bluuebunny/crossref_metadata_2025 huggingface dataset:
Trans differentiation of Chondrocytes to Osteoblasts during Endochondral Ossification in the Healing Mandible
Authors: Wong Sarah Anne , Hu Diane , Miclau Theodore , Bahney Chelsea , Marcucio Ralph
Published: November 2023

A new model of endochondral ossification has recently emerged, in which chondrocytes of the cartilage intermediate undergo a transient stem cell‐like state and ultimately trans differentiate into osteoblasts/cytes that form the new bone. This model has been established by studying long bones during development and fracture healing. However, it has yet to be determined if the same mechanism translates to mandible fracture healing. Contrary to long bones, the mandible develops primarily through intramembranous ossification; however, it heals through both intramembranous and endochondral ossification. We sought to determine whether fracture stability directs the relative contribution of each ossification mechanism during mandible healing and to determine whether chondrocytes $trans differentiate into osteoblasts/cytes during mandibular endochondral fracture healing. We hypothesized that stable mandible fractures heal through intramembranous ossification and unstable fractures heal through endochondral ossification, in a manner analogous to appendicular fractures, and that chondrocytes trans differentiate into osteoblast/cytes during endochondral ossification. To test this, we generated mandible fractures with different levels of stability by creating either 1mm trephine defects or unstable transverse fractures in the mandibular rami of C57BL/6 mice and compared the ossification process at multiple time points using histology and immunohistochemistry. Preliminary data demonstrate minimal cartilage formation in stable fractures compared to the robust cartilage formation in unstable fractures, supporting our hypothesis that mobility promotes endochondral ossification. Our data illustrate maximum cartilage formation in unstable fractures at 10 days post‐fracture. At this time point, hypertrophic chondrocytes at the chondrosseous junction, termed the “transition zone,” stained positive for the pluripotency factors Oct4 and Sox2 by immunohistochemistry. These data parallel our studies of tibia fracture repair where we have performed genetic lineage tracing experiments with inducible Col2, Oct4, and Sox2 reporter mice. Taken together these data suggest that motion during mandible fracture repair promotes endochondral ossification and that chondrocytes trans differentiate into osteoblasts by acquiring what appears to be a transient pluripotent state. Future studies will elucidate the mechanisms that regulate chondrocyte trans differentiation during mandible repair and test novel therapeutic strategies designed to treat hypertrophic non‐unions.

Support or Funding Information

NIH: NIAMS: F32AR062469 to C.B., R01‐AR057344 to T.M., NIA: R01‐AG046282 to R.M. MTF Junior Investigator Award to C.B.
Developmental and Injury‐Induced EphB3 Expression in Bone
Authors: Kamath Rajay A. D. , Szczerba Mark , Xiang Lilin , Benson M. Douglas
Published: November 2023

Defects in bone growth underlie common congenital malformations such as craniosynostoses, and contribute to osteoporosis and poor fracture repair. Thus, a thorough understanding of bone formation is vital to effective treatment of such conditions. Mounting evidence points to a central role for the Eph family of receptor tyrosine kinases and their membrane‐bound ephrin ligand in the regulation of developmental and homeostatic bone growth. However, which members of this receptor family are the central players in bone growth remains unknown. We therefore examined the expression patterns of several candidate Ephs in mouse calvaria and long bones. We used engineered mouse lines to determine the expression of Ephs B1–3 in long bones and in intact and injured calvaria. The EphB1 and EphB2 lines harbor LacZ cDNAs fused to truncated Eph coding regions. The EphB3rtTA line contains an EphB3 gene‐controlled tetracycline transactivator that drives LacZ expression from a tetOP LacZ transgene. A 3 mm defect was administered to the parietal bones of these mice, and the mice were sacrificed after one to three weeks. Their bones were then stained for beta‐galactosidase. Although EphB2 is expressed in late embryonic calvaria, we observed no significant expression in intact or injured adult bones. EphB1 was similarly absent. EphB3, however, was highly expressed in suture mesenchyme and periosteum of young adult mice, and was upregulated at the margins of calvarial injury. EphB3 was also found in the proliferative and hypertrophic cartilages of femora and tibiae. These data identify EphB3 as a likely mediator of bone growth. Our ongoing gene knockout studies will define the role of EphB3 in bone growth and healing and focus further mechanistic studies that will ultimately facilitate clinical treatments of bone pathologies.

Support or Funding Information

Supported by NIH/NIDCR R01DE022804 to MDB and the Baylor Oral Health Foundation
Stronger Composite Dental Restoration Results from Inclusion of Extracellular Matrix Protection Factor‐2 (ECPF‐2), a New Class of Matrix Metalloprotease 8 (MMP‐8) Inhibitor
Authors: Persky David William , Tuloglu Nuray , Ghanta Haritha , Kitagaito Kaz , Ciccarelli Haley , Clauss Gary , Datillo Victoria , Selim Abdulhafez , Ozer Fusun , D'Angelo Marina
Published: November 2023

Each year in the United States alone, the prevention and restorative treatment of tooth decay exceed costs of $100 billion. A considerable percentage of these costs arise from the need for retreatment and replacement of failed restorations. An innovative means of preventing the failure of dental restorations would ease the financial burden associated with retreatment procedures. Although bacterial enzymes contribute to the degradation of the layer between the composite and dentin, current research indicates a role for endogenous Matrix Metalloprotease‐8 (MMP‐8), a collagenase enzyme, in degradation of the hybrid layer, the region between the dentin and composite resin material.

