bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison between morphological and morphometric parameters of recurrent laryngeal nerve in developing spontaneously hypertensive rats
Authors: da Silva Greice Anne Rodrigues , Genari Adriana Borges , Salgado Helio Cesar , Fazan Valéria Paula Sassoli
Published: June 2021

Despite some reports on the peripheral nerve morphology in spontaneously hypertensive rats (SHR), no investigations on the recurrent laryngeal nerve (RLN), known to contain baroreceptor fibers is available in this rat strain. We aimed to describe the normal morphological and morphometric parameters of the recurrent laryngeal nerves in developing SHR. Animals were divided into three groups: Female SHR 5, 8 and 20 week‐old (n=4 in each group). The animals were anesthetized for direct measurement of blood pressure, perfused with a fixative solution and had their right and left RLN, dissected and prepared for light microscopy and morphometry. Body weight increased significantly with aging. Mean arterial pressure was significantly higher in 20‐week old animals compared to 5 week‐old, but not different between 5 and 8‐week old animals or 8 and 20‐week old ones. In general, proximal segments are larger than distal segments with a significant reduction on the myelinated fiber number from proximal to distal. Myelinated fiber size is larger on the older animals, following the increase in body weight. No significant alterations of the nerve morphology were observed in 20‐week old animals, despite the elevated arterial pressure.

Grant Funding Source: FAPESP, CNPq, CAPES and FAEPA
A bilateral pudendal nerve injury model in male rats
Authors: Shalhoub Philip J. , Brancato Sam J. , Foecking Eileen M. , Fargo Keith N.
Published: June 2021

Radical prostatectomy typically results in some degree of urinary incontinence, usually due to dysfunction of the urethral rhabdosphincter consequent to pudendal nerve damage. However, while pudendal nerve injury has been studied in relation to female incontinence, it has largely been neglected in the male literature. Here we describe an animal model of male urethral rhabdosphincter dysfunction following bilateral pudendal nerve compression injury. All subjects were young adult male Sprague‐ Dawley rats. Briefly, a horizontal incision was made between the anus and tail, and the pelvic space exposed. The motor branch of the pudendal nerve was visualized in Alcock's canal and crushed bilaterally (sham controls were not injured). Animals were allowed to survive for 2, 4, 7 or 14 days, then tested for leak point pressure prior to sacrifice. Injured animals displayed variable abnormalities of the urogenital system, usually including one or more of the following: marked urinary retention, hydroureteronephrosis, hematuria, retrograde ejaculation, and/or reduced leak point pressure. These results indicate that bilateral pudendal nerve injury in the male rat leads to a complex pathology of the urogenital system and dysfunction of the urethral rhabdosphincter. This model will be useful for future studies aimed at further elucidating the function of the pudendal nerve and urethral rhabdosphincter in males.

Grant Funding Source : VA RR&D CDA‐2 B6598W
The structure of a phalloid organ of buffalo weaver (Bubalornis niger) and its sensory nerve endings ‐a light‐and electron microscopic study.
Authors: Halata Zdenek , Birkhead Tim R
Published: June 2021

The Buffalo weaver is a unique species of bird. Males possess a phalloid organ near the cloaca in the lower abdomen. The birds live in colonies in sub‐Sahara African regions. During copulation the male rubs the phalloid organ around the outside of the female's cloaca. This can last up to 30 minutes before the male appears to experience an orgasm under intense shaking of the whole body and ejaculates. The phalloid organ is up to 17 mm long and about 5 mm in diameter. It consists of bundles of skeletal muscle fibres arranged in longitudinal direction but does not contain any sperm duct. The thin skin on the surface is sparsely covered with small feathers. Bundles of myelinated nerve fibres of about 8 μm diameter run through the connective tissue of the dermis. Using light and electron microscopy, small Herbst corpuscles are found within the dermis of the skin near the bulb of the feathers. Herbst corpuscles consist of a terminal axon placed in an inner core formed of thin cytoplasmic lamellae of terminal glial cells surrounded by a capsule of thin perineural cells. The subcapsular space between the inner core and the capsule is filled with thin collagen fibrils and intercellular substance.
Use of a natural latex membrane for nerve grafting: an experimental study
Authors: Ganga Marcos Vinícius Muniz , Fazan Valéria Paula Sassoli , Júnior Wilson Marques , da Silva Lopes Luiza
Published: June 2021

A membrane produced from a natural latex has been used successfully both experimentally (angioplasties, esophagus neoformation, reconstruction of the ocular conjuntive) and clinically (myringoplasties, treatment of skin ulcers), demonstrating angiogenic potential and leading to tissue neoformation. We evaluated the ability of this natural latex membrane in speeding up and improve the regeneration of sectioned sciatic nerve in rats. Forty adult male Wistar rats were divided in 4 experimental groups (n=10 each) and operated with the auto‐scion or the interposition of a tube made up of that membrane derived from natural latex (Hevea Brasiliensis). After 4 or 8 weeks, animals were submitted to neurological function evaluation, pace evaluation and electromyography. Afterwards, the animals were killed and the nerves were processed for paraffin embedding and observation under light microscopy. Our results indicated that the rats operated with latex have a better recovery rate than the ones operated with autologous nerve duo to the higher use rates of the operated paw. Morphologically, the regenerative capacity of the latex membrane is larger than auto‐scion of the sciatic nerve in rats.

