bluuebunny/crossref_metadata_2025 huggingface dataset:
AN ANATOMICAL MODEL OF EAR IN PLASTER (GILLOT‐BRIEUX) OF THE 20th CENTURY
Authors: LE FLOCH‐PRIGENT Patrice
Published: June 2021

A very enlarged anatomical model of ear in plaster belonged to the Gillot‐Brieux bookstore in Paris (47 rue Jacob). This model was probably designed and built in Europe at the beginning of the 20th century. Neither trade, nor date were found on the model. It laid on a black painted basis in wood which measured 48×41×4,4 cm. The model width was 41 cm and the pavilion of the ear measured 24×14 cm. It was fixed on the basis by two bolted stems. Not any part of the sample was dismantabled. The three parts of the ear (external, middle and internal) were represented on a frontal cross‐section with a very good representation of all the elements: particularly the cochlea and one of the semi‐circular canal presented on a cross‐section with a 25 mm diameter. The ossicles had vanished except the stapes. The internal carotid artery was represented in red and cross‐sectionned at its two points of penetration through the cranium (endo‐ and exo‐cranial ones). The parotid gland was represented in yellow with small embossments; its diameter was 30 mm. The vestibulo‐cochlear cranial nerve (VIII) was represented with its two components from the organs of reception. The anatomical rendering was accurate with a fair representation of the osseous structures.
Obstruction of the thoracic duct(s) causes chronic lymphedema of the upper limb in patients presenting with symptoms of thoracic outlet syndrome: MRI/MRA/MRV
Authors: Collins James Douglas , Saxton Ernestina , Gelabert Hugh , Ahn Samuel , Carnes Alfred Edward , Daniels Portia
Published: June 2021

INTRODUCTION

Obstruction of the thoracic duct(s) causes chronic upper extremity lymphedema. Lymphatics have bicuspid valves like the venous system. Metaplastic fibrosis resulting from obstruction of lymph drainage in the upper extremities impedes vertebral venous plexus / Batson's plexus circulation. The upper limb undergoes painful swelling into the axilla and compresses the brachial plexus. Patients present with tingling/numbness and weakness of the affected arm; lightheadedness; blurred vision and floaters in the visual fields, and tinnitus with whooshing sounds in the ear on the affected side and increased hair growth and nail changes in the affected limb.

METHODS

Bilateral MRI/MRA/MRV displays the sites of obstruction of lymphatics, draining veins of the neck and the subclavian and axillary arteries with binding nerve roots. Monitored multiplanar images are acquired on a 1.5 Tesla GE Signa LX unit, 44 cm field of view, 512 × 256 matrix and saline water bags to enhance signal to noise ratio and Fast Spin Echo (FSE) to display lymphedema (JNMA 1999; 91:333–341).

RESULTS

Two patients were selected: one with acquired obstruction of the thoracic lymph duct with lipoma extending into the left hemithorax, the other with developmental fibrosis of the right thoracic duct complicated by pulmonary emboli. Cannulation of the lymphatics could not be performed because of obstructed lymphatic flow.

CONCLUSIONS

MRI/MRA/MRV is the only alternative to lymphangiogram in patients with obstruction to lymph flow.
Genetic analysis of dermal bone fragmentation in a natural model system.
Authors: Gross Joshua B. , Krutzler Amanda J. , Bruns Lauren E.
Published: June 2021

Natural model systems that reside in extreme environments can provide powerful insights into fundamental biological processes. For instance, many of the genetic and cellular regulators of normal dermal bone formation have been clarified. However, the precise genetic and molecular bases for altered bone phenotypes evolving under environmental pressure remain largely uncharacterized. In this study, we evaluated a dermal bone fragmentation phenotype that occurs in the blind Mexican cavefish (Astyanax mexicanus). We investigated this phenotype using exhaustive cranial bone measurements in the context of an F2 pedigree of surface fish × cavefish hybrids. We determined that fragmentation of the suborbital bones occurs in a largely asymmetric fashion. Further, we clarified the genetic architecture for left‐sided and right‐sided suborbital bone fragmentation from our hybrid pedigree. Our results indicated that while the area of the face occupied by suborbital bones is constrained between different‐sized individuals, the pattern and severity of fragmentation could not easily be predicted by co‐occurrence of other degenerative characteristics. Our results may indicate a pleiotropic interaction between genes selected in the cave environment for constructive traits that interfere with normal patterns of bone ossification. Research supported by NIDCR (NIH) R03DE022403.

Grant Funding Source: grossja@ucmail.uc.edu
Temporomandibular joint articular disc: Providing a structural scaffold for use in tissue bioengineering of replacement constructs
Authors: ten Hove Steven Holterman , Wilson Tim , Shimizu Michael , Merrifield Peter , Galil Khadry
Published: June 2021

The temporomandibular joint's (TMJ) articular disc facilitates normal function in the healthy joint. Temporomandibular disorders (TMDs) are associated with disc derangement or damage resulting in joint dysfunction. TMD can progress to an end‐stage where surgical removal of condyle and disc are performed. No adequate prosthetic replacement exists for the disc. A dimensional model for the disc is yet to be provided. Our objective is to assess the dimensions of the articular disc providing a structural scaffold for tissue engineering of replacement constructs. Discs were dissected bilaterally from 12 cadavers (56–100yrs). Linear surface dimensions of the discs were measured using digital calipers to assess absolute and regional anteroposterior width (APW) and mediolateral length (MLL). Regional measurements were taken perpendicularly at relative ratios of the absolute dimensions (50% central; 20% from borders). All measurements (mm) were repeated twice by 2 observers to assess reliability. Mean absolute APW (15.31±1.08) and MLL (24.71±2.29) were calculated from 9 specimens along with mean values for 6 regional surface measurements and 9 regional depth measurements per disc. Within the limits of this study it is possible to provide reliable dimensions for use in creating a structural scaffold of the TMJ articular disc.