It has been demonstrated that acid etching of the tooth in preparation for administration of the adhesive bonding agent will activate endogenous MMPs in the hybrid layer. The contemporary clinical solution to hybrid‐layer degradation is the use of a nonspecific protease inhibitor, Chlorhexidine Gluconate (0.12%), at the time of treatment. Since MMP‐8 is primarily responsible for the degradation of the organic component of dentin, which is composed predominantly of type I collagen fibers. A more specific targeted protease inhibition should increase bond strength in restorations.

To address this notion, our laboratory has developed a proprietary compound, Extracellular Matrix Protection Factor‐2 (ECPF‐2), to specifically inhibit the interaction of MMP‐8 and collagen type I. Utilizing a novel tooth‐restoration model, we treated non‐carious, extracted human third molars with 5μg ECPF‐2 after acid etching and before application of the adhesive. Following placement and curing of the composite material, teeth were either immediately prepared for bond strength testing or first exposed to one‐year of artificial accelerated aging prior to the bond strength testing.

Beams (dimensions of 1mm X 1mm) were cut with a milling tool and subjected to microtensile bond strength testing with a single‐speed actuator. Teeth treated with ECPF‐2 exhibited statistically significant increased bond strength (39.6 Megapascals [MPa]) compared to Chlorhexidine treated preparations (20.3 MPa) and bond strength was improved even after one‐year accelerated aging (ECPF‐2 28.9 MPa versus Chlorhexidine 24.0 MPa) even though, as expected, the absolute values were reduced. The enhanced bond strength in ECPF‐2‐treated preparations indicates the importance of this novel inhibitor with respect to clinical and commercially‐available applications in extending the lifespan of dental restorations.

Support or Funding Information

Center for Chronic Disorders of Aging Small Grant, Philadelphia College of Osteopathic Medicine

Schonleber Foundation Grant, University of Pennsylvania
Expression of CGRP, Osteopontin and VEGF in embryonic mouse mandible
Authors: Sato Iwao , Azuma Yuri , Miwa Yoko , Hara Setuhiro , Sunohara Masataka
Published: November 2023

Objective

Neuropeptide calcitonin gene‐related peptide (CGRP) is a marker neurotransmitter related to vasculargensis in muscle differentiation and development of organ. The CGRP was first appeared at mouse embryonic day 16.5 (E16.5) and localization around blood vessels and closely cartilaginous bone matrix in the mouse limb (Bidegain et al., 1995). However, expression of CGRP and angiogenesis markers were unknown in developed mandible during craniofacial formation. Moreover, Osteopontin (OPN) detects in fibroblasts, preosteoblasts, osteoblasts, osteocytes, odontoblasts and bone marrow cells. We try to clearly the relation between CGRP, bone morphological marker, and angiogenesis marker in terminating the differentiation of mouse mandible.

Methods

We analyzed the expression and localization of CGRP, VEGF, OPN by in situ hybridization in the developing mouse mandible during embryonic days at E14.5 and E17.5. We also detected the mRNA abundance of various markers (Collagen I, VEGF, CD31, LYVE‐1, MMP‐2, OPN, and CGRP) from embryonic stages by real‐time RT‐PCR. Principal component analysis (PCA) was performed for mouse mandible in different early postnatal stages and the expression of the following 7 mRNA CGRP, OPN, CD31, LYVE‐1, VEGF, Collagen I, and MMP‐2.

Results

The antisense probe for VEGF, OPN and CGRP was detected in mesenchymal cells surrounding area of mandible (Meckel's cartilage) at E14.5, and clearly localization was seen at E17.5 in the embryonic mouse masseter by in situ hybridization. These data suggested that angiogenesis markers may effect to influence during mouse Meckel's cartilage development and morphogenesis. These findings provide direct insight into the origins and potential prevalent development affecting the mandibular formation.

Support or Funding Information

none
Bat Development: Interspecies Differences in Baculum Ossification Patterns
Authors: Herdina Anna Nele , Nugraha Taufiq Purna , Semiadi Gono , Haase Astrid , Lina Peter H. C. , Godlevska Lena , Vlaschenko Anton , Metscher Brian D.
Published: November 2023

Developmental studies are fundamental to understanding specialized morphologies. The baculum (os penis) is a heterotopic bone in the glans penis, which is probably subject to sexual selection and may play a role in reproductive isolation between closely related species. In this ongoing study, the pre‐ and postnatal development of the reproductive organs will be compared between species of the bat genera Pipistrellus, Eptesicus, and Cynopterus histologically and with 3D X‐ray microtomography (microCT) imaging. We aim to show how bacula with different shapes start to calcify at different ossification centers and to compare soft tissue development of bat reproductive organs.

Bat organ system development has mainly been studied using histological methods, dissection, or clearing and staining. The skeletal system has also been studied with x‐ray techniques including microCT. Here, we will use 1% (w/v) elemental iodine in 100% ethanol (I2E) to study the development of reproductive organs in a sample of 14 Cynopterus brachyotis (Chiroptera: Pteropodidae) embryos from the collection of the Museum Zoologicum Bogoriense (Indonesia), and several juvenile and subadult bats of the species Cynopterus sphinx (Indonesia), Pipistrellus pipistrellus (Naturalis Biodiversity Center Leiden, Netherlands), and Eptesicus serotinus (Netherlands and Ukraine). Iodine contrast staining has been used successfully in a number of studies on fetal and adult vertebrates, because soft tissues can thus be differentiated in microCT images.

