bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of Blood Cell Populations in the Development and Regeneration of a Colonial Ascidian
Authors: Houck Tyler , Boyd Megan , Balde Erick , Keeling Elena
Published: November 2023

Botrylloides violaceus is a colonial marine ascidian capable of undergoing whole body regeneration after removal of all tissues except the peripheral vasculature. As chordates, colonial ascidians are more closely related to vertebrates than other model organisms for whole body regeneration. There is evidence that one or more populations of stem cells are present in the circulating blood of B. violaceus; these cells may also be involved in asexual reproduction by budding. We are studying changes in blood composition during development and regeneration. Hemoblasts, a type of undifferentiated blood cell, have been implicated as stem cells in botryllid ascidians and are therefore of special interest. Comparisons of blood composition at several developmental stages are in progress. Preliminary data suggest that hemoblast composition of B. violaceus blood is stable during the regenerative process. Further, we show dramatically higher relative amounts of hemoblasts in the blood of B. violaceus compared to reported amounts in related species. This high percentage of hemoblasts may be associated with the readiness of B. violaceus to undergo regeneration at any stage of its life cycle.

Support or Funding Information

CSU Council on Ocean Affairs, Science & Technology (COAST); Biological Sciences Department, California Polytechnic State University
Potential Mechanisms of Action for Exogenous Ketone Enhancement of Ischemic Wound Healing in Young and Aged Fischer Rats
Authors: Kesl Shannon L , Wu Mack , Gould Lisa J , D'Agostino Dominic P
Published: November 2023

Chronic wounds represent an under‐acknowledged socioeconomic epidemic, affecting 1.8 million new patients per year and costing the US health care system upwards of $25 billion annually. This substantial cost is rapidly growing due to a disproportionate occurrence in the ever‐aging population. Ketone bodies, including β‐Hydroxybutyrate (βHB), are naturally occurring energy substrates. We previously reported that oral ketone supplementation without dietary restriction enhanced wound closure and increased blood flow in young and aged Fischer 344 rats. We hypothesized that exogenous ketone supplementation promoted wound healing via enhancement of physiological factors such as increasing proliferation, advancing migration, reducing ROS production, and resolving inflammation. Experiments in vitro with young and aged primary human dermal fibroblasts supplemented with 5mM βHB for 72 hours ahead of an oxidative stimulus (100μM tert‐butyl‐hydrogen peroxide) resulted in significantly decreased cellular ROS production, enhanced cell migration, and augmented cellular proliferation (p<0.05). Additionally, peri‐wound tissue lysates were isolated from 1,3 Butanediol (BD), βHB NA+/K+ salt mixed with MCT oil in a 1:1 ratio (BMS+MCT), and standard diet (SD) fed rats on day three and day seven post‐wounding. Ketone administration during wound healing elicited changes in inflammatory cytokines including a significant elevation in epidermal growth factor (EGF) at day 7 of wound healing compared to control (p<0.05). BMS+MCT supplementation in aged rats significantly decreased tumor necrosis factor‐ alpha (TNF‐α) levels on day 7 of wound healing compared to controls (p<0.05). Furthermore, ketone supplementation significantly reduced leptin levels in both young and aged rats. Harvested wound tissue demonstrated a change in immunofluorescence of the pro‐inflammatory (IRF‐5 labeled, M1) to anti‐inflammatory (CD206 labeled, M2) macrophage ratio and immunohistochemistry labeled CD31 blood vessel density markers compared to controls. We conclude that ketone supplementation in vivo and in vitro modifies systemic physiology to enhance wound closure.

Support or Funding Information

This study was supported by the Office of Naval Research (ONR) Grant N000140610105 (DPD), Scivation Inc, and a Morsani College of Medicine Department of Molecular Pharmacology and Physiology departmental grant.
Trabecular mapping of the human distal femur: Leveraging geometric morphometrics for studies of bone microstructure
Authors: Sylvester Adam David
Published: November 2023

Trabecular microstructure of limb bone epiphyses have been used to elucidate the relationship between skeletal form and locomotor behaviors among mammals. Such studies have often relied on the analysis of a single volume of interest (VOI) placed within the trabecular structure. Trabecular structure likely varies across continuous regions of bone and a single VOI will miss this variation, and much of the functional signal will be lost.

Here I present a method for evaluating variation in bone microstructure across articular surfaces by leveraging sliding landmarks to sample multiple VOIs. 1007 sliding semi‐landmarks were distributed across the articular surface of 10 human distal femora using standard geometric morphometric procedures. Landmarks were then used to locate the positions of 1007 VOIs in the trabecular structure immediately deep to the cortical shell. VOIs were extracted and trabecular properties were measured for each VOI using BoneJ. Two questions are addressed: 1) are microstructural variables homogeneous across the articular surface? and 2) if microstructure is not homogeneous, are values of microstructural variables patterned on the condyles in a way that reflects human walking?