Grant Funding Source: Departmental Funding
Enthesophyte on Radius
Authors: Singh Rajani
Published: June 2021

During examination of radii in the osteology lab of KGMU three (two radii of left hands and one of right hand ) out of assorted one hundred radii were having longitudinal and transverse enthesophytes. The lengths, heights and thicknesses of these longitudinal enthesophytes in the left two radii were 2.2/0.7/0.2 cm, 1.9/0.7/0.4 cm and 2.2/0.6/0.4 cm in one right radius. The lengths, heights and thicknesses of those transverse enthesophytes in the left two radii were 1.1/0.3/0.2 cm, 0.8/0.2/0.2 cm and 0.7/0.1/0.1 cm in one right radius. This extra bony growth may impinge on the surrounding structures leading to neurovascular complications and restriction of biomechanical movements of forearm. The study will be of paramount importance to anatomists, radiologists and clinicians. The defect is virgin and seen for the first time so the case has been reported.

No external support is available to the author
Situs Inversus Totalis: A Cadaveric Case Report
Authors: Campeau Lisa M , Casey Gregory Patrick
Published: June 2021

During the 2012 summer semester at the LSU Health Sciences Center, students of the cardiopulmonary gross anatomy course discovered a cadaver with a condition called situs inversus totalis. In this condition, the thoracic and abdominal organs are transposed to the contralateral sides of the body. Situs inversus totalis is a condition with an average incidence of 1:10000 to 1:20000. A cadaveric donor with this condition is extremely rare. Right versus left laterality is regulated by over one hundred genes during development. Although eight genes have been linked to situs inversus totalis, the genes causing primary ciliary dyskinesia are most commonly associated with the presence of this condition. In this case report, the donor died at the age of 73 from myocardial infarction. During initial dissection, we note that the right lung contained 2 lobes, the left lung 3 lobes, and the apex of the heart points toward the right side of the thorax. On further investigation, the aorta, superior and inferior vena cavae, azygos vein and other structures are found transposed to the contralateral side. In the abdomen, there is complete reversal of the liver, stomach, colon, appendix, and associated vasculature. The discovery of situs inversus totalis has prompted several studies in our department to contribute to the present literature.
Vagus Nerve's Topography in the Carotid Sheath: preliminary results
Authors: Yendo Tatiana Mina , Docko Ana Jung , Hojaj Flávio Carneiro , Akamatsu Flávia Emi , Andrade Mauro , Jacomo Alfredo Luiz
Published: June 2021

The knowledge of the vagus nerve's location within the carotid sheath is essencial in many surgical contexts, such as thyroidectomy. Intraoperative neuromonitoring may prevent iatrogenic nerve injury. There are only few studies about vagus nerve and its topography in the carotid sheath. This study aims to analyze the variation of the vagus nerve topography and its relation with the common carotid artery and the internal jugular vein, at the infrahyoid region.

We dissected 10 carotid sheaths from 5 cadavers and the position of the vagus nerve and its relationship with the common carotid artery and the internal jugular vein were determined. Based on the carotid sheath cross‐section, the location of the vagus nerve was classified in four quadrants: between the common carotid artery and the internal jugular vein, but anterior to the carotid artery's cross section (A), between the common carotid artery and the internal jugular vein, but posterior to the carotid artery's cross‐section (B), posterior and medial to the carotid artery's cross section (C) and posterior and lateral to the internal jugular vein's cross‐section (D).

We found 6 vagus nerve in position (A), 1 in position (B), 0 in position (C) and 3 in position (D).

The knowledge of variations and their prevalence regarding the position of the vagus nerve as described in this preliminary study may contribute to avoid iatrogenic lesions in cervical surgeries.
CUTANEOUS RECEPTORS OF THE EPITHELIUM OF LABIA MINORA IN PREPUBERTAL GIRLS
Authors: Schober Justine M , Alguacil Nieves Martin , Pfaff Donald , Mayaglou Lazarus , Aardsma Nathan
Published: June 2021

Introduction

Sparse information is available regarding terminal sensory nerve endings within the glabrous skin of the female external genitalia. Receptors sense tactile stimuli encoding stimulus position, changes in temperature, pressure and pain. Diversity of possible sensations suggests the presence of a variety of receptor types. This neuro‐histological study seeks to document presence and characteristics of cutaneous sensory receptors in female genital tissue.

Materials and Methods

Labial skin samples from ten girls who underwent surgery for labial fusion were waste tissue, obtained after informed consent from parent or legal guardian. Cajal‐type silver impregnation, immunocytochemistry against protein gene product (PGP) 9.5, and neuron‐specific enolase (NSE) was performed.

Results

Dense innervation of labia minora epidermis with intense staining along the introital versus the external border was noted. Free nerve endings were identified in papillary dermis. Sensory corpuscles: non‐capsulated, Meissner‐like corpuscles, scattered in dermal papillae interdigitated with epidermal ridges of the skin, encapsulated corpuscles protruded into epidermis from deep dermis, Merkel cell‐neurite complex was identified in stratum basale and spinosum. Encapsulated corpuscles and cells located in the inner and outer core were strongly positive to PGP 9.5.
Vascularization of the Pes Anserinus Superficialis
Authors: Hirtler Lena , Faber Mike , Weninger Patrick , Streicher Johannes
Published: June 2021

Introduction

The aim of this study was to evaluate the vascularization of the superficial pes anserinus and its inserting tendons with regard to its potential use as a vascularized graft for the reconstruction of the anterior cruciate ligament.