Preliminary results show different patterns of ossification. In Pipistrellus pipistrellus the distal part of the baculum reaches its adult shape before the proximal part. The different states of medullary cavity development found in our samples suggest the medullary cavity first forms from the ventral side of the baculum, where the branches of the base meet the shaft and that it is later replaced by a secondary medullary cavity. The baculum of Eptesicus serotinus calcifies from both lateral sides, while four different calcification centers can be shown in a developing baculum of Cynopterus sphinx.
The Birth and Evolution of Modern Anatomy
Authors: Barbieri Charles , Lugar Catherine , Nusse Gloria , Summers Orianna
Published: November 2023

Andreas Vesalius is considered by many to be the father of modern human anatomy because his work in De Humani Corporis Fabrica shifted anatomical education away from a 1200 year old, hands‐off tradition of analog dissections to a modern humanistic; cadaver center approach. De Humani Corporis Fabrica was revolutionary for artists and anatomists alike because it provided the first accurate, detailed images of human anatomical systems, at a time when most medical texts contained minimal graphical representations. An in depth analysis of Vesalius’ anatomical models reveal a lasting influence his methods have had on modern anatomical practice. With many of the current anatomical dissection methodology and presentation having evolved though Vesalius’ work.

The aim of this study was to create a bridge between modern anatomy and its origins within De Humani Corporis Fabrica, with the primary focus on the educational methods that transcended his work and became some of our current teaching methods anatomy.

One of Vesalius’ major contributions was the creation of meticulously accurate diagrams that went on to evolve into the models and diagrams that play an essential role in today's anatomical education. This primarily began with his work on the “Muscle Men” plates with the artist Titian. These plates showed his dedication to creating a medium that could innovate the education methods of his time. The “Muscle Men” are a series of anatomical depictions that when placed side‐by‐side show a progressive dissection of a man in the Euganean Hills surrounding the Padua University in Italy. This method of showing progressive dissections is now commonplace in modern anatomy education, but was absent before Vesalius’ work. To show how powerful these can be, the images were digitally stitched together then colorized to show the original panoramic view of the euganean hills.

Vesalius’ method of dissection evoked a fundamental shift in anatomical study has carried over 500 years, into the modern anatomical laboratory where instructor‐led student dissections play a major role in teaching anatomy. De Humani Corporis Fabrica provided detailed descriptions of the tools and methods used for the dissections. While many of the protocols and tools have been refined over time their initial incorporation into dissection was largely wrought out of his work.
a Sample of the “Tabulae Sceleti et Musculorum Corporis Humani” by Albinus and Wandelaar, Leyden, 1747
Authors: LE FLOCH‐PRIGENT PATRICE P , FROMANGER Marine , GILLOT Jean‐Bernard
Published: November 2023

An original edition of the Albinus “Tabulae sceleti et musculorum…” belonged to the “Librairie scientifique Alain Brieux ‐ Gillot” in Paris, in November 2015. This large (in plano) volume was very complete of the very famous 12 plates of the Homo perfectus. The 17 plates of the anatomical details and the engraved dedication were missing here. We have analyzed the anatomical veracity of each plate. This book was published two hundred years after the Vesalius “ de Humani fabrica”. Bernard Siegfried Albinus (1697–1770) was hired as a Professor in Leyden when he was 24 years old. He began to work with the artist Jan Wandelaar (1690–1759) from 1725 until the publication in 1747 of this monument of the gross‐anatomy. The foreground of the plates are marvelous, however we will focus on their anatomical aspects. One time again, one can regret that the drawings of Leonardo Da Vinci were not still known at this time, as in this occurrence would had happened, the work of the extraordinary team Albinus ‐ Wandelaar would had gone scientifically still further.
Long term muscle involvement after neonatal pain
Authors: Alves Júlia Aparecida , Simões Ana Leda B , Dias Fernando José , Carmo Elisabete C , Machado Nathalia B , Sanada Luciana S , Fazan Valeria Paula Sassoli
Published: November 2023

Nociceptive stimuli applied soon after birth play an important role in the development of pain in adult life. Previous studies in our laboratory have shown that pain in the neonatal period can cause a number of changes both in the central nervous system and the musculoskeletal [1], but experimental substrates for these changes still need investigation. We designed an experimental study to explore whether pain in the neonatal period can cause long term changes in the locomotor system. The pain groups (male and female Wistar rats, N = 10 per group) were stimulated with a needle on the right hind paw, twice a day, since birth, for 15 consecutive days. The control groups (male and female Wistar rats, N = 10 per group) were stimulated with a cotton swab. Final experiments were performed 180 days after birth. In order to evaluate the motor function of the hind paws, grip strength tests were conducted. Maximum and average (mean of three consecutive measures) grip strength were studied. Afterwards, rats were anesthetized and the medial gastrocnemius (MG) muscles were obtained by surgical biopsy. The muscle fragments were frozen and stained with the techniques for nictotinamide adenine dinucleotide (NADH) and mitochondrial succinate dehydrogenase (SDH). For muscle morphometry, the number of fibers was counted and their area and diameter were measured. Specific statistic tests were applied and differences were considered significant when p<0.05. The grip strength was higher in males compared to females in both, control and pain groups, but smaller on pain groups, in both genders, compared to controls. Preliminary morphometric results showed that the female pain group had a higher average area of the MG fibers than the control group, while no differences between pain and control groups were found on males. The comparison between genders showed that male controls have larger average number of fibers compared to the females. On the pain groups, females showed larger fiber area compared to males. Our results are suggestive that pain in the neonatal period caused a reduced grip ability that was maintained in the long term after the painful stimulus was ceased, with a slightly better performance on older females. Neonatal pain was also responsible for increasing the average size of muscular fibers in the MG in females, with no apparent effect in males. Increasing the number of animals for the histological studies will help the identification of the type of muscle fibers that are being related to the grip ability alterations observed in this model of pain.