Grant Funding Source : FAPESP, CNPq and FAEPA
Neurovue as a tracer for human nervous tissue
Authors: Pendola Martin , Caceres Noel , McGillicuddy Michael , Marquez Samuel
Published: June 2021

Neurovue is a lipophilic neuronal tract tracer designed for fixed tissues. Although it has been used in perfused animal brains, its use in either human or immersion fixed tissues has not been well documented. Diffusion properties of Neurovue in immersion fixed human optic nerves was studied. Brains were collected from cadavers in the Anatomy Department of SUNY Downstate Medical Center. Brains were immersion fixed in 10% formalin for three days. A Neurovue filter strip was then placed on the right optic nerve. Brains were then re‐immersed in fixative with the filter strip still affixed to allow the tracer to diffuse into the tissue. Neurovue diffusion was grossly evident after only two weeks of exposure to the tracer. After four weeks of exposure, samples were sliced into 5 um sections and examined under fluorescent microscopy at 4× and 10× magnification. Results indicate that Neurovue was successfully transported across the specimen, and there was visible contrast with unlabeled tissue. The tracer traveled a path from the right optic nerve to the contralateral side of the optic chiasm, following the expected course of optic nerve tracts. This study shows that Neurovue can be used as a marker in human fixed tissue without utilizing perfusion procedures. Future studies will evaluate Neurovue in labeling human cortical connections, and the feasibility of its use in macroscopic visualizations.
Influence of painful procedures in the neonatal period in motor and sensory functions of Wistar rats during development
Authors: Carmo Elisabete Cássia do , Machado Nathalia Leilane Berto , Sanada Luciana Sayuri , Fazan Valéria Paula Sassoli
Published: June 2021

We aimed to evaluate motor and sensory functions in animals exposed to painful stimulation after birth. Wistar rats were separated in: male pain group, 15 days (N=14); male control group, 15 days (N=11); female pain group, 15 days (N=10); female control group, 15 days (N=10); male pain group, 30 days (N=14); male control group, 30 days (N=17); female pain group, 30 days (N=10); female control group, 30 days (N=9); male pain group, 90 days (N=16); male control group, 90 days (N=10); female pain group, 90 days (N=13); female control group, 90 days (N=9); male pain group, 180 days (n=16); male control group, 180 days (N=17); female pain group, 180 days (N=17) and female control group with 180 days of life (N=18). Pain groups were stimulated with a needle while control groups with a cotton swab, both on the right paw, twice a day for 15 days. The grip strength was measured using the Grip Strength Meter. Perimeter of the hindpaw was measured with a caliper rule associated with tape. Sensory function was accessed using Von Frey monofilaments and a calibrated forceps. Control groups had lower body weight than the pain groups. The grip strength, the perimeter of the hind paws, the calibrated forceps and the analysis with the Von Frey of the pain groups showed lower scores than the control groups. Our data suggest that the painful stimulation on neonatal period can influence the motor and the sensory function.

Grant Funding Source: FAPESP, CNPq and FAEPA
Videoscopic images of unique septal and medial papillary muscle complexes recorded from reanimated human hearts
Authors: Bateman Michael G , Iaizzo Paul A
Published: June 2021

The continued rapid development of right‐sided cardiac transcatheter therapies has led to an increase in the application of such technologies; thus generating a renewed interest in the nuances of right heart anatomies. The use of Visible Heart® methodologies in over forty reanimated human hearts (deemed not viable for transplant) has allowed us to capture unique footage of the internal anatomy of the right ventricle (Hill et al., 2001). Footage was recorded using 4 and 6mm endoscopes (Olympus Corporation, Tokyo, Japan) while the hearts are operating in a functional four‐chamber working mode. Presented here is a selection of images of unique tricuspid septal and medial papillary complexes recorded from within the right ventricle displaying the wide variety of morphologies and anatomies of the valve apparatus. Such varied and complex anatomies could potentially alter, or be altered by the delivery of right‐sided transcatheter therapies: e.g., increasing the possibility of damage to the functional anatomy of sub‐valvular apparatus. We consider here that such images can be employed as educational tools for both clinicians and device designers to better understand the dynamic internal anatomies of the human heart. This project was funded in part by Medtronic, Inc.
Morphological pattern of mouse intrinsic cardiac nerve plexus
Authors: Pauza Dainius H. , Rysevaite Kristina , Saburkina Inga , Jokubauskas Marius , Pauziene Neringa
Published: June 2021

The study was aimed to determine the 3D organization and immunohistochemistry of intrinsic neural plexus in mouse heart. In a whole heart preparation, intrinsic neural structures were revealed by a histochemical staining for acetylcholinesterase, while the double labeling of tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), substance P (SP) and calcitonin gene related peptide (CGRP) was applied to identify immunohistochemically the distribution of adrenergic, cholinergic and peptidergic neural components, respectively. Our findings demonstrate that extrinsic cardiac nerves access the ganglionated nerve plexus of the mouse heart hilum at the right and left cranial veins. Nerves and bundles of nerve fibers extend epicardially from this plexus to atria and ventricles by 5 neural pathways or subplexuses. Generally, the examined hearts from 56 mice contained 19±3 ganglia, in which 1.082±160 neurons reside. Majority of intrinsic neurons (83%) are ChAT(+), 3% ‐ TH(+); 14% – bifenotypic, i.e. they were immunoreactive both for ChAT and TH. The TH(+) epicardial nerve fibers are predominant on the dorsal and ventral sides of left atrium, whereas the most ChAT(+) axons proceed on the heart base toward the large intrinsic ganglia and within epicardium of the root of the right cranial vein. Peptidergic nerve fibers were abundant in the epicardium and within intrinsic ganglia adjacent to the heart hilum. In conclusion, although majority of nerves and neural bundles in the mouse heart are mixed, some of them express either adrenergic or cholinergic phenotype. This study was supported by the Research Council of Lithuania (No. MIP‐11184).
Application of four‐dimensional computed tomography angiography in the study of vascular structure of the deep inferior epigastric artery perforator
Authors: Wenting Li , Borong Fang
Published: June 2021