Results suggest that trabecular microstructural variables are not homogenous across human femoral condyles. Bone volume fraction and trabecular thickness were greatest on the most inferior portions of the condyles, suggesting these areas reflect loading of the joint during normal walking. The technique presented here may have applications for reconstructing locomotion behavior of extinct animals as well as evaluating bone strength in clinical practice.
Cranial Index and Pattern of Head Shapes in Children Aged 0 – 36 months from Sokoto, Nigeria
Authors: Musa Muhammad A , Zagga Abdullahi Daudu , Danfulani Mohammed , Hamid Ahmed , Usman Jibrin Danjuma , Abdulhameed Aliu , Dahiru Mohammed
Published: November 2023

This study investigated the pattern of head shapes in children with normal brain development using cranial index values. Head Length (HL), Head Width (HW) and Cranial Index (CI) of 210 normal growing children (100 males and 110 females) aged 0 – 36 months old seen at the Institute of Child Health Center, Usmanu Danfodiyo University Teaching Hospital, Sokoto over the period of six months were determined using internationally accepted landmarks and procedures. The pattern of head shapes was investigated using the cranial index values obtained from the study participants. The overall mean and standard deviation values for head length, head width and cranial index were 13.79±1.79 cm, 10.95±1.39 cm and 79.49±3.42 respectively. A statistically significant increase (p = 0.0203) was observed in the head width of males when compared to that of females. Meanwhile, there was no statistically significant difference observed in the mean head length and mean cranial index values between the sexes. The dominant head shape type observed in males was Mesocephaly (31.90%) and Brachycephaly (26.19%) in females while the least observed head shape pattern was Hyperbrachycephaly (1.42%) in males and Dolicocephaly (0.47%) in females. The head measurements, cranial index and pattern of head shapes evaluated in this study are good indicators of skull and brain growth, thus can be of clinical importance in determining the timing and etiology of brain insults, especially in the first three years of life.

Support or Funding Information

Nil

Mean ± SD of Head Measurements and Cranial Index of Study Participants.

Head Measurements
N
Minimum Value
Maximum Value
Age Range (months)
Mean ± SD

Head Length (cm)
210
10.50
17.20
0–36
13.79±1.79

Head Width (cm)
210
8.30
14.30
0–36
10.95±1.39

Cranial Index (%)
210
68.39
89.29
0–36
79.49±3.42

Keys:

N ‐ Number of Study Participants

SD ‐ Standard Deviation

Comparison between the Overall Mean ± SD of Head Measurements and Cranial Index in Relation to Sex.

Head Measurements
N
Males (N = 100)
Females (N = 110)
p‐value

Head Length (cm)
210
14.30±1.53
13.33±1.89
6.0560

Head Width (cm)
210
11.18±1.15
10.73±1.55
0.0203*

Cranial Index (%)
210
78.26±3.15
80.61±3.27
2 9992

Keys:

N ‐ Number of Study Participant

Statistically significant difference between sexes

cm ‐ Centimeter

% ‐ Percentage
Morphometric Characterization of Maxillary and Mandibular Interdental Spaces at the Mucogingival Junction: Implications for Safe Orthodontic Miniscrew Insertion
Authors: Yang Jackie , Tassi Ali , Wilson Tim D , Hosein Yara , Galil Khadry
Published: November 2023

Interdental alveolar bony spaces of the maxillary and mandibular dental arches are common target site for orthodontic miniscrew insertion. The mucogingival junction is a clinically important anatomical landmark as miniscrews are inserted into the attached gingiva above it. Although numerous studies have measured interdental spaces to determine suitable minscrew insertion sites, none focus on the clinically relevant mucogingival junction. Further, there is little information regarding the potential relationship between craniofacial characteristics (linear mandibular measurement and dental arch shape) on size of the interdental spaces. The aim of this study is to assess the dimensions of interdental spaces at the mucogingival junction, and to investigate its relationship to the jaw size and dental arch shapes. Twenty bisected cadaveric heads were obtained from the Body Bequeathal Program in the Department of Anatomy and Cell Biology at Western University. Mandibular size of each specimen was measured using a digital caliper by two measurers on different days which demonstrated high interrater reliability for the linear mandibular measurements (r2= 0.99; p<0.001). Currently, the classification of the dental arch shape (oval, tapered, square) for each specimen using orthodontic templates is taking place. Lastly, the mandibular and maxillary arches will be isolated from the heads and cut just above the mucogingival junction towards crown of tooth along its transverse plane. A dissecting microscope will be used to measure interdental spaces (buccolingual and mesiodistal) and cortical bone thicknesses. It is expected that interdental spacing would be widest distally, and narrowest mesially. It is hypothesized that more tapered dental arches will exhibit smaller interdental spacing, while square dental arches would correlate to larger interdental spacing. The resulting data will benefit the orthodontists when deciding on safe placement site and size of orthodontic miniscrews.
Production Of Interleukin 1 Beta In Primary, Serum‐Free Cultures Of Human Osteoarthritic Chondrocytes
Authors: Lozano‐Torres Xiomara , Buckley Andrea Lynn , Miller Lawrence , Bundens Grace , Datillo Victoria , Milton LaBraya , Behling Kathryn , Chmielewski Sarah , Cho Ellen , Selim Abdulhafez , George‐Weinstein Mindy , D'Angelo Marina
Published: November 2023

Osteoarthritis (OA) is characterized by a loss of cartilage extracellular matrix (ECM) and an increase of cytokine production in the synovial joint. Notably, Interleukin‐1 beta (IL‐1β), elevated in OA cartilage, induces a number of responses from chondrocytes, including release and activation of matrix metalloproteases (MMPs). Enhanced degradation of the ECM, a hallmark of OA pathology, is likely due, in part, to increased cytokine production.