Support or Funding Information

FAPESP, CNPq, CAPES and FAEPA
Cadaveric study of the anatomical and histological relationships between the Achilles and plantaris tendons
Authors: Goral Joanna , Inouye Sandra E , Belford Zachary , Leland Eric , Ferrel Benjamin , Cheung Andrew , Meyer Alice , Fanter Nathan
Published: November 2023

Rationale

The purpose of this study was to investigate whether the proximity in anatomical locations of the Achilles (AT) and plantaris (PT) tendons promotes histopathological changes common to both tendons and their shared paratenon. The Achilles and plantaris tendons (ATs and PTs, respectively) share close anatomical locations in the foreleg, which has led to a proposed association between pathological findings in these tendons. Previous studies have identified such an association in specimens with known tendinopathy. This study aimed to determine whether similar correlation could be found in a group of cadavers without any record of tendon pathology.

Materials and methods

The subjects in this study were 20 donor cadavers (11 females and nine males) fixated in formalin, provided by the Department of Anatomy at Midwestern University. The mean age was 81 (±12). Twenty left, and 19 right legs were dissected. The ATs and PTs were assessed based on their gross anatomical characteristics, then samples of the tendons were harvested from two sites, one where the ATs and PTs ran closely together and a second where the PT was unassociated with the AT. The collected specimens were fixed, processed for paraffin embedding, sectioned, and stained with hematoxylin and eosin for histological analysis. Tissues were examined for the presence of inflammatory cells and for the evidence of stress‐induced conversion of the tendons’ dense connective tissue into fibrocartilage.

Results

In four specimens (10.2%) the ATs and PTs ran separately, in eight (20.5%) specimens they were fused, and in three specimens the plantaris muscle was absent (7.7%). In the rest of the specimens the ATs and PTs were closely associated, but not fused. Histologically, no distinct evidence of inflammation was found at either the related or unrelated AT and PT sites. A presence of fibrocartilage with chondrocytes within the AT was noted in six (15.4%) specimens.

Conclusion

The results of this study did not demonstrate any significant histopathologic changes in the areas of close contact between the ATs and PTs. The presence of fibrocartilage in the Achilles but not in the plantaris tendons suggests that the AT may be more affected by compression‐induced stress than the PT. These findings suggest that within the control group, close contact between AT and PT does not result in interrelated histopathologic changes. However, it is possible that histopathology may be observed in patients with mid‐portion Achilles tendinopathy.

Support or Funding Information

Zachary Belford and Benjamin Ferrel were supported by the Midwestern University Research Fellowship Program. Eric Leland and Andrew Cheung were supported by the Midwestern University work‐study program.
Computer‐Assisted Anatomical Dissection (CAAD) of the Renal Plexus to Refine Renal Denervation in the Treatment of Refractory Hypertension
Authors: Bessede Thomas , Lebacle Cedric , Timoh Krystel Nyangoh , Martinovic Jelena , Diallo Djibril , Zaitouna Mazen , Benoit Gerard
Published: November 2023

Context

RadioFrequency (RF) renal denervation is an emerging treatment of refractory arterial hypertension with various possible approaches: endovascular, endourologic, laparoscopic. Results and adverse events remain controversial. Theoretical safety and efficiency of all of the approaches deserve to be addressed with special insight into the anatomy of the renal plexus and hilar structures. The objective was to describe the location and distances between nerve fibers and renal vasculature.

Methods

Sagittal serial sections were performed in 4 human fetuses and 10 adult bodies were dissected. In fetuses, sections were stained by Masson's trichrome, HE, and immunostained by PS100 neuronal antibody. The nature of renal plexus fibers was investigated by adrenergic (TH) and somatic (PMP22) labelings. In cadavers, the distances between nerve fibers and arteries were measured at the renal ostium, at mid‐artery, at the hilum of the kidney, and were statistically compared (one‐way repeated measures ANOVA). 3D reconstructions were performed from the digitized serial sections obtained in fetuses.

Results

The renal plexus can be described as a guying contained in an hourglass shape centered on the renal artery. The isthmus of this hourglass corresponds to the hilum of the kidney and is a mandatory waypoint of afferent and efferent renal plexus. At this level, the nerves are at the closest location of arterial branches (11 mm at the renal ostium, 6.5 mm at mid‐artery and 2 mm at the hilum of the kidney; p<0.001). The sectorial distribution of the nerves around the renal artery is heterogeneous and fibers predominate on the superior aspect. Nerve fibers are autonomic (PMP22−) and adrenergic‐sympathetic (TH+). With an animated 3D reconstruction, it is possible to analyze the relationship between renal plexus, vasculature and urinary tract, and the potential risks of various denervation techniques.