To explore the vascular structure of the deep inferior epigastric artery perforator with the four‐dimensional computed tomography (CT) angiography. 10 abdominal specimens harvested from fresh cadavers were cannulated with trocar and injected with contrast medium in the deep inferior epigastric artery at different levels. During the perfusion of the contrast medium in the flap, the flap was scanned by three‐dimensional CT (four‐dimensional defined as three dimensional CT plus time). The CT data were sent to CT workstation and the images were processed and reformatted. The deep inferior epigastric artery perforator terminates in the subdermal vascular plexus, with bifurcating superior to the Scarps fascia and under the skin. The anastomosis in the abdominal midline occurs in the subdermal vascular plexus. The zone overlying the rectus muscle in the pedicle side has the best blood supply. The four‐dimensional CT is a useful method to study the vascular structure of the deep inferior epigastric artery perforator in the superficial fascia.

Grant Funding Source: Institute of Basic Medical Sciences£¬Chinese Academy of Medical Science Funding(2008PY02)
Immunohistochemical Localization of Collagen Types I, III and IV in Fibrotic Livers of Elderly Cadavers
Authors: Mak Ki Mark , Chu Edward
Published: June 2021

Elderly cadavers with diverse causes of death demonstrated a high prevalence of liver fibrosis, as assessed by Sirius red stain (Mak and Hoo, FASEB J 2011 25: 870.2). The staining, however, did not reveal the chemical types of collagen (Col). Therefore, this study examined localization of Col I, III and IV (the principal Col produced in liver fibrosis) in fibrotic foci of elderly cadavers by immunohistochemistry, using Col I, III or IV antibodies. Col I stained the fiber bundles in fibrotic central veins, perisinusoidal fibrotic foci, stroma of portal tracts, and expanding and bridging fibrous septa. Col III showed a similar localization as Col I. In fibrotic central veins, Col IV stained the endothelial basement membranes and mesenchymal cells. The sinusoidal lining and perisinusoidal cells displayed variable Col IV staining. In portal tracts, Col IV stained the stromal cells and basement membranes of bile ducts and vasculature. Col IV staining also revealed ductular and capillary‐like structures in the matrix of expanding fibrous septa and more strikingly at the septal‐parenchymal and portal‐parenchymal borders.

Conclusions

In elderly cadavers, Col types I, III and IV localize to the fibrotic foci and are reliable markers of liver fibrosis. As liver fibrosis becomes more prominent with aging, fibrotic lesions can be better characterized by combining Sirius red staining with Col I, III and IV detection.
Novel Dose Dependent Positive Feedback Loop in Endothelial Cells: A Response to rhBMP2 Treatment
Authors: Hussein Khaled , Helwa Inas , Zakhary Ibrhaim , Elrefai Rami , Al-Shabrawey Mohamed , Sharawy Mohamed
Published: June 2021

Recently, we reported that Endothelial Cells (ECs) could be a potential source of endogenous BMP2 which is essential for successful bone regeneration in vivo. The aim of this study was to investigate the capability of ECs to respond to exogenous BMP2 by secretion of endogenous BMP2.

Methods

Human Umbilical Vein Endothelial Cells were treated with serial concentrations of rhBMP2 (25, 50 and 100ng/ml of rhBMP2) for only 3 hours and the cultured cells were maintained for 12 hours. Cell lysates were collected at 5 minutes, 3hours, 6 hours and 12 hours time points after rhBMP2 treatment. ELISA for BMP2 was done to detect its secretion in supernatant at the 6 and 12 hours time points. Real time PCR was done to detect mRNA of BMP2, BMP2R1B and SMAD1/5. Western blot analysis was done to detect the phosphorylation of SMAD1/5/8.

Results

Exogenous rhBMP2 significantly induced BMP2 secretion only in the 100ng rhBMP2 treatment at the 6 and 12hours time points consistent with a gradual upregulation of BMP2 mRNA. BMP2R1B, SMAD1 and SMAD5 gene expression were also significantly upregulated. SMAD1/5/8 was consistently phospholyated in the 5 minutes and 3 hours time points.

Conclusion

ECs respond to 100ng/ml rhBMP2 treatment by secreting BMP2 via a SMAD dependent pathway. These results suggest that ECs treated in‐vitro with rhBMP2 could be used as a source of BMP2 in‐vivo to optimize bone regeneration in challenging clinical situations.

Grant Funding Source: Ministry of Higher Education, Egypt
A donor model assessing the effect of errant drill passes during placement of femoral nail interlock screws
Authors: Mulchandani Neil , Cataldo Steven , Russo Christen , Hip-Flores David , El-Gendi Hebah , Hayes Westley , Saha Subrata , Márquez Samuel
Published: June 2021

Current orthopaedic practice to fix femoral shaft and intertrochanteric fractures is intramedullary (IM) nailing. Technical errors in nailing often result in errant drill passes (EDP) during placement of distal interlock screws. This study determined if EDP create stress risers that significantly alter the biomechanical strength of the bone. Ten mm IM femoral nails were placed in 16 paired adult cadaveric distal femur specimens. Matched specimen pairs were randomized into two groups (n=16/group). Nails were statically locked distally with two 4.5 mm bicortical screws in both groups, with specimens in Group 2 being exposed to a 4.2 mm drill hole placed anterior and inferior to the nail, representing an EDP. Using the Instron 8874 Axial‐Torsion Testing System, torsional load to failure and rotational angle at failure were recorded. Student t‐tests revealed no significant decrease in load to failure for the distally locked group with an errant drill pass (p=0.435) or any differences in rotational angle at failure between groups (p=0.581). Introducing an errant drill hole while distally locking a femoral nail did not significantly decrease the specimens’ load to failure indicating that bone strength may remain uncompromised. This suggests that although these technical errors may occur intraoperatively, post‐operative outcomes may remain unaffected.