In this study, osteoarthritic human chondrocytes isolated from total knee arthroplasty were reared in three‐dimensional, serum‐free, alginate cultures. The sides of least and greatest pathology of the femoral condyles and tibial plateau were determined by gross inspection in the surgery suite and histological assessment. Medial and lateral cells isolated from femoral condyles and tibial plateau were cultured separately. Chondrocytes were plated at 2.5×106 cells per ml of alginate and incubated in 2 milliliters of serum‐free medium for five days. Conditioned media was collected on days 2 and 5 of culture, concentrated by centrifugation and IL‐1β measured by immunoassay.

IL‐1β production was detected in medial and lateral chondrocyte cultures; however, the concentration of this cytokine was highest in the cultures established from the region of greatest pathology. Collagens type I and II degradation products were also increased in cultures of cells from the side of greatest pathology. These results demonstrate a correlation between elevated levels of IL‐1 β, collagen degradation and extent of pathology in primary cultures that maintain the phenotype of human articular chondrocytes. This culture system provides a platform with which to further define the feedback loop between chondrocyte produced cytokines and MMPs in OA.

Support or Funding Information

Center for Chronic Disorders of Aging Pilot Grant, PCOM
An original Letter of Georges Cuvier to Charles Bell: 1832, january 24th about the models of anatomy in “papier mâché” by the Dr Auzoux
Authors: LE FLOCH‐PRIGENT PATRICE P , FROMANGER Marine , GILLOT Jean‐Bernard
Published: November 2023

A unique, original (1832, january 24th) letter written by Georges Cuvier to Charles Bell about the models of anatomy in “papier mâché” by the Dr Auzoux belonged in november 2015 to the Scientific Library Alain Brieux ‐Gillot in Paris. We have translated this letter in english and analyzed the relationships between Cuvier and Bell which were celebrities of their times. This presentation of Dr Auzoux who travelled in England in 1832 to the english intelligentsia was a great help for the international development of his young firm (Saint Aubin d'Escroville and Paris). His production in “papier mâché” largely dismantled (clastic anatomy) was mainly human, animal but also plant's anatomical models always in accordance with the scientific knowledge of his time.
Is Diabetic Glomerular Basement Membrane Thickening Hyperglycemia Driven? Or Is It a Part of the Normal Aging Process
Authors: Carlson Edward , Bleth Jordan , Miller Cory , Laturnus Donna , Young Laurie Kim , Schaff Elizabeth
Published: November 2023

Most microvascular basement membranes, including glomerular basement membranes (GBMs) are thickened in diabetes, and though the molecular mechanisms are elusive, hyperglycemia is nearly universally believed to play a major role. However, we and others have also shown that GBMs thicken with age, and in at least one spontaneous diabetic animal model (BB Wistar rats), GBMs thicken beyond normal age‐related thickening in the absence of hyperglycemia. Accordingly, in the current study, electron microscopic morphometric techniques were employed to determine whether age‐related or glycemia‐related GBM thickening may be the dominant parameter. Four murine genotypes including 150‐ and 450‐day‐old FVB background (normoglycemic), NMT, (transgenic normoglycemic), OVE (hyperglycemic), and OVE‐NMT (transgenic hyperglycemic) were prepared for TEM examination and determination of true GBM thickness by the “gold standard”, orthogonal intercept technique. Our data indicated that for age‐matched animals, increased glycemic level was a consistent predictor of increased GBM thickness. As examples, GBMs from both 150‐ and 450‐day‐old diabetic mice were approximately 24% thicker than age‐matched normoglycemic animals. Although these data strongly suggest that glycemia is an important parameter in GBM thickening, our unbiased morphometric analyses also show that regardless of blood sugar levels (including normoglycemics), substantially increased (88–100%) GBM thickening is recapitulated in 450‐day animals compared to glycemia‐matched 150‐day‐old mice. Likewise, numerous other cellular and extracellular parameters are augmented by increased duration, and are amplified further by glycemic levels. Alternatively, these parameters may be diminished by curtailed time and/or mitigated by reduced oxidative stress. In summary, both time and level of glycemia are major factors in early GBM thickening, and both must be considered in any therapeutic intervention in the progression of diabetic or non‐diabetic basement membrane disease.
THE ANATOMY OF PEDIATRIC LAPAROSCOPIC AND THORACOSCOPIC PROCEDURES ‐ NEWER CONCEPTS AND PROCEDURES
Authors: Ghosh Tamojit
Published: November 2023

Laparoscopic procedures in pediatric surgery can be be divided grossly into abdominal and thoracic procedures. The basic procedures in laparoscopy require an understanding of body wall surface and muscle anatomy. Laparoscopic pediatric procedures have revolutionized the treatment of tracheo‐oesophageal fistulas with oesophageal atresia, duodenal atresia, anorectal malformations, appendicitis, hirschsrpung disease and inguinal hernias.