Conclusion

CAAD in fetuses identified the renal hilum as the theoretical preferential site for renal denervation. To optimize endovascular denervation in future clinical studies, a compromise should be sought between the size of the RF probe and the arteries’ diameter. There is an opening to laparoscopic surgical denervation.

Support or Funding Information

None
The anatomy of the human myometrium: A stereological approach
Authors: Dockery Peter , Sweeney Eva Marie , Denis Crankshaw J , John Morrison J
Published: November 2023

In this presentation we illustrate the usefulness of the stereological approach in providing quantitative anatomical information of the human myometrium in pregnancy. Altered uterine contractile performance has been associated with maternal obesity and advanced maternal age. The aim of this study was to investigate the microanatomy of the human myometrium in pregnancy, and to evaluate a potential influence of maternal body mass index (BMI) and age.

Study Design

Biopsies of human myometrium were obtained from 57 women at cesarean section (n = 26, n = 13, n = 18 normal, overweight and obese BMI, respectively). Samples were obtained using a systematic random protocol and processed for light and electron microscopy. A number of absolute and relative stereological were used to document the microanatomy. All material was obtained with informed consent and appropriate ethics committee approval.

Results

There was found to be significant differences in smooth muscle mitochondrial volume, length density of blood vessels and volume density of smooth muscle nuclei with age. The only BMI related change was found to be in the volume density of connective tissue.

Conclusion

The results of our studies suggest that there may be structural changes in the myometium in older women during pregnancy. These results highlight the usefulness of the stereological approach in trying to untangle the structural sequelae of the altered uterine contractile performance has been associated with maternal obesity and advanced maternal age.

Support or Funding Information

Studentship from NUIGalway
Optical Clearing and 3D‐Histology of Murine Testis
Authors: Kaufman Jason A. , Castro Monica J. , Rodriguez‐Sosa Jose Rafael
Published: November 2023

Optical clearing techniques (e.g., CLARITY, Scale, SeeDB, and 3DISCO) provide unprecedented opportunities to study large tissue samples at histological resolution, eliminating the need for physical sectioning while preserving the three‐dimensional structure of intact biological systems. Although these methods have been used primarily to investigate nervous system tissues, there is significant potential for applying optical clearing to reproductive tissues. In testicular biology, for example, the study of spermatogenesis and the use of spermatogonial stem cells offer high‐impact applications in fertility medicine and reproductive biotechnology. Therefore, the objective of our study is to apply optical clearing and immunofluorescence to testicular tissue in order to reconstruct its three‐dimensional microstructure in intact large samples. We used Triton‐X detergent‐based clearing in combination with Refractive Index Matching Solution to achieve optical transparency of fixed mouse testes. An antibody against smooth muscle actin (SMA) was used to label peritubular myoid cells of seminiferous tubules while an antibody against ubiquitin C‐terminal hydrolase (UCHL1) was used to label Sertoli cells and spermatogonia in the seminiferous epithelium. Specimens were then imaged using confocal fluorescence microscopy. Our results demonstrate that we were able to successfully clear testicular tissue and utilize immunofluorescent probes. Even with a basic confocal microscope and conventional objective lenses, we were able to image through as much as 500 microns of cleared tissue (the mechanical limit of our microscope). Moreover, we successfully visualized the histological compartments of testicular tissue in three‐dimensional reconstructions. The anti‐SMA antibody labeled the peritubular myoid cells in striking detail and exhibited their characteristic polygonal morphology. The antibody against UCHL1 provided clear visualization of mouse Sertoli cells and spermatogonia. When complemented with DAPI nuclear staining, the progressive stages of spermatogenesis could be observed proceeding from the basal compartment to the adluminal compartment of the seminiferous epithelium. In summary, optical clearing combined with immunofluorescence and confocal imaging offers a powerful new method to analyze the cytoarchitecture of testicular tissue at histological resolution while maintaining the large‐scale perspective of the intact system.

Support or Funding Information

This research was generously funded by the Arizona Alzheimer's Consortium and Midwestern University.
Studies of Bilateral Asymmetry of the Upper Limb in Adult Males in Ilorin Metropolis, Nigeria
Authors: Oyewopo Adeoye Oyetunji , Gbadamosi Ismail , Ezra Lambe , Arogundade Tolulope , Jaji‐Sulaimon Rukayat
Published: November 2023

BACKGROUND

The region of the upper extremity has long been of interest to researchers especially when it comes to handedness. The upper limb or upper extremity is the region of the body extending from the deltoid region to the hand, including the arm, axilla and shoulder. There are several theories of how handedness develops in individual humans. This study aims to investigate the bilateral asymmetry of the upper limb in adult males in Ilorin metropolis.

MATERIALS AND METHODS

A total of 40 male subjects with equal number of left and right hander between the ages of 18 and 23 within the Ilorin metropolis were used. Various anthropometric measures which include the length of the whole arm; lengths of upper and lower arm, widest circumferences of upper and lower arm of their upper limbs were estimated with aid of a measuring tape.

RESULTS

All the parameters in individuals using right showed an increase in right hand readings compared to the left except in the width of the palm that shows increase in the left hand palm width than the right hand but for the left handed individuals, all the parameters showed a decrease in right hand readings compared to the left.

CONCLUSION

Statistical analysis of anthropometric measures from the upper limbs of adult males in Ilorin metropolis has clearly indicated that the bilateral asymmetry of the upper limb is an indication of handedness.