Methods

Twelve fresh cadaveric knees were used in this study. The inferior medial genicular artery was identified at its origin from the popliteal artery and incised to be perfused with a methylene blue solution. Its intra‐ and peritendinous course was evaluated macroscopically and histologically.

Results

This study proved that the superficial pes anserinus is a highly vascularized structure. The vascularization is potentially able to supply the inserting tendons of the semitendinous and gracilis muscles retrogradely.

Conclusion

The results of this study indicate that the option of a pediculated cruciate ligament reconstruction with both tendons of the semitendinous and gracilis muscles inserting at the superficial pes anserinus is theoretically possible. A pediculated reconstruction of the anterior cruciate ligament could decrease postoperative morbidity of the transplant and therefore enshorten the vulnerable healing period after surgery.

Grant Funding Source: N/A
Transplanting normal endothelial cells into tumor mass retards the tumor growth by vascular reconstruction and vascular normalization
Authors: Kitahara Shuji , Nishio Miwako , Morikawa Shunichi , Nakada Kazuko , Yoshii Asuka , Saeki Kumiko , Ezaki Taichi
Published: June 2021

Normalization of tumor vessels using molecular targeted therapy has been attracting attention as a potential new treatment for cancer. Therefore, we hypothesized that reconstruction of normal blood vessels in tumors may lead to improvement of hypoxic conditions, contributing to potential improvements in the delivery of anticancer drugs. In this study, we sought to determine whether transplanting normal endothelial cells into tumor‐bearing mice would trigger vascular reconstruction. Tumor cells were injected into the dorsal subcutis of SCID mice. After 2 weeks, the tumor‐bearing mice were injected cisplatin intraperitoneally to regress tumors and tumor vessels for the first step. Two weeks later, human endothelial cells (HMVECs) or human ES‐derived endothelial cells were transplanted into necrotic regions of the tumor to induce normal angiogenesis for the second step. Microscopic observations revealed that the transplanted HMVECs formed anastomoses with the host mouse vasculature, and vessels with blood streams were detectable after 14 days. Blood vessels reconstituted by HMVECs exhibited the normalization of tumor vasculature, resulting in a significant reduction in the tumor volume. Thus, this mouse vessel reconstruction model is a useful model for the future development of new cancer therapies by the construction of reliable drug delivery systems and the improvement of hypoxic conditions.
The Endoplasmic Reticulum Stress and Unfolded Protein Responses in Sulfur Mustard induced Mouse Skin Wound Repair
Authors: Chang Yoke‐chen , Wang James D , Gordon Marion K , Gerecke Donald R
Published: June 2021

The endoplasmic reticulum stress (ERS) pathways activate the unfolded protein response (UPR) to mediate cell survival against severe stress in an attempt to prevent ERS mediated apoptosis. Sulfur mustard (SM), a chemical warfare vesicating agent, causes significant delayed wound repair in skin. This study explored the contribution of UPR and ERS to SM cutaneous injury in mice. We performed time course studies using the mouse ear vesicant model and showed histopathology changes over time. RT‐PCR demonstrated at least a 2‐fold increased of the ER stress survival gene, GRP78; UPR gene, ATF6; and proapoptotic gene, CHOP as time progressed from 24h to 168h post‐SM exposure. Western blot analysis demonstrated increased GRP78, CHOP, and full length and cleaved (activated) forms of ATF6 as time progressed. This was confirmed by immunofluorescence which showed significant nuclear staining of cleaved ATF6 in the hyperplastic epidermis at 168h post exposure. Double immunofluorescent labeling for GRP78 and CHOP at 72h post exposure showed abundant cytoplasmic staining of GRP78 in the basal, proliferating epidermis. In contrast, the expression of CHOP was more numerous in the migrating keratinocytes. This suggests that the migrating cells are targeted for apoptosis while the basal, proliferating cells are targeted for survival. The continued proliferation of the basal cells and their subsequent conversion to migrating cells may in part explain the delayed wound repair frequently observed in SM skin injury.

Grant Funding Source: NIH grants AR055073, EY09056, and ES005022.
The mechanics of acceleration and deceleration in primate quadrupeds:implications for primate locomotor evolution.
Authors: Pinkard Henry , Schmitt Daniel , Johnson Laura E , Miller Charlotte E.
Published: June 2021

The ability to accelerate and decelerate is critical for animal survival and may be a selective priority determining body form in mammals. Studies of quadrupedal mammals have shown that pitching moments around an animal's center of mass (COM) limit maximum acceleration and deceleration and that the ability to resist such moments is influenced by limb length, COM position, and the ability to shift weight to the fore‐ or hindquarters. Primates have relatively long, gracile forelimbs and have higher vertical ground reaction forces on their hindlimbs than forelimbs, unlike most mammals. Primates should then differ from other mammals in that they will be effective in producing acceleration but limited in producing deceleration. This hypothesis was tested with lateral video and single limb forces recorded during accelerating and decelerating steps for two strepsirhine primate species: Eulemur fulvus and Lemur catta. Results show that primates are limited in maximum acceleration and deceleration by their limb length and that they adopt specific mechanical solutions including loading the trailing limb relatively more, initiating decelerations with the hindlimbs rather than the forelimbs, and increasing contact time to limit instantaneous vertical force increases. These solutions are consistent with other unusual primate gait characters associated with adaptations to arboreal locomotion.
Ontogeny of Pan troglodytes and Macaca mulatta Metatarsal Cross‐sectional Properties and Comparisons to the Miocene Catarrhine Proconsul
Authors: Organ Jason M. , Dunsworth Holly M
Published: June 2021