Grant Funding Source : Not Applicable
Reduction of muscle fiber size, muscle IGF‐1, and increased myostatin in the leptin receptor‐deficient POUND mouse
Authors: Hamrick Mark , Arounleut Phonepasong , Bowser Matthew , Fulzele Sadanand , Pollock Norman , Isales Carlos M. , Erion Joanna , Stranahan Alexis
Published: June 2021

Functional leptin receptors are expressed in skeletal muscle, but the role of leptin signaling in muscle tissue is not well understood. We examined skeletal muscle in POUND mice, which lack all functional leptin receptor isoforms, and in db/db mice, which express the short form but not the long form of the leptin receptor. POUND mice and db/db mice both showed reductions (~30%) in muscle fiber diameter within the predominantly fast‐twitch extensor digitorum longus (EDL) muscle. ELISA assays were used to quantify levels of insulin‐like growth factor 1 (IGF‐1), which enhances myogenesis, and myostatin, which constrains muscle growth, in muscle homogenates. POUND mice exhibited greater reductions in EDL muscle IGF‐1 expression compared to db/db mice. Likewise, EDL muscles from POUND mice showed a 170% increase in myostatin levels compared to controls, whereas db/db mice showed a 70% increase in myostatin protein expression. These findings suggest that progressive deletion of various leptin receptor isoforms yields increasingly severe alterations in muscle fiber protein synthesis in predominantly fast‐twitch skeletal muscle fibers. Funding for this research was provided by the National Institute on Aging (P01AG036675‐ 01A1).
Cthrc1 is Involved in the Activation of Adult Stem Cells in Skeletal Muscle
Authors: Miller Ryan , Dupuis Johnathan , Schuenke Mark , Lindner Volkhard , LeClair Renee
Published: June 2021

Collagen triple helix repeat containing‐1 (Cthrc1) is a novel protein highly expressed after vascular injury, however the global functionality of the protein remains largely unknown. To determine the role of Cthrc1 in skeletal muscle we have characterized the skeletal muscle phenotype in Cthrc1 overexpressing transgenic (Tg15), knock out (KO) and wildtype (WT) mice. Preliminary characterization of the tibials anterior from KO and WT 12‐week old males identified a reduction in Type I muscle fiber size (KO vs. WT) with no change in distribution of Type I and Type II muscle fibers (as shown by cytochrome oxidase staining). Immunohistochemistry of the gastrocnemius from Tg15 and WT animals showed colocalization of activated Pax7+ satellite cells with both Cthrc1 and myogenin expression, suggesting that Cthrc1 correlates with stem cell activation. Tg15 muscles showed signs of active fiber regeneration, both basophilic cytoplasm and nuclear chains, possibly as a result of increased Cthrc1 expression leading to an increased number of activated stem cells within the tissue. Muscle stem cell populations dual‐labeled for Sca‐1 and CD31 were evaluated by flow cytometry; KO animals showed a significant reduction in the Sca‐1+/CD31− cell population when compared to wildtype animals. C2C12 myoblasts cultured in Cthrc1 conditioned media show increased transcript levels of muscle regulatory factors (MRF) Myf5 and myogenin when compared to controls; consistent with our animal data. The difference between Sca‐1+ MSCs, coupled with the influence of Cthrc1 on MRF expression suggests that Cthrc1 may affect MSC activation as well as proliferation. Cthrc1's potential to act as an MSC activator suggests it may play a significant role in skeletal muscle repair and regeneration offering new therapeutic approaches to fibrotic and muscle wasting diseases.
The myomere‐myoseptal intersections in a lamprey (Petromyzon marinus) and a shark (Squalus acanthias)
Authors: Wood Bradley Morgan , Andermann Roy J. , Homberger Dominique G.
Published: June 2021

An accurate description of the myomere‐myoseptal intersections of the axial musculature of piscine vertebrates is a necessary basis for a mechanical explanation of how contractile forces are transmitted from one myomere to the next via myosepta. In a lamprey, the tendons of superficial muscle fiber bundles join a myoseptum on each side, but in opposite directions, at shallow to right angles. In a shark, the tendons of superficial muscle fiber bundles traverse a myoseptum to connect to muscle fiber bundles partly directly across and partly diagonally across. Hence, contractile forces of the superficial muscle fiber bundles may be transmitted to each other across the myosepta. The construction of an accurate biomechanical model of the myomeric‐myoseptal intersections may be relevant for a range of questions, such as those pertaining to (1) the biomechanics of serially arranged contractile systems that are also found in the apterial and feather muscles of birds and the sarcomeres of myofibrils; (2) the interactions between the central nervous system and the axial musculature during undulatory locomotion; and (3) the evolutionary changes in the axial musculature of Petromyzontiformes and Chondrichthyes.

Funding: LSU Foundation to DGH.