Abdominal laparoscopy requires gas insufflation, the use of surface ports in various sites and and a keen understanding of upper and lower abdominal anatomy. Iatrogenic abdominal injuries include vascular trauma, retroperitoneal heamorrhage, bladder injuries and stomach injuries especially when it is overinflated. Most procedures use a camera port with 2 or more ports for instrumentation. Lower abdominal laparoscopies have a risk of hernias, damage to the inferior epigastric blood vessels and bowel damage. A detailed knowledge of the abdominal body wall musculature and underlying vascular structures is required. The anatomy reinforcing single port laparoscopy procedures is presented in this work too. Transuterine and cul‐de‐sac approaches are revisited and explained.

Thoracospic pediatric surgical procedures have a risk of injury to the lung, pleura and thoracic wall vasculature. Newer anatomical approaches through the posterior thoracic wall ports are also proposed and explained.

To correct spinal and vertebral defects newer sites of laparoscopic port entry are proposed with an eye to access and maximum utilization of small working space in such procedures.

Support or Funding Information

Funding received from the Ministry of Health, Saudi Arabia and the Indian Institute of Morphology, Kolkata.
An expansion of the anatomy of the anterolateral ligament of the knee: Morphological variation and an alternate dissection approach
Authors: Parker Matthew F , Smith Heather F
Published: November 2023

In 2013, Claes and colleagues documented and described in detail the anterolateral ligament (ALL) of the knee, demonstrating its existence in 97% of their cadaveric samples (40 out of 41). In this study, we sought to further examine the anatomy of this ligament, document its morphological variation in an additional sample of cadaveric knees, and assess the feasibility of its dissection in preserved cadaveric specimens.

We dissected the knee joints of 53 (26m/27f) preserved cadaveric knees (paired, when possible), and documented the presence/absence, course, and morphological characteristics of the ALL, as well as collected quantitative data on its length and width. The majority of specimens in our sample (51 out of 53 = 96%) were found to possess a discrete ALL. In our sample, we found support for the hypothesis that the primary function of the ALL is to limit internal (medial) rotation of the tibia. However, we discovered noticeable variation in the insertion of the ligament compared to the originally described pattern. Most notably, it was often found to insert more laterally than has been previously described, closer to the fibular head. This variant would likely result in decreased rotational stability of the knee in individuals with this pattern of insertion. A paired‐samples t‐test revealed highly significant differences between the length of the ALL at extension compared to 90 degrees flexion (p < 0.001), supporting the idea that the ALL is active during knee flexion.

In addition, the originally described dissection technique for identifying and following the ALL requires flexion of the knee, a state which is often not possible in stiff, preserved cadavers. We confirmed the feasibility of an alternate dissection technique in which the quadriceps femoris tendon is incised, for use on specimens in which flexion of the undissected knee is not possible. This technique would be useful for dissection of preserved cadavers used in gross anatomy laboratories, and we discuss the applications of such an approach in student‐led dissections.

Support or Funding Information

This research was supported by Midwestern University faculty start‐up funds to HFS.
Case Report of a Bilateral Absence of the Semimembranosus Muscle
Authors: Morrison Nina , Hervey Shane , Runyeon Jon
Published: November 2023

Bilateral absence of the semimembranosus muscle was discovered during a routine dissection of a 62‐year‐old Caucasian male cadaver. The origination of the semimembranosus muscle is from the posterior superior lateral quadrant of the ischial tuberosity of the pelvis and its insertion is into the posterior surface of the medial condyle of the tibia[2]. Semitendinosus and biceps femoris muscles were observed bilaterally in the posterior compartment of the thigh. The dissection presented hypertrophy in the remaining hamstring muscles—the semitendinosus and biceps femoris. Congenital absence of skeletal muscle in the lower extremity is rare, as few have been reported previously in the literature [1, 2, 3]. Knowledge of muscle variation is important to not only anatomists, but also many types of clinicians when diagnosing and treating patients.

Support or Funding Information

Department of Human Physiology

University of Oregon
Anatomical Variation and Morphological Relationships of the Human Pharyngeal Recess: Clinical Implications
Authors: Sorrento Cristina Maria , Ramalanjaona Benjamin , Pagano Anthony , Lawson William , Márquez Samuel
Published: November 2023

The pharyngeal recess (of Rosenmüller) has been identified as the most common site for development of nasopharyngeal carcinomas, a disease involving pathological change of stratified squamous epithelial cells lying in the cleft between the torus tubarius and the superior pharyngeal constrictor muscle. The morphological relationships of the lateral pharyngeal recess are of great clinical importance to the otolaryngologist. This study measured variation in pharyngeal recess depth, torus tubarius width, width of the medial and lateral laminae of the Eustachian tube at its pharyngeal orifice, and choanal height between hormion and the posterior nasal spine. Donor heads from NYU School of Medicine anatomy course were bisected (n=16, 9 males and 7 females). A Shapiro‐Wilk test of normality was performed on the pharyngeal recess depth values and a non‐significant result was yielded, confirming that the data appeared normally distributed. Next, the correlation of each measure to pharyngeal recess depth was calculated and the only strong correlation was with medial lamina width scaled over total torus tubarius width (r=−0.6120, P=0.012; both measured at the level of the roof of the cartilaginous Eustachian tube pharyngeal orifice). However, the one individual that exhibited a near lack of a pharyngeal recess also possessed a ratio of medial lamina width/torus tubarius width that was identical to another individual with one of the deepest recesses. Males and females did not exhibit any significant (P<0.05) differences in recess depth when scaled over torus tubarius width. These results indicate that variation in pharyngeal recess depth is not gender determined but rather related partially to the dimensions of the torus tubarius and cartilaginous Eustachian tube laminae. Further morphometric analysis is required to fully explain all of the factors influencing pharyngeal recess depth.
The relationship between the parotid fascia and the deep fascia of the infraorbital region: a new concept in descriptive and applied anatomy
Authors: Amodeo Chiara Andretto , Delmas Vincent , Casasco Andrea , Keller Gregory S
Published: November 2023

This study aims to demonstrate that a relationship exists between the parotid fascia and the deep fascia of the infra orbital region, recently described by the first author, that continues from the temporal region and covers the mimetic muscles that lift the upper lip.

