Pilot Grant
Investigating the Role of the Human Parvovirus B19's Main Viral Protein, NS1, in Viral Replication and Interactions with Host DNA
Authors: Hernandez Christian Joab , Sanchez Jonathan Lares , Horton Nancy C.
Published: November 2023

Human Parvovirus B19 (B19V) is a ubiquitous virus that infects the majority of the human population. B19V is a non‐enveloped, single stranded DNA virus with a viral genome composed of 5594 nucleotides. Infection with B19V has been associated with a multitude of diseases, such as hydrops fetalis, arthropathy, hepatitis, cardiomyopathy, erythema infectiosum (Fifth disease), and possible onset of a variety of autoimmune diseases. The viral genome of B19V encodes for five known, possible six, protein products: VP1 and VP2 that compose the capsid, NS1, the main viral protein, and two to three smaller protein products of indistinct function. The main replicative protein, NS1, is a multiple domain protein with a nuclease domain, helicase domain, and a C‐terminal domain. Through its multi‐functional capabilities, NS1 has been hypothesized to play several roles in the triggering autoimmune disease via formation of covalent DNA‐NS1 adducts and/or host gene transactivation. In order to understand the mechanism behind NS1's ability to bind viral and host genomic DNA, biochemical experiments were carried out to build a model for NS1's role in viral genome replications and host gene transactivation capabilities.

Support or Funding Information

Supported by: the National Science Foundation under Grant MCB1410355, and National Institutes of Health via Grant S10OD013237
A conserved active site residue favours metal ion binding by destabilizing apo‐protein.
Authors: Miller Anne‐Frances , Wang Ting
Published: November 2023

The active sites of Fe‐containing SOD and Mn‐containing SOD (FeSOD and MnSOD respectively) are organized around a single redox‐active ion, Fe or Mn, which alternates between its 3+ and 2+ states during turnover. The proteins are homologues, and some members of the family support activity with either Fe or Mn (Fe&MnSODs), while others bind either metal ion but display activity with only one. A second‐sphere Gln/His residue is the most commonly conserved difference between FeSODs and MnSODs, which supply the Gln from postion 69 or 146, respectively. Mutation of Gln69 of E. coli FeSOD to Glu or His produced large changes in the E° and catalytic activity indicating that the residue at position 69 has a large influence on the affinity for at least one of the Fe oxidation states (Yikilmaz et al 2006, Biochemistry 45:1151). Analogous studies of MnSOD have found that replacement of Gln146 results in changes in activity and redox tuning, but the Q146E‐MnSOD mutant has evaded study because it fails to bind Mn. We have compared the stability and metal ion binding of a series of mutants of the MnSOD protein wherein different amino acids occupy position 146. These studies shed light on the relationship between metal ion binding and protein stability in this series of related variants of MnSOD. In particular, Q146E‐apoMn‐SOD displays a melting temperature elevated over that of WT‐apoMn‐SOD by 30 °C, and other mutations produce intermediate behaviour. Thus the WT apoMn‐SOD is less thermally stable than many of the variants. We propose that the WT protein conserves a destabilizing amino acid at position 146 as part of a strategy for favouring metal ion binding, as well as redox tuning.
Recruitment of Drp1 in Mitochondrial Fission
Authors: Egner John M , Bakkum Amber , Gardner Alex , Hill Blake
Published: November 2023

Intracellular membranes are dynamically shaped by proteins including fission and fusion events important for vesicular transport, organelle trafficking, and cellular homeostasis. Yet how proteins interact with membranes to control these events is largely unknown. We are interested in addressing this question through mechanistic studies of a dynamin superfamily member central to the division, or fission, of mitochondria. Mitochondrial fission is driven by the GTPase mechanoenzyme, Dynamin‐related protein 1 (Drp1). At the mitochondrial surface, Drp1 is thought to assemble into rings around a future site of scission in a GTP‐dependent manner. Curiously, Drp1 is primarily localized to the cytoplasm and how it is recruited to the mitochondrial surface is unknown. Several proteins resident to the mitochondrial surface have been implicated in Drp1 recruitment and subsequent assembly, but the mechanism remains unclear. We are addressing these problems using biochemical, structural, and cell biological approaches. Our results to date will be presented and suggest a new model for Drp1 activity in mitochondrial fission.

Support or Funding Information

This project was supported by the Clinical & Translational Science Institute (CTSI) through the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number UL1TR001436. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Project also supported by NIH grants NIH R01‐GM067180, and NIH R01‐HL128240.
Capillary Dilatometry, In Conjunction with UV Spectrophotometry, Measures Conversion of Insulin to Polymeric Protein Fibrils (Amyloid)
Authors: Carman Peter J. , Kahn Peter C.
Published: November 2023

Insulin, which is produced by the pancreas, is a hormone that permits muscle, fat, and liver cells to absorb glucose from the blood. Diabetics who produce insufficient levels of insulin may take the hormone through injection. Repeated insulin injection often causes localized amyloidosis, a poorly understood process characterized by the misfolding of insulin into insoluble polymeric protein fibrils. The dose circulating in the blood is then less than the administered dose with attendant medical consequences. In this work, we followed the misfolding of insulin to its amyloid state through the measurement of the volume change of the reaction. This thermodynamic parameter, the change in volume per mole of native insulin polymerized to amyloid, was measured by capillary dilatometry in conjunction with UV spectrometry. The change in volume is due to water expulsion from the surface of native insulin upon amyloid formation. Preliminary experiments indicate volume change of 200–400 mL/mol of insulin polymerized. The data are being used to estimate the number of water molecules expelled, and we are investigating the possibility of determining the entropy change upon polymerization. Fluctuations from one experiment to the next suggest the presence of additional variables not yet accounted for. Electron microscopy of the amyloid product shows distinct fibrils. Accordingly, future directions include further electron microscopy to analyze how amyloid fibril size affects the amyloid conversion rate.