Catarrhines differ in foot use, function, and anatomy between species and within species through ontogeny. For example, adult Pan and Macaca differ in hallucial anatomy and also in the relative distribution of ground reaction forces. Further, they experience different growth in hindlimb proportions, which may correlate with differences in locomotor ontogeny. These differences should be reflected in metatarsal (MT) cross‐sectional geometries (CSG). To test this hypothesis we measured the CSG of MTs 1–5 in a cross‐sectional ontogenetic sample of 80 M. mulatta and 74 P.troglodytes. Most of the CSG were collected with biplanar radiography and the latex cast method (LCM). However, an additional 28 juvenile M. mulatta individuals, too small for LCM, were recently scanned with microCT. Midshaft CSG were calculated using ImageJ. As expected, CSG are significantly different in both absolute and relative magnitude and patterning across the rays between the two species as well as the age groups within them, particularly in the hallux where Pan shows greater bending and torsional resistance. These results provide insight on foot use in fossils. MTs from six Proconsul individuals ranging from infant to adult were scanned with microCT. Proconsul MT CSG are similar in magnitude to those of Macaca, but share relative hallucial patterning with Pan. This research was funded by the Leakey Foundation and Penn State University.

Grant Funding Source: N/A
Herpes Simplex Virus type 1 replication proteins disable ATR signaling by binding to substrates that would normally recruit 9–1‐1 and topBP1 to activate ATR
Authors: Weller Sandra K , Mohni Kareem N
Published: June 2021

During HSV‐1 infection, ATM but not ATR is activated leading to the phosphorylation of Chk2 but not Chk1. Furthermore, although hydroxyurea normally activates ATR, Chk1 is not phosphorylated in HU‐treated HSV‐1 infected cells. The ss DNA binding protein, ICP8, and three subunit Helicase/Primase (H/P) are required to inhibit ATR signaling. These four viral proteins are necessary and sufficient to disable ATR signaling in transfected cells. ATR is generally activated by substrates which contain ssDNA adjacent to dsDNA. ICP8 and H/P also recognize this type of substrate. We suggest that these four viral proteins prevent ATR activation by binding to the DNA substrate and obstructing loading of the Rad9‐Rad1‐Hus1 checkpoint clamp (9–1‐1). In fact, when ICP8 and H/P are expressed by transfection, the recruitment of the 9–1‐1 is prevented which in turn prevents the recruitment of TopBP1 and thus effectively disables ATR signaling. In addition, we show that 9–1‐1 becomes chromatin bound in response to DNA damaging agents in uninfected controls, but is excluded from chromatin in the presence of HSV‐1 replication proteins. The observation that viral proteins can exclude cellular DNA repair proteins from the sites of DNA damage represents a novel mechanism of ATR inhibition.
Characterization of a Putative Nuclear Localization Signal in the Fanconi Anemia D2 Protein
Authors: Boisvert Rebecca Anne , Rego Meghan A , Mauro Maurizio , Howlett Niall G
Published: June 2021

Fanconi anemia (FA) is a rare disease characterized by bone marrow failure and increased cancer susceptibility. FA is caused by mutation of any one of 15 known genes. The FA proteins function collectively to repair DNA interstrand cross‐links (ICLs) and to prevent cellular transformation. Following exposure to ICLinducing agents, e.g. mitomycin C (MMC), the FA core complex catalyzes the mono‐ubiquitination of the FANCD2 and FANCI proteins, targeting them for chromatin localization. Currently, very little is known about the structure, function, and regulation of the FANCD2 and FANCI proteins, and, in particular, how these proteins are recruited to chromatin. In this study, using an in silico approach, we have identified a highly conserved bipartite nuclear localization signal (NLS) in the amino‐terminus of FANCD2. When fused to the coding sequence of GFP, the FANCD2 NLS promoted exclusive nuclear expression of GFP. Using deletion and site‐directed mutagenesis strategies, we have generated FANCD2 mutants lacking 57 (ΔN57) or 100 (ΔN100) amino‐terminal residues, and a missense mutant changing amino acids K4, R5, R6 to N4, N5, N6 respectively (3N). Our preliminary studies demonstrate that the NLS is required for FANCD2 and FANCI monoubiquitination, and their localization to and retention in chromatin. Consequently, the NLS mutants fail to rescue the MMC hypersensitivity of FA‐D2 (FANCD2−/−) cells. Our results corroborate our hypothesis that FANCD2 and FANCI enter the nucleus as a heterodimer primarily via the FANCD2 NLS, lending important insight into the regulation of a major tumor suppressor network.
XRCC4 Protein Interactions with XRCC4‐like Factor (XLF) Create an Extended Grooved Scaffold for DNA Ligation and Double Strand Break Repair
Authors: Hammel Michal , Rey Martial , Yu Yaping , Lees‐Miller Susan , Tainer John
Published: June 2021

The XRCC4‐like factor (XLF)‐XRCC4 complex is essential for nonhomologous end joining, the major repair pathway for DNA double strand breaks in human cells. Yet, how XLF binds