Grant Funding Source: N/A
An intrinsically unstructured domain in MBD2 recruits the histone deacetylase core complex of NuRD and modifies kinetics of DNA binding
Authors: Williams David C , Desai Megha , Ginder Gordon D , Walavalkar Ninad M
Published: June 2021

MBD2 specifically recognizes methylated DNA and recruits the NuRD chromatin remodeling complex to silence expression of the associated gene. We recently solved the structure of a coiled‐coil interaction between MBD2 and p66α that recruits the Mi2 nucleosome remodeling protein to the complex (Gnanapragasam et al., Proc Natl Acad Sci. 2011, 108:7487). In subsequent work, we have shown that a region between the methyl‐cytosine binding (MBD) and the coiled‐coil domains of MBD2 stably binds the histone deacetylase core complex of NuRD comprised by HDAC1/2, MTA1/2, and RbAp46/48. NMR and CD studies demonstrate that this region is largely unstructured even in the context of full‐length protein and while bound to methylated DNA. Immunoprecipitation studies show that the critical determinant of protein binding resides within two small molecular recognition fragments (MoRFs) that together are sufficient to recruit the core deacetylase complex. Previous analyses showed very rapid association and dissociation rates for the isolated MBD. In contrast, incorporation of the intrinsically unstructured domain reduces the association rate and markedly prolongs dissociation from methylated DNA. Therefore, this intrinsically unstructured domain functions both to recruit a large portion of the NuRD complex and stabilize interaction with methylated DNA. Supported by ACS IRG‐73‐001 (DCW) and NIH R01‐DK029902 (GDG).
SREBP1c is regulated by E3 ligase RNF20/BRE1A upon hormonal changes
Authors: Lee Jae Ho , Lee Gha Young , Jang Hagoon , Kong Jinuk , Park Yoon Jeong , Kim Jae Bum
Published: June 2021

Sterol regulatory element binding protein 1c (SREBP1c) is a key transcription factor to regulate de novo lipogenesis. However, molecular mechanisms involved in suppression of SREBP1c are largely unknown. In this study, we reveal that SREBP1c is negatively regulated by ubiquitination upon fasting signals. To better understand which factors are involved in ubiquitination of SREBP1c, we take advantages of affinity purification and mass spectrometry. In this analysis, RNF20/BRE1A is identified as a novel SREBP1c E3 ligase. Here we show that RNF20/BRE1A binds to and ubiquitinates SREBP1c, leading to its degradation. Previously, it has been well established that PKA signaling is activated by fasting signals. Interestingly, we find out that PKA activator such as forskolin enhances expression of RNF20/BRE1A and stimulates ubiquitination of SREBP1c via RNF20/BRE1A. In accordance with these, RNF20/BRE1A negatively regulates the transcriptional activity of SREBP1c and expression of SREBP1c target genes. In contrast, knockdown of RNF20/BRE1A increases expression of SREBP1c and lipogenic genes. Taken together, these data suggest that RNF20/BRE1A would be a negative regulator of lipogenesis through degradation of SREBP1c upon fasting.

J. H. Lee was supported by Hi Seoul Science (Humanities) Fellowship funded by Seoul Scholarship Foundation.
Regulation of microRNA Expression by AUF1 Through Dicer
Authors: Johnson Jessica Michelle , Tominaga Kumiko , Gorospe Myriam , Abdelmohsen Kotb
Published: June 2021

MicroRNA (miRNA) biogenesis is a tightly regulated process that involves several enzymes, including Drosha and Dicer. Although Dicer is required for the processing of all precursor microRNAs into mature microRNAs, the regulation of Dicer expression is virtually unknown. It was hypothesized that the RNA‐binding protein (RBP) AUF1 is capable of binding Dicer mRNA, causing mRNA destabilization and thereby lowering Dicer protein levels. Using immunoprecipitation of AUF1 ribonucleoprotein complexes and biotin pulldown‐in vitro binding assays, AUF1 downregulation increased Dicer mRNA stability and elevated Dicer protein levels. Conversely, AUF1 overexpression lowered Dicer mRNA and protein levels. As result of these changes in Dicer abundance, microRNA levels were also affected, with AUF1 overexpression decreasing microRNA abundance. In summary, AUF1 negatively regulates microRNA levels by lowering Dicer mRNA biosynthesis. These findings implicate microRNAs function as important regulators of gene expression and play a role in multiple cellular processes. MicroRNA expression or function is significantly altered in many disease states, including cancer, inflammatory diseases, metabolic diseases, and fibrosis.
Altered states of Pax3 phosphorylation contribute to regulation in melanocytes and melanoma
Authors: Iyengar Aditi S , Hollenbach Andrew D.
Published: June 2021

Melanoma is the deadliest form of skin cancer with an alarming increase in incidence, dismal prognosis and resistance to chemotherapy. Thus, a better understanding of the mechanisms controlling the aberrant expression of melanoma factors will assist in developing urgently needed alternative approaches to melanoma treatment. One potential target is the developmentally crucial transcription factor Pax3, which is up‐regulated by 2500 fold in melanoma. Our lab has shown that in mouse myoblasts Pax3 is phosphorylated at three serines (Serines 201, 205 and 209) in a region that mediates protein‐protein interactions. Additionally, Pax3 phosphorylation patterns vary during the course of differentiation with phosphorylation of Ser205 occurring only in proliferating cells. In this study, we demonstrate for the first time that Pax3 is phosphorylated in melanocytes as well as in melanoma cells. In contrast to melanocytes, Pax3 is almost exclusively phosphorylated at Ser205 in melanoma cells, an event that is exclusive to proliferating cells. Also, we show that phosphorylation of Pax3 enables its interaction with factors Lef1 and SOX10. Our results suggest that Pax3 phosphorylation regulates the expression of downstream targets by altering its ability to interact with co‐regulatory proteins and an aberrant change in Pax3 phosphorylation contributes to the development of melanoma. (Funding: NCI R01 CA138656)
The structure and kinetics of an unusual DNA polymerase I from the thermophilic bacterium, Rhodothermus marinus
Authors: Omattage Natalie Saranga , Gomez Miguel Angel Rubio , Kornberg Emily , Duffany Brooke , Dow Robert , Spicer Lori , Wu Eugene
Published: June 2021

