bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparisons of Forelimb Muscular Anatomy in Humans, Macaques, and Chimpanzees: An Evolutionary Perspective
Authors: Kenyon Brittany , Eriksen Amandine , Sirianni Joyce E.
Published: June 2021

The forearm extensor muscles are physiologically organized into three functional groups; one group is made up of muscles that extend and abduct or adduct the hand at the wrist. Two of these muscles are Extensor Carpi Radialis Longus (ECRL) and Extensor Carpi Radialis Brevis (ECRB). Both originate from the distal end of the humerus; ECRL inserts onto the base of metacarpal (MC) 2 and ECRB inserts onto the base of MC3. Previous research has demonstrated that 24% of human forearms include an extra extensor muscle, which has important clinical implications.

Here, we test the hypothesis that this extra extensor muscle is present in a quarter of human forearms, but not in other primates due to locomotor constraints. Our sample includes prosected human (n=25), macaque (n=90), and chimpanzee (n=2) cadavers. All cadavers (n=117) were surveyed for the presence or absence of accessory extensor muscles, noting the origin and insertion of extra muscles.

Results indicate the presence of an additional extensor, most likely Extensor Carpi Radialis Accessorius (ECRA) in 5% of the human sample, and complete absence in both the macaque and chimpanzee samples. Given the similar anatomy among these three species, it is noteworthy that accessory extensor muscles do not appear in non‐human primate forearm muscles, especially in the chimpanzee, which is closely related to humans. However, based on patterns in human evolution, these results are somewhat expected. Throughout primate, including human, evolution, locomotor patterns have changed and allowed for adaptations in the wrist and forelimb. Macaques are terrestrial quadrupeds and thus have stable wrists. Brachiating animals, like chimpanzees, also have stable wrists to support their body weight while suspended. Both of these locomotor patterns are reflected in the musculoskeletal anatomy of these animals. Humans have evolved more flexible wrists, since their forearms are unnecessary for bipedal locomotion. Thus, as a byproduct of primate evolution, species such as humans develop extra extensor muscles for use in tool making and other needs, whereas other primates do not in order to maintain wrist stability for more important demands like locomotion.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vertebral Regionalization Facilitates Functional Diversification of the Mammalian Axial Skeleton
Authors: Jones Katrina Elizabeth , Angielczyk Kenneth , Pierce Stephanie
Published: June 2021

The mammalian vertebral column is subdivided into semi‐autonomous modules – regions – which vary strongly in morphology along the column and between species. Although regional differentiation is an important component of the mammalian bauplan, very little is known about the impact of regionalization on vertebral function, nor the evolutionary steps which underlie its origin. To examine the link between regionalization and function, we conducted ex‐vivo biomechanical testing on presacral intervertebral joints of two species: Felis catus – a mammal with strongly‐defined vertebral regions, and Salvator merianae – a lizard with poorly‐defined regions. Regionalization patterns in biomechanical parameters (e.g., stiffness, range of motion) were compared to those in vertebral morphology to test the hypothesis that vertebral regionalization facilitates functional diversification of the axial skeleton.

Results indicate tight correlations between presacral morphological and functional regions in Felis. By contrast, subtle morphological regions in Salvator were only associated with transitions in vertebral function at the cervico‐dorsal boundary. Further, form‐function relationships along the column were stronger in Felis and were highly differentiated between vertebral regions. Taken together, these results suggest that evolutionary differentiation of presacral regions in mammals has resulted in both functional diversification and enhanced plasticity in form‐function relationships in the axial skeleton.

Support or Funding Information

AAA Postdoctoral fellowship, NSF EAR 1524523

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential growth of seals and sea lions with paedomorphic and peramorphic adult skull morphologies
Authors: Asquino Nicole A. , Dewar Eric W.
Published: June 2021

Links between behavior and skull morphology have been widely demonstrated in carnivorans. The skulls of more‐aggressive species tend to be more peramorphic, with greater crest development for the emplacement of muscles, compared to the more paedomorphic rounded skulls of less‐aggressive species. Are these two endpoints the result of differential growth patterns in dimorphic species?

To answer this, we looked at skulls of seals and sea lions (n = 58, including 22 males, 14 females and 22 unknowns, usually juveniles) across 8 species. We categorized the individuals into ages from immature (teeth erupting, less fused sutures, smaller size), sub‐adult (teeth erupted but not worn, more sutural fusion), and adult (teeth with wear, fully‐fused sutures, and larger size). We compared these individuals by observing the placement of 26 bony landmarks using a geometric morphometric approach.