XRCC4 and impacts nonhomologous end joining functions has been enigmatic. Here, we report the XLF‐XRCC4 complex crystal structure in combination with biophysical and mutational analyses to define the XLF‐XRCC4 interactions. Crystal and solution structures plus mutations characterize alternating XRCC4 and XLF head domain interfaces forming parallel superhelical filaments. XLF Leu‐115 (“Leu‐lock”) inserts into a hydrophobic pocket formed by XRCC4 Met 59, Met‐61, Lys‐65, Lys‐99, Phe‐106, and Leu‐108 in synergy with pseudo‐symmetric beta‐zipper hydrogen bonds to drive specificity. XLF C terminus and DNA enhance parallel filament formation. Super‐helical XLF‐XRCC4 filaments form a positively charged channel to bind DNA and align ends for efficient ligation. Collective results reveal how human XLF and XRCC4 interact to bind DNA, suggest consequences of patient mutations, and support a unified molecular mechanism for XLF‐XRCC4 stimulation of DNA ligation.
Molecular delineation of CENP‐E in mitotic spindle plasticity using chemical probe syntelin
Authors: Yao Maomao , Liu Xing , Yao Xuebiao
Published: June 2021

Mitotic motor CENP‐E plays key roles in chromosome congression and spindle checkpoint satisfaction. We have recently identified and characterized syntelin, a novel selective CENP‐E inhibitor (Ding et al., 2010. Cell Res. 20, 1386–1390). Cells treated with syntelin progress through interphase, enter mitosis normally with a bipolar spindle and lagging chromosomes around the poles. Syntelin is an allosteric inhibitor which tightens CENP‐E‐microtubule interaction by slowing inorganic phosphate release. To delineate the role of CENP‐E in reorganization of interpolar microtubules into an organized central spindle, metaphase synchronized cells were exposed to syntelin and other mitotic motor inhibitors. Syntelin does not perturb interpolar microtubule assembly but abrogates the anti‐parallel microtubule bundle formation. Real‐time image shows that CENP‐E inhibited cells undergo central spindle splitting and exhibits chromosome instability phenotypes. Interestingly, inhibition of CENP‐E did not alter the interaction between CENP‐E and PRC1 but perturbed temporal assembly of PRC1 to the midzone. Surprisingly, inhibition of CENP‐E perturbs the temporal control of PRC1 dephosphorylation which led to a persistent phosphorylation of PRC1 and an inhibition of central spindle assembly. These findings reveal a previously uncharacterized role of CENP‐E motor in temporal control of central spindle assembly.
HO‐2 deletion impairs wound closure in Human Corneal Epithelial (HCE) cells by altering EGFR and FAK‐ mediated signaling pathways
Authors: Halilovic Adna , Lin Daohong , Joseph Gregory , Shkolnik Brian , Schwartzman Michal L
Published: June 2021

Heme oxygenase (HO) represents an intrinsic cytoprotective and anti‐inflammatory system. Inhibition of HO activity significantly impairs wound healing in human corneal epithelial (HCE) cells. HO‐2, but no HO‐1, knockdown using virus‐mediated shRNA plasmid transfection impaired HCE wound healing by 35% and 25% at 24h and 48h after injury. HO‐2 knockdown inhibited HCE migration, measured using a modified Boyden Chamber, by 50% and 40% in control and EGF (10ng/mL) treated media, respectively. The HO‐2 knockdown‐induced inhibition of migration was associated with a 70% decrease in p‐ERK and 35% decrease in p‐Akt levels. Moreover, HO‐2 knockdown reduced p‐EGFR by 45% in injured HCE cells. Immunofluorescence of HO‐2shRNA and control transfected cells revealed a cytoplasmic distribution of p‐FAK. Upon injury, p‐FAK was localized to the nucleus in HO‐2 knockdown cells, whereas, control cells displayed typical p‐FAK as focal adhesion points in the membrane. In summary, HO‐2 knockdown altered EGFR and FAK‐mediated signaling pathways that are crucial for cell migration and proliferation.
Activation of AMP kinase plays a role in the increased apoptosis in the renal proximal tubule in cystinosis
Authors: Taub Mary , Cutuli Facundo
Published: June 2021

In cystinosis, renal proximal tubule (RPT) function is compromised, due to mutations in ctns, which encodes for the transporter cystinosin, which removes cystine from lysosomes. Altered RPT function in cystinosis has been attributed to decreased ATP, as well as increased apoptosis. In this report, the role of AMPK was examined. AMPK was activated in primary rabbit RPT cells with a cystinosin knockdown, using cystinosin siRNA. The activation of AMPK was associated with a 50% decrease in ATP and a 1.7‐fold increase in the ADP/ATP level. Cisplatin‐induced apoptosis also increased in primary RPT cells with a cystinosin knockdown. The role of AMPK in the increased sensitivity to cisplatin was examined. The increased sensitivity to cisplatin was prevented in primary RPT cells with a cystinosin knockdown by the AMPK inhibitor Compound C. The effect of siRNAs against AMPKα1 and AMPKα2 was also studied. The siRNAs knocked down AMPKα, and prevented AMPKα activation by 5‐aminoimidazole‐4‐carboxamide‐1‐β‐d‐ribofuranoside (AICAR). The siRNAs against AMPKα1 and AMPKα2 also prevented the increased sensitivity to cisplatin in the primary RPT cells with a cystinosin knockdown. These results suggest that signaling through AMPK plays a role in the enhanced apoptosis in the RPT in cystinosis. Funded by the Cystinosis Research Foundation to MLT.
Retention of anatomical knowledge: Impact of current curricular approaches
Authors: Drake Richard L , McBride Jennifer M. , Pawlina Wojciech
Published: June 2021