Support or Funding Information

NIL
Nerve‐Muscle Perforation Variations in the Upper Limb: A Review with a Description of Three New Variants
Authors: Miller Joseph M.A. , Trelease Robert
Published: November 2023

Muscle perforation by somatic nerves is well known in the body. In the upper limb, three such muscle perforations are classically described: the coracobrachialis perforated by the musculocutaneous nerve (MCN), the pectoralis minor perforated by the medial pectoral nerve, and the supinator perforated by the deep radial nerve. It is less well recognized that there are other muscle perforation variations in the upper limb. This study reviews known muscle perforation variations of the upper limb by somatic nerves and introduces three new observed variants. Bergman et al. (1988) catalog the following four muscle perforation variations: pectoralis minor perforated by the lateral pectoral nerve, the coracobrachialis perforated by the lateral cord, the short head of the biceps perforated by the MCN, and the brachialis perforated by the MCN. As a result of our own cadaveric studies, we discovered the following variations: 1) a third head of the biceps perforated by the MCN, 2) long head of the biceps perforated by the MCN, and 3) pronator teres perforated by the median nerve. These variations are of intrinsic anatomical interest. Clinically, they expand the possible etiologies of compression/entrapment syndromes encountered in the upper limb.

Support or Funding Information

We wish to thank the UCLA Donated Body Program and our donors who gifted their remains for scientific research.
Crystal Structure of the Cancer Genomic DNA Mutator APOBEC3B Catalytic Domain
Authors: Aihara Hideki , Shi Ke , Carpenter Michael A. , Kurahashi Kayo , Harris Reuben S.
Published: November 2023

Background

APOBEC3 family DNA cytosine deaminases provide overlapping defenses against pathogen infections as part of innate immunity. APOBEC3B is the only family member that is predominantly nuclear‐localized, and recent studies have demonstrated its major role in cancer mutagenesis.

Methods

We used the E. coli‐based rifampicin resistance mutation assay and in vitro DNA deamination assay to guide the generation of a highly soluble and catalytically active APOBEC3B catalytic domain construct, and used X‐ray crystallography to analyze its structures.

Results

We have determined the first high‐resolution structures of APOBEC3B catalytic domain in multiple crystal forms. The structures revealed a tightly closed active site conformation, which suggest that the binding of a single‐stranded DNA substrate is regulated by adjacent flexible loops. Replacing one of these loops adjacent to the active site (loop 1) with the corresponding loop from the highly homologous cytoplasmic deaminase APOBEC3A generated a hyperactive enzyme, consistent with tight regulation of APOBEC3B activity. We also determined a nucleotide (dCMP)‐bound crystal structure that suggests a possible mode of APOBEC3B‐DNA interaction and informs a multi‐step model for the binding of single‐stranded DNA.

Conclusion and Significance

A comparison between our APOBEC3B structure and those of other cytoplasmic APOBEC3 members with more open active site conformations suggests that the active site accessibility of APOBEC3B may be regulated in a manner that maximizes innate antiviral activity and minimizes damages to genomic DNA. The novel nucleotide‐binding site on the surface of the APOBEC3B catalytic domain is accessible even when the active site is in the closed conformation, and thus it could serve as an initial contact point for a single‐stranded DNA substrate. Given its role in cancer mutagenesis and being a non‐essential enzyme, APOBEC3B is an attractive target for anti‐cancer drug development. Our high‐resolution structures provide a framework for further mechanistic studies and development of APOBEC3B inhibitors to suppress cancer evolution to minimize drug resistance and metastasis.
Investigation of Structure‐Activity Relationships for Small‐Molecule Inhibitors of Human DNA Polymerase Eta
Authors: Zafar Maroof K , Maddukuri Leena , Eddy Sarah , Ketkar Amit , Penthala Narsima R , Crooks Peter A , Eoff Robert L
Published: November 2023

Efficient DNA replication by high‐fidelity polymerases (pols) can be perturbed by both endogenous and exogenous sources of DNA damage, resulting in accumulation of replication intermediates that activate DNA damage response pathways. DNA damage and replication stress are hallmarks of cancer and cells have intrinsic mechanisms that rely on replication stress response pols to assist in replication fork progression following DNA damage. Many chemo‐ and radio‐therapies rely on targeting DNA, thereby, blocking DNA replication to limit tumor growth. These genotoxic drugs can be rendered ineffective through Translesion DNA synthesis (TLS), involving Y‐family pols, namely human polymerases eta (η), kappa (κ), iota (ι) and Rev1. Most importantly, both in vitro and in vivo studies have highlighted the role of hpol η in bypassing lesions caused by doxorubicin and platinum based drugs. Therefore, targeted inhibition of these specialized pols will improve the ability to destroy cancer cells that rely on these enzymes to survive treatment with genotoxic anti‐cancer drugs. The main objective of our study was to identify small‐molecule inhibitors of hpol η and determine if these molecules show synergy with genotoxic anti‐cancer drugs in an effort to ameliorate existing cancer therapeutics to improve patient outcomes. Catalysis by hpol η was measured in the presence of eighty‐eight small molecules at a single dose of 20 μM from a novel library of more than 100 indole thio‐barbituric acids (ITBA) and indole barbituric acid (IBA) derivatives. From this initial study, we identified several potent inhibitors of hpol η activity. Michaelis‐Menten kinetic analysis and in‐silico docking approaches were employed to determine the mechanism of action for the most potent inhibitors. One of these compounds, PNR‐7‐02, an ITBA derivative with both N‐napthoyl moiety and 5‐chloro substituent on the indole ring inhibits hpol η activity with an IC50 value near 8 μM. In silico docking and Michaelis‐Menten kinetics revealed that PNR‐7‐02 likely inhibits hpol η activity through a partial competitive/allosteric mechanism of action. PNR‐7‐02 inhibited hRev1 and hpol lambda (λ) with low micromolar IC50 values. Another ITBA derivative with a 4‐(2‐ethoxyethyl) morpholine substitution at position 5 on the indole ring (PNR‐9‐59) inhibited hpol η with an IC50 of 13 μM. PNR‐9‐59 showed improved drug‐likeness as well as improved specificity for hpol η against A‐, B‐ and X‐family pols and also within Y‐family pols. In summary, our work has improved the potency and drug‐likeness of small‐molecule inhibitors of hpol η, and it stands as the first detailed analysis of structure‐activity relationships for inhibitors of a TLS pol involved in resistance to chemotherapeutics.