Methods

24 hemifaces were dissected to show the relationship between these two deep fascial layers, a preauricular incision was performed, the Superficial musculo‐aponevrotic layer was dissected and reflected. The dissection was then performed deep to the parotid fascia to reach the anterior border of the parotid gland.

Another incision was placed in the temporal area, the temporoparietal fascia was elevated, the superficial layer of the deep temporal fascia was incised and dissected toward the zygomatic arch and the infraorbital region.

Results

in all specimens a relationship was present between the parotid layer and the superficial layer of the deep temporal fascia, that continued from the temporal region to the infraorbital one, after crossing the zygomatic arch.

The two layers were in continuity in the superior third of the parotid gland, at the anterior border of the gland where the branches for the zygomaticus major, minor and the other muscles that lift the upper lip leave the gland to reach the muscles.

Conclusions

a relationship exists between the deep fascia of the infraorbital region, this relationship was observed in the superior third of the anterior border of the parotid gland at the level of the emergency of the branches of the facial nerve for the muscles that lift the upper lip and it was connected to the ligaments present in this area.
Subcutaneous tissue depth over intraosseous infusion sites in a cadaveric model
Authors: Glazier Megan , Reed James Christopher
Published: November 2023

In an emergency setting, it is important to be able to quickly and safely deliver life‐saving fluids and medications to the patient. This is achieved by introducing a peripheral intravenous (IV) catheter into one of the patient's peripheral veins. When emergent intravenous access is not available an intraosseous (IO) infusion is performed. To accurately perform an IO infusion the provider must precisely palpate and identify the associated anatomical landmark for correct placement. A thicker layer of subcutaneous tissue over the insertion site can make this process difficult, leading to an increased risk of misplacement. Misplacement of an IO needle subsequently can lead to extravasation and compartment syndrome if left unchecked. The purpose of this study was to relate subcutaneous tissue depth with body mass index (BMI) and percent body fat in order to better understand IO placement sites in relation to these body composition factors. Presented here are the results of an systematic examination of 22 cadavers, representing ages 54–95 and both sexes for IO sites with the least tissue indicating the most desirable locations to perform IO infusion. Using height and weight, BMI was calculated and skinfold measurements were taken to detemine percent body fat. Subcutaneous tissue depth was determined by taper gauge measurements and then used to correlate depth and body composition factors.
Prevalence of pathology in the cadaveric heart, lungs, liver and kidneys
Authors: Pontiff Mattie , Rae Guenevere
Published: November 2023

The purpose of this project was to investigate the macroscopic and microscopic appearance of pathology of the heart, lung, kidney and liver in the cadaver population. In addition to the description of the common pathologic incidences, this project also served to separate the characteristics into three categories pertaining to the state of cadaveric tissue: normal, abnormal or artifact. Cadaveric organs (n= 39 hearts, 97 lungs, 76 kidneys and 44 livers) were grossly and histologically examined. The gross characteristics that appeared abnormal were statistically correlated with the histologic confirmation of pathologic changes in the tissue. The significant correlations between the gross and histologic observations indicate the most common pathologic incidences that can be grossly observed are: replacement scarring, interstitial fibrosis and necrosis (heart); chronic obstructive lung disease, acute bronchopneumonia and plexogenic arteriopathy (lung); glomerulosclerosis, arteriosclerosis and acute tubular necrosis (kidney); portal fibrosis, fatty changes and cirrhosis (liver). Correlative analysis was used to determine the presence of artifact in cadaveric organs caused by the embalming process that may resemble pathologic states such as discoloration of both the myocardial wall and cortical surface of the kidney. These results indicate that there are certain disease conditions that are prevalent among the cadaver “population” that may be used for clinical, teaching and research purposes.
Olive Leaf Metabolites as Covalent Human Topoisomerase II Poisons
Authors: Vann Kendra R , Sedgeman Carl A , Gopas Jacob , Golan‐Goldhirsh Avi , Osheroff Neil
Published: November 2023