Over the past decade significant curricular changes have altered medical student education related to the anatomical sciences. The purpose of this study was to measure retention of anatomical knowledge as student's progress through medical school. The anatomy program at the Cleveland Clinic Lerner College of Medicine is unique not only in its presentation style, but also its duration. In the first year the course meets for 2 hours every Monday for 28 weeks. This is much shorter than the current national average. However, in the second year, the students have an anatomy session once during each of the second year courses where the anatomy presented during the first year is revisited. Additionally, when the medical students participate in their clinical rotation in surgery, they are again able to review the anatomy during a 2 hour afternoon session. Does revisiting anatomy during later years in medical school impact the retention of anatomical knowledge? To determine this, students were asked to complete an assessment tool consisting of multiple choice and fill‐in‐the‐blank questions at various times during their medical school career. The results of this ongoing study will be discussed.
A histone acetylation switch regulates H2A.Z deposition by the SWR‐C remodeling enzyme
Authors: Watanabe Shinya , Peterson Craig
Published: June 2021

The conserved histone variant H2A.Z plays key roles in regulation of gene expression, DNA repair, and centromere function. H2A.Z deposition is controlled by members of the SWR‐C subfamily of chromatin remodeling enzymes that catalyze the nucleosomal exchange of canonical H2A with H2A.Z. Nucleosomes that harbor H2A.Z are dynamic in vivo, but the regulatory mechanisms are not clear. Here we report that acetylation of histone H3 lysine 56 alters the substrate specificity of SWR‐C, leading to promiscuous dimer exchange where either H2A.Z or H2A can be exchanged from nucleosomes. Our work also suggests that a conserved SWR‐C subunit may function as a “lock” that prevents removal of H2A.Z from nucleosomes. Collectively, our study provides a novel example of a histone modification that regulates a chromatin remodeling reaction and provides mechanistic insights into how nucleosome turnover can be controlled by chromatin regulators.
The role of the small GTPase Arf6 and its potential regulation by miRNA‐31 during early embryogenesis
Authors: Dumas Megan Elissa , Stepicheva Nadezda , Song Jia L.
Published: June 2021

microRNAs are small, non‐coding mRNAs that repress protein translation and/or mRNA degradation. microRNA‐31 is one of the most abundant miRNAs in the sea urchin embryo, and it is essential for proper cell specification. We bioinformatically identified Arf6 to be a potential target of microRNA‐31. Arf6 mediates membrane trafficking between the plasma membrane and endosomal compartments, including receptor endocytosis and actin rearrangement. The function of Arf6 has not been examined in early development, and we hypothesize that Arf6 is regulated by microRNA‐31 and may direct cell movement and specification of mesenchymal cells. Quantitative, real time PCR and Western blotting were used to characterize Arf6. Arf6 protein levels in microRNA‐31 knockdown embryos are increased compared to the control, suggesting that Arf6 may be regulated by microRNA‐31. We are currently testing the function of Arf6 with morpholino antisense oligonucleotides, constitutively active (Q67L) and dominant negative (T27N) Arf6 mutants. The Arf6 knockdown phenotypes include thickened filopodia, exogastrulation and vegetal cell detachment, indicating that Arf6 is critical for proper early development. This research provides insight into the function of Arf6 during early embryogenesis, its regulation by miRNA‐31, and the role of miRNAs during early development. This work is funded by the University of Delaware Research Fund.
RNAseq analysis of embryonic and postnatal stages of mouse retina reveal transcriptome diversity and novel alternatively spliced isoforms
Authors: BANDAY ABDUL ROUF , Congdon Sean , Seesi Sahar Al , Mandoiu Ion , Kanadia Rahul N
Published: June 2021

Mammalian retina exhibits a highly organized laminar structure consisting more than fifty cell types which are produced and eventually differentiated in specific manner during development. Several gene expression studies have been performed in retina. However, the landscape of alternatively spliced isoforms on global level at embryonic and postnatal stages of retinal development has not been done.

Here we employed high throughput RNAseq from illumina HiSeq 2000 platform for retina samples of embryonic day, E16 and postnatal day, P0. The sequence reads were analyzed by using combination of tools such as Bowtie, IsoEM, Cuflinks and Seqmonk.

We have identified 18021 and 17320 transcripts in E16 and P0 time points, respectively. Comparison of the E16 and P0 transcriptomes showed differential expression patterns and levels (as FPKM values) of transcripts. qRT‐PCR analysis of genes such as Pax6 further confirmed the results. Our results suggest that regulation of alternative spliced isoforms may be the key to the differentiation of retinal cell‐types during development. Our approach of global transcriptome characterization would advance the findings in cellular networks of proteins required for specific functions and transitions required in the developing retina. In all, this analysis will provide a database of alternatively spliced isoforms of genes expressed in the retina. Funding from NEI K99/ROO‐5ROOEYO1954704.
The role of NK1 receptor antagonist L‐703, 606 in the experimental traumatic brain injury
Authors: Duan Hubin , Hao Chunyan , Li Shuzhen , Fan Yimin
Published: June 2021

Background and purpose

L‐703, 606 is a NK1 receptor antagonist. the purpose of the research is to explore the the role of L‐703, 606 in the experimental traumatic brain injury£TBI£©.

Methods

45 male Wistar rats were randomly divided into control group, L‐703, 606 intervention group (group L) ,TBI trauma group (group T). L‐703, 606 was injected into the tail vein of rats in group L after TBI. HE staining were used to observe the pathological changes in each group. The gene and protein expression of substance P(SP), calcitonin gene related peptide (CGRP) and nuclear transcription factor‐kappa B (NF‐¦ÊB) were detected by immunohistochemical staining and RT‐PCR.