Support or Funding Information

This work was supported by NIH grants GM 084460 and CA 183895 (to R.L.E.).
Biochemical and Biological Investigation of Human Polymerase Kappa Activity on G‐Quadruplex DNA
Authors: Eddy Sarah , Maddukuri Leena , Tillman Magdalena , Smith Rachel , Ketkar Amit , Zafar Maroof K , Eoff Robert L
Published: November 2023

Faithful replication of genomic information is crucial for maintenance of cellular function and integrity. DNA can adopt non‐B‐form structures in vivo, such as G‐quadruplex DNA (G4 DNA). G‐quadruplexes represent highly stable guanine‐rich secondary structures that can hinder DNA replication, impeding B‐family polymerases (pols), which are the enzymes responsible for faithfully copying information contained within DNA sequences. The accurate replication of G4 DNA is important, as these structures regulate transcription of oncogenes and telomere dynamics. Y‐family translesion pols have been implicated in successful G4 DNA replication; thus, we hypothesized that these enzymes may facilitate efficient bypass of G4 DNA structures. The primary goal of the current project is to investigate the role of human Y‐family pol kappa (hpol kappa) in replication of G‐quadruplex sequences with in vitro enzymatic assays and experiments in cell culture. Using fluorescence anisotropy, we find that hpol kappa binds G4 DNA with a 5.6‐fold preference over control, non‐G4 DNA. Results from pol extension assays are consistent with the notion that G‐quadruplexes represent a barrier to DNA replication by pols, and kinetic analysis reveals that activity of hpol kappa is diminished to 12% on highly stable G4 DNA substrates as compared to control, non‐G4 substrates up to ten nucleotides away from a G4 structure. These results are in contrast to a previous study from our group that showed the activity of another Y‐family pol member, hpol eta, is essentially unperturbed ten nucleotides from G4 DNA and can maintain >20% activity even when copying a quadruplex‐associated guanine. Nevertheless, experiments in HeLa cell culture have revealed that stabilization of G4 DNA structures with pyridostatin (PDS) resulted in a ~40% decrease in cell viability for cells depleted of hpol kappa by siRNA compared to scrambled siRNA treatment (P = 0.0455). In summary, our in vitro kinetic analysis does not support a model in which hpol kappa promotes direct bypass of G4 DNA. Instead, the sensitivity of hpol kappa‐deficient cells to PDS may be related to the role of hpol kappa in activation of the replication stress response through ATR‐mediated phosphorylation of Checkpoint kinase 1 (Chk1).

Support or Funding Information

This work was supported by NIH grants GM 084460 and CA 183895 (to R.L.E.).
In silico exploration of the mechanism of translesion synthesis by DinB
Authors: Perez Elevit , Benitez Benedetta Sampoli
Published: November 2023

In prokaryotic organisms, environmental stress elicits the up‐regulation of a variety of genes involved in the so‐called SOS response. One major aspect of this response is the increase of mutation frequency. Some of the mutations may confer unique characteristic to the organism that will be beneficial for adapting to the environment, therefore accelerating evolution during stress. Y‐family DNA polymerases are a class of error‐prone polymerases able to perform DNA translesion synthesis. E. coli DNA Polymerase IV (also called DinB from the name of the gene) is a Y‐family DNA polymerase implicated in stress‐induced mutagenesis. As other DNA polymerases, DinB has the general architecture of a right hand, with palm, thumb and fingers subdomains. In addition, Y‐family polymerases have an extra domain, namely the “little fingers” and have a more solvent accessible active‐site than their high‐fidelity replicative counterparts. Kinetics studies have shown that DinB is particularly efficient at bypassing damaged guanosines, such as the N2‐furfuryl‐dG, and even more efficient in lesion bypass on damaged DNA than in replication of undamaged DNA. Recent hydrogen exchange experiments have suggested that the protein undergoes a conformational change in the presence of certain DNA damages on the templating strand, but not others, implying a possible mechanism of bypass discrimination. However, the exact nature of such movement is not well characterized. Using molecular dynamics (MD) simulations we explored different DNA/protein/incoming nucleotides combinations to dissect the protein conformational change. We were also able to identify the important residues implicated in this process. All systems were prepared starting with the available crystal structure, solvated and equilibrated using CHARMM‐GUI. MD simulations were run for 10 ns with the NAMD simulation package and multiple runs were done for the same system to ensure reproducibility of the data.