DNA polymerase I employs a multistep mechanism for sorting correctly paired nucleotides from mismatches. We aim to characterize reaction intermediates during nucleotide selection to better understand how this class of enzymes achieves high DNA replication fidelity. DNA polymerase I from R. marinus contains an unusual and disruptive proline in the mobile O‐ helix near the active site. To characterize this unusual enzyme, we solved the structure of the large (5′‐to‐3′ deficient) fragment of the R. marinus DNA polymerase I (RF) to 2.95 Å (R = 0.246) using multi‐wavelength anomalous dispersion. Alignment with homologous Escherichia coli Klenow Fragment (KF) DNA polymerase confirmed that the active sites of each structural domain were conserved. In order to study the polymerase activity in isolation, we eliminated the 3′‐to‐5′ exonuclease activity using site‐directed mutagenesis of an aspartic acid involved in binding DNA (D497A). Unexpectedly, mutation of an aspartic acid involved in binding a catalytic magnesium ion (D421A in RF) failed to abolish exonuclease activity despite its ability to do so in the KF (D424A). Enzyme kinetic assays of the 3′‐to‐5′ exonuclease and polymerase activities of RF and its mutants indicate that RF is a highly unusual member of the DNA polymerase I family. Work funded by University of Richmond School of Arts & Sciences.
E. coli and S. cerevisiae clamp loaders, γ complex and RFC, actively close clamps
Authors: Hayner Jaclyn , Bloom Linda
Published: June 2021

Sliding clamps and their respective clamp loaders perform integral functions in DNA metabolism and are therefore found throughout all kingdoms of life. Clamp loaders, members of the AAA+ ATPase family, use ATP binding and hydrolysis to load sliding clamps onto DNA where the clamps then serve as a platform for enzymes that act on DNA. In E. coli, the β clamp is loaded by the γ complex clamp loader, and in S. cerevisiae, PCNA is loaded by the RFC clamp loader. Using fluorescent assays developed in our laboratory to report on clamp closing and release, we have shown that the rate of clamp closing is about 10 times faster than the rate of clamp release for both systems, indicating that these clamp loaders actively close clamps. When ATP hydrolysis is blocked, the closing and release rates become similar, indicating that the mechanism of closing before release is ATP hydrolysis‐dependent. ATP hydrolysis experiments were performed using fluorescently labeled phosphate binding protein to determine the rate of ATP hydrolysis and a temporal correlation between ATP hydrolysis and clamp closing. These experiments identify a common mechanistic feature in the clamp loading reaction and help to understand how these enzymes efficiently load clamps for replication and repair.

This work supported by NIH grant GM 082849 and 5 T32 CA009126‐32 Training Grant in Cancer Biology
Complex formation between LL‐37 and CpG DNA: thermodynamic properties and effects in a prostate cancer progression model
Authors: Craig Maria Lee , Dodson CarolAnn , Gupta Angela Satya , Deeble Paul D
Published: June 2021

LL‐37, a DNA‐binding peptide of innate immunity, is a molecule of interest in the anti‐DNA autoimmune response and in prostate tumor progression. LL‐37 binds to DNA and shuttles it across the cell membrane, leading to activation of Toll‐like receptor 9 (TLR9) pathways. We have used electrophoretic mobility shift assays (EMSA), western blotting, and isothermal titration calorimetry (ITC) to investigate the thermodynamic properties of complex formation between LL‐37 and CpG DNA sequences known to activate TLR9. We observe that LL‐37 binds to both CpG and control (GpC) DNA sequences with estimated Ka's of 104–105M−1. In EMSA, LL‐37 is aggregated in the absence of DNA but exhibits an increase in gel mobility upon DNA binding, suggesting that CpG binding disrupts LL‐37 aggregation. Increases in LL‐37 concentration lead to reduced complex mobility and decreased ethidium bromide staining, both indicative of further deposition of LL‐37 on the DNA. We have also extended our binding studies to poly A/T and poly G/C sequences. In related cellular studies, we have used cell scratch wound and soft agar assays to test the efficacy of CpG/LL‐37 treatments on migration and anchorage‐independent growth in a prostate cancer progression model (PC‐3 cell line). This work was supported by the Virginia Foundation of Independent Colleges Summer Undergraduate Research Fellowships and NIH EARDA grant 5G11HD037064‐08.
The use of molecular approaches to identify changes in soil microbial communities during storage
Authors: Freeman Victoria Hope , Patel Niraj , Larson Sabreena , Bailey Cheryl
Published: June 2021

The purpose of this project is to identify the time interval during storage in which the greatest change in microbial community within soil samples of varying soil types occur. Three storage conditions, air drying, 4° C, and −20°C will be tested, and DNA extracted from stored soil at time intervals of 0, 1, 2, 4, 7, and 14 days. the extracted DNA will undergo PCR to amplify the v3 region of the 16S rDNA. The PCR product will be processed using Capillary Electrophoresis‐ Single Stranded Conformational Polymorphism (CE‐SSCP) on a genetic analyzer. The resulting microbial community fingerprints will be compared using multi‐variant statistical analysis using the StatFingerprint component of the R Software package. Comparisons will be made to determine the time interval that undergoes the most change as well as the storage condition that produced the most change overall. Detailed results will be presented. This project is funded by The NASA Nebraska Fellowship Grant.
Genetic and Proteomic Dissection of TDP43 Toxicity in S. cerevisiae
Authors: Higgins Matthew James , Krogan Nevan J , Farese Robert V
Published: June 2021