Results

The gene and protein expression of SP, CGRP and NF‐¦ÊB in rat cerebral cortical lesions of group L and group T was increased compared with that of control group. Above indicators of group L was significantly reduced compared with that of group T. Only mild congestion, a small amount of inflammatory cell infiltration showed in group L. (P <0.05)

Conclusion

L‐703, 606 has a protective effect on the brain injury in rats. The possible mechanism maybe reducing the inflammatory reaction induced by SP and CGRP which released from cerebral cortical lesion.
Stem cells, pluripotency and nuclear reprogramming
Authors: Jaenisch Rudolf
Published: June 2021

The recent demonstration of in vitro reprogramming using transduction of 4 transcription factors by Yamanaka and colleagues represents a major advance in the field. However, major questions regarding the mechanism of in vitro reprogramming need to be understood and will be one focus of the talk.

During cellular reprogramming only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells and impeded identification of events at the single‐cell level. We utilized two different gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. We show that Esrrb, Utf1, Lin28, and Dppa2 have a better potential to predict cells that will become iPSCs, compared to Fbxo15, Fgf4, and Oct4, which were previously suggested to be markers of reprogramming. Our data suggest that stochastic gene expression early in reprogramming is followed by a late sequential phase with Sox2 activation upstream in a gene expression hierarchy. Finally, we demonstrate that subsets of downstream factors derived from the sequential phase can activate the pluripotency circuitry.

A major impediment in realizing the potential of ES and iPS cells to study human diseases is the inefficiency of gene targeting. Using Zn finger or TALEN mediated genome editing we have established efficient protocols to target expressed and silent genes in human ES and iPS cells. Finally, our progress in using iPS cells for therapy and for the study of complex human diseases will be summarized.
Binding of trans‐ regulatory elements to the 3′UTR adenine/uridine rich elements of human interleukin‐3 mRNA
Authors: Hernandez Marimar , Gonzalez Jose Antonio , Martínez Marina , Gonzalez Pablo , Gonzalez Carlos Ivan
Published: June 2021

Human Interleukin‐3 is a lymphokine that supports a broad range of hematopoietic cells. Aberrant expression of IL‐3 has been implicated in chronic inflammation, cancer and other human diseases. IL‐3 is a member of a class of transiently expressed mRNAs that harbor Adenosine/Uridine‐Rich Elements (ARE) in their 3’‐UTRs. Previous results from our laboratory have suggested a translational role for the hIL‐3 ARE. To understand the hIL‐3 AREs role in translational regulation, we conducted site‐directed mutagenesis of the ARE cluster. Firefly luciferase reporters harboring these ARE mutations were transfected into HeLa and T cells. In order to identify the ARE‐ Binding Proteins (ARE‐BPs) involved in this regulatory process electrophoretic mobility shift assays (EMSAs) and UV‐catalyzed crosslinking were performed using cytoplasmic Jurkat cell extracts. The luciferase chimeras lacking the hIL‐3 ARE showed an increase in luciferase activity. ARE mutations showed specific clusters involved on translational repression. EMSA analysis demonstrated that HuR is a component of the hIL‐3 ARE‐specific protein complex and its binding is modulated upon T‐cell stimulation. Together, these results suggest that HuR and specific ARE clusters play a role in the post‐transcriptional regulation of human IL‐3 mRNA upon T cell stimulation.
Hsp90 chaperone machinery coexpression increases functional glucocorticoid receptor protein expression, receptor•hsp90 heterocomplex formation, and steroid binding activity
Authors: Biette Kelly M. , Belyaev Alexander S. , Murphy Patrick J. M.
Published: June 2021

While hsp90 client proteins such as the glucocorticoid receptor (GR) are highly expressed using a baculovirus expression system, the majority of overexpressed protein is misfolded and nonfunctional. Nonaggregated‐GR forms a functional GR•hsp90 heterocomplex with steroid binding activity. We hypothesized baculoviral co‐overexpression of the complete hsp90 chaperone machinery (i.e. hsp90, hsp70, Hop, hsp40, and p23) would decrease overexpressed client protein aggregation and increase functional client protein concentration and activity. A fusion protein containing wild‐type GR ligand binding domain (GST‐LBD) was produced using a baculovirus expression system in the presence or absence of coinfected baculovirus expressing the hsp90 chaperone machinery. Western blot and steroid binding analysis demonstrated baculoviral coexpression of hsp90 chaperone machinery elicited a >;2‐fold increase in client protein expression and a >;7.5‐fold increase in steroid binding activity. Coexpression of the hsp90 chaperone machinery increased formation of functional GST‐LBD•hsp90 heterocomplexes and steroid binding capacity following in vitro reconstitution. These data show baculoviral coinfection with hsp90 chaperone machinery increases overexpressed GR LBD folding and function. We speculate this methodology may be applicable to baculoviral expression of many, if not all, hsp90 client proteins.
Identifying interaction partners of the Arabidopsis thaliana deubiquitinase‐associated WD40‐repeat proteins LRS1 and WDR20
Authors: James Sean Ryan , Erickson Patti T. , Erickson Floyd L.
Published: June 2021