Topoisomerase II plays many essential roles in genome maintenance. To carry out its physiological functions, the enzyme generates transient double‐stranded breaks in the DNA to help resolve topological problems that naturally occur. Thus, while essential to cell survival, topoisomerase II has the potential to fragment the genome. Beyond its critical cellular functions, human topoisomerase II is the target for a number of widely prescribed anticancer drugs that act by increasing levels of enzyme‐mediated DNA strand breaks. Many of these “topoisomerase II poisons” are derived from natural sources. Identification and characterization of topoisomerase II poisons from natural sources can provide novel drug scaffolds. Therefore, a series of water‐soluble extracts from Mediterranean plants were screened for activity against human topoisomerase IIα. An extract from Phillyrhea latifolia L., a member of the olive tree family, significantly enhanced DNA cleavage mediated by human topoisomerase IIα. P. latifolia L. contains a number of phenolic metabolites, including antioxidants associated with several human health benefits. Oleuropein, hydroxytyrosol, and verbascoside enhanced topoisomerase IIα‐mediated DNA cleavage ~4‐fold. Analysis of topoisomerase II‐mediated DNA cleavage carried out in the presence of oleuropein, hydroxytyrosol, and verbascoside indicated that these compounds displayed hallmark characteristics of “covalent topoisomerase II poisons.” First, the ability of the metabolites to poison topoisomerase II was enhanced substantially in the presence of an oxidant, but was abrogated in the presence of a reducing agent. Second, the metabolites required the N‐terminal region of topoisomerase IIα to enhance DNA cleavage. Third, the metabolites inhibited topoisomerase II activity when incubated with the enzyme prior to the addition of DNA. Phenolic olive plant metabolites retained the ability to act as topoisomerase IIα poisons in complex dietary herbal formulations, such as commercial olive leaf extracts and extra virgin olive oils. These results strongly suggest that olive plant metabolites can act as dietary topoisomerase II poisons. These studies may provide a platform for developing new anticancer or chemopreventative therapies. Current studies are focused on further elucidating the interaction of covalent poisons with topoisomerase II. Results from ongoing mutagenesis studies may provide additional insight into the mechanism by which covalent poisons interact with human topoisomerase II.

Support or Funding Information

GM033944, GM062459, GM08320, ICA (Israel), Deutsche Forschungsgemeinschaft, and R. H. Holzer Foundation.
A nucleolar role for the Fanconi anemia pathway protein, FANCI, in rDNA transcription
Authors: Sondalle Samuel B. , Longerich Simonne , Sung Patrick , Baserga Susan J.
Published: November 2023

Fanconi anemia (FA) is a disease of DNA repair because proteins mutated in patients with FA are impaired in removing interstrand DNA crosslinks. We propose a new nucleolar role for the FA protein, FANCI, in ribosome biogenesis based on extensive preliminary data. Ribosome biogenesis is the process of making ribosomes that initiates in the cell nucleolus with the transcription and processing of the pre‐ribosomal RNA (rRNA) followed by the assembly of mature ribosomes. Immunoaffinity purification of FANCI from HeLa cell nuclear extract followed by mass spectrometry revealed co‐purification with nucleolar proteins required for ribosome biogenesis, including PES1. Using subcellular fractionation, we have found that FANCI is indeed enriched in the nucleolus. In particular, the deubiquitinated form of FANCI is by far the most predominant form of nucleolar FANCI, even under conditions of DNA damage by mitomycin C, which stimulate monoubiquitination of FANCI. Immunoprecipitation revealed that deubiquitinated FANCI, but not its binding partner in DNA repair, FANCD2, is co‐immunoprecipitated by the nucleolar protein, PES1. To probe a possible nucleolar function for FANCI, we depleted FANCI in human cells and observed reduced transcription by RNA polymerase I (RNAPI). In addition, FANCI is co‐immunoprecipitated by the large subunit of RNAPI, indicating a physical and functional role for FANCI in efficient pre‐rRNA transcription. Finally, our results demonstrate novel regulatory mechanisms by deubiquitinases on the ubiquitination state of FANCI in the nucleolus. While our model does not contest the well‐established underlying DNA repair defect observed in cells from FA patients, it does suggest a dual pathophysiology for FA that includes defects both in DNA repair and in ribosome biogenesis.

Support or Funding Information

This work is funded by the NIGMS and NCI.
Participation of Distal RRRY Motif in Strand Displacement DNA synthesis by A‐Family DNA Polymerases
Authors: Modak Mukund J , Ketkar Amit , Singh Kamal
Published: November 2023

Strand displacement synthesis of DNA (SDSD) is an essential activity during Okazaki fragment maturation process. We have previously shown that residues in fingers subdomain and RRRY motif of E.coli DNA polymerase I participates in SDSD and 3′ – 5′ exonuclease activity, respectively (J. Biol. Chem. 2008, 283: 17979–17990). We further investigated the function of the conserved RRRY motif in the function of DNA polymerase I enzymes with or without associated 3′‐5′ exonuclease activity. Using DNA polymerase I from M. tuberculosis, which lacks 3′ – 5′ exonuclease activity and E.coli DNA polymerase I, which contains the 3′ – 5′ exonuclease activity, we now show that the RRRY motif is essential for SDSD. A detailed biochemical properties of individual arginine to alanine mutant enzymes showed that the first and the third arginine (R796 and R798) in M. tuberculosis polymerase I are essential for the SDSD, while, the 2nd arginine (R797) and 4th tyrosine (Y799) are not required. Similar properties were displayed by the mutant derivatives of equivalent residues in E.coli polymerase I. The loss of strand displacement DNA synthesis activity resulted from the significant reduction in the DNA binding affinity by mutant enzymes. Two most affected mutant enzyme species were further found to be defective in the formation of the enzyme‐DNA‐dNTP ternary complexes. We further find that displaced strand cleavage is mediated by 3′ – 5′ exonuclease activity of E.coli DNA polymerase I, in RRRY‐dependent manner. Our data, for the first time, demonstrate dual function of a conserved motif despite its distal location from the active sites.