The RNA binding protein transactivator region DNA‐binding protein 43 (TDP43) was identified in ubiquitin‐positive aggregates in patients with frontotemporal dementia (FTD), and subsequent studies have demonstrated that TDP43 is associated with several neurodegenerative diseases including amytrophic lateral sclerosis (ALS) and Alzheimer disease. The mechanism by which TDP43 contributes to neurodegenerative disease is not understood. Similarly, whether TDP43‐toxicity results from a gain‐ or loss‐of‐function is unclear and will likely require a molecular understanding of how TDP43 functions as a protein‐ and RNA‐scaffold under normal and toxic conditions. To gain insight into the mechanism of TDP43 toxicity, we performed a synthetic genetic array screen in Saccharomyces cerevisiae by using the deletion and decreased abundance by mRNA perturbation (DAmP) gene collections. We have identified a variety of genes whose deficiency suppresses or enhances TDP43 toxicity, including several involved in pathways of RNA metabolism. Additionally we have identified many proteins associated with TDP43 expressed in S. cerevisiae, some of which participate in pathways similar to those that were identified in the genetic screen. Constructing a more detailed proteomic and genetic network of modifiers of TDP43 toxicity will allow a better understanding of TDP43 normal and pathogenic function and, hopefully, enable more directed approaches to therapies targeting neurodegenerative diseases involving TDP43.

Funding to RVF Jr. from the Consortium for Frontotemporal Dementia Research
Allosteric inhibition of a stem cell RNA‐binding protein by an intermediary metabolite
Authors: Ryder Sean P , Clingman Carina C , Deveau Laura M , Massi Francesca
Published: June 2021

Gene expression and metabolism are coupled at numerous levels. Cells must sense and accurately respond to the nutrients in their environment, and specialized cells must synthesize the metabolic products required for their function. Pluripotent stem cells have the ability to differentiate into a wide variety of specialized cells. Exactly how metabolic state contributes to stem cell differentiation is the subject of intense investigation. Here, we show that the RNA‐binding activity of the stem cell translational regulator Musashi‐1 (MSI1) is allosterically inhibited by non‐esterified 18–22 carbon cis ω‐9 monounsaturated fatty acids. Inhibition is direct and highly specific. The fatty acid binds to the N‐terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi‐family proteins are critical for development of the brain, blood, and epithelial lineages, and play an important role in maintaining the viability of oligodendrocyte progenitor cells (OPCs). We identify stearoyl‐CoA desaturase 1 as a MSI1 target, revealing a feedback loop between ω‐9 fatty acid biosynthesis and MSI1 activity. We propose that other RRM proteins could act as metabolite sensors to couple gene expression changes to physiological state.
Role of the Cofactor in the Catalytic Mechanism of the glmS Ribozyme
Authors: Claverol Julia Viladoms , Fedor Martha J.
Published: June 2021

The glmS ribozyme is the first natural self‐cleaving ribozyme that requires a cofactor. A cofactor can greatly expand the number of reactions that RNA can catalyze because it contributes chemical diversity that is otherwise not available with only four nucleotides. Although it has been proposed to serve as a general acid, the role of the cofactor in the glmS catalytic mechanism is unclear.

We surveyed different molecules for their ability to support self‐cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of each cofactor. Self‐cleavage of the glmS ribozyme also depends on the concentration of the free base form of the cofactor, evidence that the reaction is subject to general base catalysis. Mechanistically, this behavior may correspond to true general base catalysis, with the cofactor accepting a proton, or to specific base general acid catalysis, with a hydroxide ion accepting a proton and the cofactor donating one.

We are currently acquiring pre‐steady state kinetics of glmS ribozyme self‐cleavage promoted by several cofactors that differ in intrinsic Brønsted base strength and binding affinity to apply linear free energy relationships. The results of these experiments will provide insight into how the cofactor contributes to the glmS catalytic mechanism.

This work was supported by NIH RO1 GM046422.
Gene Expression Profiling of Oxidative Stress on Vascular Tissue in Puerto Rican Diabetic Patients Undergoing Coronary Artery Bypass Grafting
Authors: Albino-Rodriguez Elinette , Soltero Ernesto , Ferder Leon , Dutil Julie
Published: June 2021

In the US, Puerto Rican Hispanics have the highest prevalence of Type II diabetes. Type II diabetes is the main risk factor for coronary artery disease (CAD), and patients with advanced symptomatic CAD are referred for coronary artery bypass grafting (CABG). High blood sugar causes generation of free radicals that leads to oxidative stress in several tissues. This study aims to evaluate the molecular differences in oxidative stress related gene expression levels in the left internal thoracic artery and saphenous vein conduits of diabetic patients undergoing CABG. RNA extraction and gene expression levels from mammary artery and saphenous vein were compared between diabetic and non‐diabetic patients for 84 genes related to oxidative stress and antioxidant defense. The expression level of the Glutathione peroxidase 1 (GPX1) gene was significantly higher in conduits of diabetic patients with respect to non‐diabetic patients. Our results suggest that oxidative stress modulates certain antioxidant defense in type 2 Diabetes, improving our understanding of the molecular pathways that are modulated in the conduits of diabetic patients undergoing CABG. Research supported by RCMI Grant #5G12RR003050.
Genetic deletion of an instability motif in the Tristetraprolin (TTP) transcript: Implications for the treatment of systemic inflammation
Authors: Patial Sonika , Stumpo Deborah J , Lai Wi S , Ward Toni W , Blackshear Perry J
Published: June 2021