The lateral root stimulator 1 (LRS1) protein of Arabidopsis thaliana contains a WD40‐repeat domain similar to those found in proteins involved in protein trafficking, ubiquitin modification, and proteasome targeting. The human homolog of LRS1, ubiquitin‐specific protease associated factor ‐1 (UAF1), binds the ubiquitin– specific proteases USP1, USP12, and USP46. USP1 is activated by UAF1 binding, which is required to regulate the Fanconi anemia DNA damage repair pathway. In C. elegans, USP‐46 regulates glutamate receptor abundance by targeting it for lysosomal degradation. Co‐immunoprecipitations have shown that LRS1 forms a complex with the A. thaliana homolog of WDR20 and ubiquitin binding protein 3 (UBP3), a USP1/12/46 homolog. To elucidate targets of the LRS1/UBP3/WDR20 complex we screened a yeast two‐hybrid expression library with LRS1 as bait. We identified ten potential interacting partners, including a zinc finger RING/ FYVE/PHD‐ domain containing protein, a leucine‐rich repeat containing protein, and a kinase involved in hormone signaling. The interaction strengths were analyzed by plating serial dilutions on selective media and β‐galactosidase assays. Ongoing experiments include expression library screening with WDR20 and UBP3 to identify additional interaction partners. This research was supported by Guerrieri and Henson Undergraduate Research programs.
Completing our understanding of tetracycline biosynthesis: the enzymatic basis of the F420‐dependent final reduction step.
Authors: Tang Yi , Wang Peng
Published: June 2021

Tetracyclines are important broad‐spectrum antibiotics. While the discovery of tetracyclines date back to the 1940s, our understanding of the biosynthesis of tetracyclines has only been investigated in recent years. One unknown step in the pathway is the conversion of 5a, 11a‐dehydrotetracycline to either oxytetracycline in Streptomyces rimosus or tetracycline in Streptomyces aureofaciens. The reduction of the 5a–11a double bond is likely catalyzed by a F420‐dependent enzymes, however, the enzymatic basis for this reaction remains unresolved. Using a heterologous expression platform as a starting point, we discovered a small, previously unknown enzyme in both the oxy and ctc pathways that is responsible for this reduction. In vitro reconstitution using this small enzymes, in conjunction with the anhydrotetracycline oxygenase, showed that anhydrotetracycline can be converted to the final products. We also provide biochemical and structural explanation for the differential formation of oxytetracycline and tetracycline from the respective pathways. The result of this work therefore completes our understanding of the tetracycline pathways and pave the way for engineered biosynthesis of analogs.
Effect of glutathione on the phospholipase A2 activity of peroxiredoxin 6
Authors: Zhou Suiping , Dodia Chandra , Shuvaeva Tea , Sorokina Elena , Harper Sandra , Speicher David W , Feinstein Sheldon I , Fisher Aron B
Published: June 2021

Peroxiredoxin 6 (Prdx6) is a unique non‐seleno 1‐Cys peroxidase with both peroxidase and phospholipase A2 (PLA2) activities. We have shown that phosphorylation of Prdx6 at Thr177 increases its PLA2 activity and broadens its pH‐activity spectrum (Biochem. J. 419:669, 2009). Our recent study indicates that a change in the conformation of Prdx6 upon its phosphorylation is the basis for enhanced enzymatic activity at pH 7.4 (Biochemistry. 51:5521–30. 2012). Here, we studied the effect of glutathione (GSH) on the PLA2 activity of Prdx6 using biochemical and biophysical approaches. With 3H‐DPPC as substrate, basal Prdx6 PLA2 activity was 0.17 ± 0.1 nmole/min/mg and increased to 49 ± 1 nmole/min/mg upon addition of 5 mM GSH. Addition of other thiols (GSSG, DTT) had no effect. The enhancing effect of GSH on the PLA2 activity of Prdx6 depended on the redox status of substrates and phosphorylation status of the enzyme. GSH has no effect on PLA2 activity if the substrate is oxidized by Cu(2+)‐ ascorbate or if the enzyme is phosphorylated. By isothermal titration calorimetry (ITC), GSH interacts with Prdx6. Circular dichroism (CD) indicates that GSH induces a change of Prdx6 conformation. Thus, a change in the conformation of Prdx6 in the presence of GSH is the basis for enhancing effect of GSH on PLA2 activity of non‐phosphorylated Prdx6 in the presence of reduced substrate.

This work was supported by NIH grants HL102016 and HL19737
Mutant Chronicles: Mutagenesis, Expression, Purification and Characterization of LigAB Mutants
Authors: Ngu Abraham , Barry Kevin , Taylor Erika
Published: June 2021

Lignin is an environmentally sustainable low‐impact starting material for making biofuels. The Taylor Lab at Wesleyan University investigates the pathways by which lignin is converted into central metabolites to help establish a path for the microbial conversion of lignin into a biofuel. LigAB, an extradiol dioxygenase from Sphingomonas paucimobilis SYK‐6, is one enzyme involved in this pathway, and is the focus of my research. The crystal structure of LigAB has revealed that phenylalanine‐103 (F103α) of LigAB is located proximal to the substrate‐binding site and is believed to contribute to the substrate specificity of the LigAB enzyme. F103α has been mutated to various smaller residues to test the hypothesis that it is functioning to limit the size of the substrate binding pocket, thus providing substrate specificity. The glutamic acid‐242 (E242β) residue, however, is essential to the coordination of the iron (II) ligand. Mutagenesis of E242β is anticipated to abolish iron binding, rendering the enzyme inactive. The histidine‐127 (H127β) and histidine‐195 (H195β) residues have been mutated to test their putative assignment as catalytic residues involved in acid‐base chemistry. Characterization of these mutants allows for better understanding of the substrate specificity and reaction mechanism of LigAB, which helps us learn about LigAB's function and about how to increase its promiscuity.