Support or Funding Information

None
The Roles of Telomerase in Regeneration during Aging and the Telomeric DNA Sequence Identification in Aeolosoma viride
Authors: Chen Chi Fan
Published: November 2023

Telomeres are the functional chromosome elements that protect the ends of eukaryotic linear chromosomes from degradation and fusion. Incomplete telomere DNA replication results in length shortening during every round of cell division. On the other hand, the enzyme telomerase can extend the ends of telomeres and prevent this situation to happen in certain tissues. With essential function for genome integrity and cell survival, it has been reported that short telomeres and defective telomerase activity are involved in several age‐related diseases. Moreover, recent studies suggest that telomerase may be important for tissue renewal and regeneration after injury. Aeolosoma viride is a kind of fresh water annelid. It has strong ability in regeneration and is easy to manipulate for experiments. After anterior four segments decapitation, A. viride can regenerate its lost parts within five days. However, the regeneration successful rate decreases when the animal gets older and older. The regulation of telomerase gene expression in regeneration and how aging affecting this process are not well understood, so we would like to explore this mysterious aspect. In this study, we cloned the gene of telomerase protein component, avi‐tert, from our A. viride transcriptome database. The results of in situ hybridization and quantitative PCR showed that avi‐tert would express at the regenerating site, the blastema, where cell proliferation is undergoing. Besides, to identify the telomere repeat sequence, we searched the telomerase database (http://telomerase.asu.edu/) and chose two possible sequences, which are TTAGGG and TTAGG, according to the phylogenetic relationship of our species. Then, we extracted the genomic DNA for dot blotting assay and terminal restriction fragment (TRF) analysis. The results suggested that the telomere sequence of A. viride is most possible to be “TTAGGG” (same as human's) rather than “TTAGG” (in some invertebrates). In the next step, we will measure the gene expression profile in the animals of different ages and try to determine whether there are any changes during regeneration process.
Human Y‐family DNA polymerase kappa is more tolerant of amino acid substitutions in the active site loop region than its E. coli ortholog DinB
Authors: Antczak Nicole M , Ippoliti Paul , Walsh Jason , Packer Morgan , Beuning Penny
Published: November 2023

More than 100,000 lesions are generated on DNA in each cell per day due to such agents as reactive oxygen species and ultraviolet radiation. Y‐family DNA polymerases are specialized to carry out translesion synthesis by replicating damaged DNA and are conserved in all domains of life. There are two Y‐family polymerases in E. coli, DinB (Pol IV) and UmuD′2C (Pol V), one in the archaeon Sulfolobus solfataricus, Dpo4, as well as several in humans including Pol κ (hPol κ), which is a DinB ortholog. Both DinB and hPol κ are known to bypass minor groove adducts of guanine whereas Dpo4 can bypass both minor and major groove adducts. Using primer extension assays on DNA substrates containing the minor groove adduct N2‐furfuryl‐dG as well as undamaged templates combined with site‐directed mutagenesis to construct polymerase variants, we found that loop 1 residues of DinB near the catalytic site are important for activity as well as the selection of the correct nucleotide. There is a similar trend in the loop 1 region of hPol κ. In single nucleotide incorporation assays, wild‐type and variants of both polymerases show a tendency to insert incorrect dT as well as the correct dC opposite the N2‐furfuryl‐dG adduct. This is also seen for wild‐type and variants of hPol κ on an undamaged DNA template. These observations suggest that the loop 1 region of both polymerases is important in correct nucleotide incorporation during translesion synthesis. Furthermore, although both DinB and hPol κ are inhibited by major groove modifications, hPol κ is more active than DinB on DNA with major groove adducts. In order to probe this further, we used primer extension assays with DNA substrates containing the minor groove adduct N2‐furfuryl‐dG, major groove adduct N6‐furfuryl‐dA, as well as undamaged templates, with loop swap constructs of DinB and hPol κ, we found the loop regions of DinB and hPol κ near the catalytic site are important for bypass of different types of DNA damage and insertion of the correct nucleotide. Specifically, hPol κ is more tolerant of substitutions in its active site loops than DinB is. By creating loop swaps, an increase in the understanding of the preferential bypass of minor groove adducts by DinB and hPol κ can be accomplished.

Support or Funding Information

Support from American Cancer Society Research Scholar Grant RSG‐12‐161‐01‐DMC.
Age‐related epigenetic modifications contribute to dysfunction of male germline progenitor cells
Authors: Lee Tin‐Lap , Liao Jinyue , Chan Wai‐Yee
Published: November 2023