Support or Funding Information

This work was partly funded by the Rose M. Badgeley Residuary Charitable Trust.
Endocrine Disrupting Chemical, Bisphenol‐A, Induces Breast Cancer Associated Homeobox Containing Gene HOXB9 Expression in vitro and in vivo
Authors: Deb Paromita , Bhan Arunoday , Hussain Imran , Ansari Khairul I. , Bobzean Samara A. , Saha Debabrata , I Perrotti Linda I. , Mandal Subhrangsu S.
Published: November 2023

HOXB9 is a homeobox‐containing gene that plays key roles in mammary gland development and is associated with breast and other types of cancer. As HOXB9 is expressed in hormone‐sensitive mammary gland development and is linked with control of steroid hormone regulated renin‐angiotensinogen system, here we study the mechanism of transcriptional regulation of HOXB9 upon exposure to Estradiol (E2), and Bisphenol A (BPA) which may suggest novel therapeutic targets for breast cancer treatment. The induction of HOXB9 expression upon exposure to E2 and BPA was observed by Q‐PCR both in vivo and in vitro. To identify a potent Estrogen Receptor Binding site in the HOXB9 promoter region, a dual luciferase reporter assay was conducted. ERs and ER coregulators that are recruited to the HOXB9 promoter region were examined by Chromatin Immunoprecipitation (ChIP) assay, followed by Q‐PCR. Here, we demonstrate that HOXB9 expression is transcriptionally regulated by E2, in vitro and in vivo. We also demonstrate that endocrine disrupting chemical BPA induces HOXB9 expression in cultured human breast cancer cells (MCF7) as well as in vivo in the mammary glands of rat. Luciferase assay showed that estrogen‐response‐elements (EREs) at HOXB9 promoter are induced by BPA. Estrogen‐receptors (ERs) and ER‐coregulators such as MLL‐histone methylases (MLL3) bind to the HOXB9 promoter EREs in the presence of BPA, modify chromatin (histone methylation and acetylation) and lead to gene activation. In summary, our results demonstrate that BPA exposure alters the epigenetic programming of the HOXB9 promoters leading to its endocrine disruption in vitro and in vivo.
Functional Role of UPF2 Phosphorylated Regions in Translation Termination in Yeast
Authors: Haddock Luis Antonio , Colón‐Morales Edgardo M , Lasalde Clarivel , Lin Qishan , González Carlos I
Published: November 2023

Eukaryotes have evolved a unique mechanism for recognizing premature termination codons (PTC) present in the mRNA. These PTC‐containing mRNAs are recognized and degraded by a mechanism known as nonsense‐mediated mRNA decay (NMD), which prevents the formation and accumulation of non‐functional or truncated proteins that can be the starting point for several human diseases, such as Cystic Fibrosis and Duchenne Muscular Dystrophy. One of the proteins involved in NMD is the up‐frameshift protein 2 (UPF2), which has been shown to be phosphorylated and essential for the accuracy of translation termination. However, the functional role of Upf2 phosphorylated residues in the translational termination accuracy is still unknown. To test this, we generated deletions of several UPF2regions containing phosphorylated residues and measured the growth rate of a Saccharomyces cerevisiae strain that harbors the can1‐100 allele in the presence of canavanine. Results demonstrated that deletion of the regions in Upf2 corresponding to amino acids (aa) 50–59 and 421–430, showed less growth due to a higher sensitivity to canavanine caused by a faulty translation termination. This suggests that these Upf2 regions are crucial for translation termination accuracy and provide a foundation for future studies to determine the role of Upf2 phosphorylation in the fidelity of translation termination in eukaryotes.
Eukaryotic translation initiation factor eIF4G utilizes different domains to coordinate with eIF4A, eIF4B and eIF4E in binding 3′ Cap‐independent translation element of Barley Yellow Dwarf Virus
Authors: Zhao Pei , Goss Dixie
Published: November 2023

Barley Yellow Dwarf Virus (BYDV) lacks a 5′ (7‐methyl guanosine) cap as well as a 3′poly A tail. Like many plant viruses, BYDV contains a cap independent translation element (CITE) in the 3′ untranslated region of the viral mRNA. BTE (Barley Yellow Dwarf Virus like cap‐independent element) is one of the well characterized CITEs. BTE mediated translation primarily depends on eukaryotic initiation factor eIF4G. BTE binds to eIF4G; however, the details of BTE initiated translation are still unclear. Three eIF4G deletion mutants with different domain organization were used to investigate BTE interaction with eIF4G: eIF4G601‐1196 is the eIF4G truncated fragment containing amino acids from 601 to 1196, including eIF4E, central eIF4A, eIF4B binding domains and the possible BTE binding region; eIF4G601‐1488 is a longer fragment with one additional C‐terminal eIF4A binding domain; eIF4G742‐1196 is a shorter deletion mutant lacking the eIF4E binding sequence. eIF4G601‐1196 binds BTE as efficiently as wild type eIF4G and supports translation. Translation initiation factor eIF4A and eIF4B with ATP (helicase complex) stimulate eIF4G601‐1196 binding with BTE but not eIF4G601‐1488, suggesting that the helicase complex function relies on the eIF4G central eIF4A binding domain, not the C‐terminal eIF4A binding domain. eIF4E, upon binding with eIF4G mutants which have eIF4E binding region, significantly increases the binding to BTE. These data suggest eIF4E has a significant role in eIF4G‐RNA binding even in the absence of the cap structure.

Support or Funding Information

NSF MCB 1513737