TTP is an RNA binding protein that binds to AU‐rich elements (AREs) in the 3’ untranslated regions of specific mRNAs such as TNFα leading to mRNA deadenylation and decay. Mice deficient in TTP develop a systemic inflammation characterized by erosive arthritis. This phenotype is almost completely due to an excess of TNFα, since the administration of anti‐TNFα antibodies can almost completely prevent the development of this phenotype. The TTP transcript also contains an ARE within its 3’UTR, which may be responsible for the extreme lability of TTP mRNA and we have recently demonstrated that TTP can use some of these sites to regulate its own expression. In this study, we used “knock in” technology to delete the ARE of the TTP mRNA from the mouse genome. Deleting the ARE of the TTP mRNA enhanced its stability as well as increased the levels of TTP protein. LPS induced expression of TNFα mRNA and protein was reduced in macrophages from TTPΔARE mice. In addition, LPS induction of other proinflammatory cytokine mRNAs, such as those encoding IL6 and IL1β, was also found to be significantly reduced. These results suggest that an attempt towards increasing TTP levels in cells could represent a novel therapeutic strategy in the treatment of inflammatory diseases. Moreover, TTPΔARE mice might represent a useful model system for examining the effects of modestly increased but normally regulated TTP levels in systemic inflammatory diseases.
Computational prediction of alternative splice site selection
Authors: Busch Anke , Hertel Klemens J.
Published: June 2021

Alternative splicing results in the production of multiple mRNA isoforms from a single pre‐mRNA. Regulation of this process relies on the activity of multiple RNA signature elements that act in concert to mediate splice site recognition. We have developed an algorithm that evaluates numerous RNA sequence features in combination to predict all major types of alternative splicing with a remarkable accuracy/sensitivity. To do so we trained support vector machines (SVMs) to distinguish between different types of exons and different types of alternative splicing. We show that each class of alternative splicing is characterized by different sets of the approximately 220 RNA features examined. For example, the strength and frequency of competing splice sites as well as the phylogenetic conservation up‐ and downstream of the splice sites turned out to be the most influential factors to differentiate between constitutive and alternative splice sites. Our results show that the SVM approach forms the foundation to delineate mechanistic differences between the types of alternative splicing and the regulation of alternative splicing at various biological conditions.
Identification of the activator binding residues in protein kinase C theta C1B domain
Authors: Rahman Ghazi M , Sanker Sreejesh , Lewin Nancy , Prasad B.V.V. , Blumberg Peter M. , Das Joydip
Published: June 2021

PKCtheta is a Ser/ Thr kinase, which is critically involved in immunity. Diacylglycerol (DAG) and phorbol esters (PE) bind to C1A and C1B domain to activate nPKC and cPKC. Selective PKCtheta inhibition may manage autoimmune disorders. Design of PKCtheta inhibitor targeting C1 domain requires the knowledge of C1 domain structure and its activator binding residues. In this direction we solved crystal structure of PKCtheta C1B and identified possible activator binding residues by molecular docking of a library of DAG and PE analogs into PKCtheta C1B as receptor. Five residues were identified, which are capable of binding to DAG/ PE analogs. To determine the role of these residues in activator binding Y239A, T243A, W253G, L255G and Q258 in isolated C1B domain were designed, expressed and purified from E. coli and their binding affinity (Kd/Ki) for Phorbol 12, 13‐Dibutyrate (PDBu) and Dioctanoylglycerol (DOG) were measured by radioactive PDBu binding assay. All the mutants showed significantly reduced binding affinity for both PDBu and DOG whereas Q258 showed the maximum reduction than the wild type (WT). Same mutations in full length PKCtheta were expressed in HEK293 cells and showed reduced Phorbol 12‐Myristate 13‐Acetate and DOG induced membrane translocation compared to WT. Our studies indicated that these residues are important for activator binding and will aid in future design of the PKCtheta inhibitor.
Investigating Synaptophysin Targeting to the Photoreceptor Synapse
Authors: Baker Sheila A , Brucato Martha F , Arshavsky Vadim Y , Hengel Sarah R , Pan Yuan , Laird Joseph G
Published: June 2021

Synaptophysin (SYP) is the most abundant protein found in synaptic vesicles. It functions to regulate the endocytosis phase of the synaptic vesicle cycle. However, it is not known how newly synthesized SYP is targeted from the neuronal soma to the synapse so that it can carry out this function. The photoreceptor is an excellent neuron in which to tackle this problem, due in part to its large size and clear compartmentalization. The objective of this study is to test the hypothesis that SYP contains a sequence specific targeting signal that directs the targeting of this protein to the synaptic terminal in photoreceptors. Our approach was to generate constructs consisting of a membrane‐associated YFP as a reporter fused to various regions of SYP, and express those constructs in the rod photoreceptors of transgenic Xenopus laevis. The subcellular localization of these proteins in tadpole photoreceptors was assessed by confocal microscopy. The YFP reporter was found throughout the cell but strongly accumulated in the outer segment, as we have previously described for membrane proteins lacking targeting information in this cell type. We found that only the cytoplasmic C‐terminus of SYP was capable of restricting the localization of the reporter to the inner segment plasma membrane and synaptic terminal. Future studies are aimed at determining which of SYP's binding partners regulates its trafficking in photoreceptors.