Ageing is a complex time‐dependent biological process that takes place in every cell and organ, eventually leading to degenerative changes that affect normal biological functions. In the past decades, the number of couples to conceive children at advanced ages has increased significantly. It is widely recognized that advanced maternal age poses higher birth defect risk due to oocyte ageing. However, the effect of advanced paternal age on male germ cell quality is largely overlooked, and the impact has been implicated in a variety of disease conditions affecting the offspring. Mammalian spermatogenesis is a tissue‐specific, stem cell‐dependent process that relies on self‐renewal and differentiation of male germline stem cells to support continual production of spermatozoa. Currently, the mechanisms regulating these processes remain largely unknown, and emerging evidence has suggested that epigenetic profiles shift dramatically during aging. Epigenetic modifications on DNA methylation and demethylation pathways through formation of 5‐methylcytosine (5mC) and 5‐Hydroxymethylcytosine (5hmC) have profound influences on developmental programs, but its role remains elusive in male germ cell development. In this study, we examined the dynamics of DNA methylation and gene expression in undifferentiated and differentiating cell populations in early male germ cell development by performing RNA‐seq and whole genome 5mC/5hmC profiling at single‐‐base resolution. We defined signaling and transcriptional difference involving key self‐‐renewal pathways in undifferentiated and differentiated cell populations and found that differentially methylated genes were enriched in various age‐related cellular developmental processes. We also observed methylation changes associated with OCT4 and GDNF, which are both important for undifferentiated spermatogonia maintenance. Finally, 5hmC was enriched specifically in proximal upstream and downstream regions relative to TSS and its presence correlates positively with gene expression, we found genes enriched in undifferentiated populations display higher promoter 5hmC levels, indicating 5hmC is associated with stemness‐‐related genes and stem cell capacity.

Support or Funding Information

Hong Kong Research Grant Council (RGC) ‐General Research Fund project no. 469713, 468312
Regulation of kcnd2 by microRNA‐30e in rat trigeminal ganglions following chronic constriction injury of infraorbital nerve
Authors: Dong Yingchun
Published: November 2023

Background

Trigeminal neuralgia is a severely debilitating orofacial neuropathic pain and is difficult to treat since the pathophysiology is not well clear.

Objective

The present study aimed to detect the expression of voltage‐gated potassium channel gene Kv4.2 (kcnd2) and microRNA‐30e in rat trigeminal ganglions (TGs) after chronic constriction injury of infraorbital nerve (ION‐CCI), and explore the mechanism of the decreased expression of kcnd2 under microRNA‐30e regulation.

Method

The rats are divided randomly into normal control and ION‐CCI group. The expressions of kcnd2 and microRNA‐30e were detected in rat TG. Luciferase assay was performed to detect whether the expression of kcnd2 was regulated directly by microRNA‐30e.

Result

The expression of kcnd2 was decreased while microRNA‐30e expression was increased significantly in ipilateral TG after ION‐CCI compared to the control. The decreased expression of kcnd2 is negatively correlated with the increased microRNA‐30e expression and positively correlated with the head withdrawal threshold reflecting pain. Luciferase assay showed that binding of microRNA‐30e to the 3′UTR of kcnd2 gene suppressed the transcriptional activity, and downregulation of microRNA‐30e significantly increased the kcnd2 mRNA level in F11 cells.

Conclusion

Our results show that microRNA‐30e is negatively correlated with the development and maintenance of orofacial neuropathic pain after ION‐CCI via regulating the activity of kcnd2. MicroRNA‐30e may be a target for the therapy of trigeminal neuralgia.

Support or Funding Information

National Natural Science Foundation of China (No. 81100768) and Key Project supported by Medical Science and Technology Development Foundation, Nanjing Department of Health (No. YKK11040; QRX11123).
ACA11, a Small Nucleolar RNA Activated in Multiple Myeloma, Stimulates Proliferation by Inactivating NRF2 and Increasing Redox Signaling
Authors: Mahajan Nitin , Weber Jason D. , Maggi Leonard B. , Tomasson Michael H
Published: November 2023

Multiple myeloma (MM) is a malignancy of antibody secreting plasma B cells. The t(4;14) translocation is detected in 20% of MM and is associated with shortened patient survival. Previously, we identified a novel orphan box H/ACA class small nucleolar RNA (snoRNA), that is located within intron 18–19 of the MMSET gene, and is expressed co‐transcriptionally with MMSET in MM cells harboring the t(4;14) translocation. H/ACA small RNAs are an evolutionarily conserved class of noncoding RNAs (ncRNA) involved in a diverse range of processes including posttranslational modifications, pre‐ribosomal RNA processing, and telomere maintenance. RNA‐Seq analysis performed on CD138+ tumor cells isolated from t(4;14) positive and negative MM patients revealed a unique signature of upregulated genes implicated in mitochondrial function and oxidative phosphorylation. We hypothesized that ACA11 helps to drive MM by increasing oxidative stress. We found that acute overexpression of ACA11 led to the increased levels of reactive oxygen species (ROS) in human myeloma cell lines, human foreskin BJ fibroblasts, murine fibroblasts and primary splenic B‐cells. Cells overexpressing ACA11 showed enhanced cell proliferation, colony formation, and elevated ERK1/2 phosphorylation, a downstream consequence of oxidative stress. In addition, nuclear factor erythroid‐derived 2‐related factor 2 (NRF2), which regulates anti‐oxidant signaling, was inactivated in ACA11 overexpressing cells. Together our results suggest that overexpression of ACA11 suppresses the ability of NRF2 to induce target anti‐oxidant genes, resulting in increased oxidative stress and ultimately cell proliferation. These ACA11‐dependent changes likely contribute to the malignant phenotype in cancers that overpass the MMSET gene.

Support or Funding Information

The work is supported by NIH R01‐CA174743 to MHT and LBM