We found that paedomorphic adults have juvenile‐looking skulls, e.g., a short or absent sagittal and nuchal crest, a narrow zygomatic width, and shorter skull length between the braincase and rostrum. Peramorphic adults had more growth resulting in traits including taller sagittal and nuchal crests, a wider zygomatic width, and a longer skull length. The brain case varied little between paedomorphic and peramorphic end points. The increase in skull length happens first through growth, and the width of the zygomatic arch and height of crests increases later, likely in response to the increase of size of the jaw‐closing muscles.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endocast of the Paleocene Carnivore Dipsalidictis krausei (Mammalia, Creodonta)
Authors: Ahrens Heather E , Passero Marita
Published: June 2021

The North American mammal fauna experienced dramatic turnover during the Paleogene Period; of note, was the decline and extinction of the once dominant carnivore clade Creodonta and simultaneous diversification of Carnivoramorpha. The role of Carnivoramorpha in the extinction of Creodonta is unclear, though many authors have hypothesized that creodonts were outcompeted by carnivoramorphan taxa. In particular, some have suggested that carnivoramorphans had a higher degree of encephalization, as well as other adaptations of the brain, that may have contributed to their success over creodonts. Given that the endocranial anatomy of creodonts has not been examined since in the mid‐twentieth century, our objective was to describe the endocast of the exceptionally‐preserved, late Paleocene creodont Dipsalidictis krausei. Our new digital endocast was then compared to previously described endocasts of several Paleogene carnivores, including Protictis schaffi, Thinocyon velox, Tritemnodon agilis, and Vulpavus palustris. The midbrain of Dipsalidictis krausei was exposed dorsally and the associated expansion of the forebrain had not reached the same extent as observed in modern carnivorans. However, the expansion was similar to that of the later Eocene creodonts, Thinocyon velox and Tritemnodon agilis, as well as the Paleocene carnivoramorphan Protictis schaffi. Within the forebrain, the olfactory bulb and piriform lobe were relatively large, potentially indicating a greater reliance on olfactory signaling in Dipsalidictis krausei even than other carnivores. This first complete endocast of the creodont clade Oxyaenidae, demonstrates that oxyaenid neuroanatomy was similar to other early Paleogene carnivore species. Further, this suggests that early members of those clades retained a plesiomorphic condition and that divergence in encephalization and neuroanatomy occurred later in time.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence for Morphological Constraint on Bony Ear Canal Length in Modern Humans
Authors: Smith Christopher M. , Laitman Jeffrey T.
Published: June 2021

Throughout hominin evolution there is a trend towards a decreased length of the ear canal. This change in length is likely due to many factors that relate to reorganization of the cranial base among hominins with a more anteriorly placed foramen magnum, coronally oriented petrous pyramids, and an enlarged brain compared to extant African great apes. Functionally, ear canal length has been postulated to influence the detection of certain sound frequencies and may also alter the accuracy of sound localization. Therefore, a change in length of the ear canal could have effects on sensory perception throughout the hominin clade. However, the factors driving this change are rather unclear.

This preliminary study tests four null hypotheses stating there is no statistically significant correlation between the length of the bony ear canal and: 1) foramen magnum position, 2) foramen magnum width, 3) petrous pyramid length and 4) facial width. All of these factors have the potential to influence the length of the ear canal, but previous studies have not used quantitative methods to determine statistical correlations. We used 3D geometric morphometric landmark data, Principal Component Analyses and ordinary least squares regressions to determine correlations between specific measurements (see above) of the basicranium of modern humans, African apes and four hominin specimens (OH 5, KNM‐ER 406, STS 5 and AL 444‐2).

Results show that there is a significant correlation between foramen magnum width and bony ear canal length in modern humans, specifically (R2=0.626, p<0.001), refuting the null hypothesis that there is no significant correlation between ear canal length and foramen magnum width. Interestingly, there was no significant correlation between facial width and bony ear canal length, except perhaps in extinct hominin taxa, likely due to their robust masticatory system. These results indicate that there may be a functional constraint on the length of the bony ear canal in modern humans, limiting the variability of ear canal length and its role within sound perception and localization. Future work should focus on increasing the sample sizes and using the timing of cranial base ossification patterns to aid in understanding if the growth of the foramen magnum is actually driving the medial constraint of the ear canal in humans.

Support or Funding Information

This research was supported by The Graduate Center, City University of New York through a Graduate Center Fellowship and would not be possible without the support of the New York Consortium of Evolutionary Primatology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anatomical variations of the sacroiliacal joint
Authors: Guzman‐Lopez Santos , Teran‐Garza Rodrigo , Quiroga‐Garza Alejandro , Elizondo‐Omaña Rodrigo Enrique
Published: June 2021

Introduction

The sacroiliac joint (SIJ) is the union between the spine and the pelvis. It is a diarthroid joint and complex due to having different types of joint classifications. The presence of an accessory articular facet was described for the first time in 1753, considered the first report of a sacroiliac anatomical variant. Since then different anatomists have reported this variant in skeletons and corpses with variations in their incidence. In 1999, the first classification was made using a Greek population reporting 5 other anatomical variants using computed tomography (CT) and correlating this to lumbar pain, reporting a high incidence when anatomical variants were present. Only one other study analyzes the joint using this classification in a Turkish population. There are no reports of other population, much less Latin American. Our aim was to identify the prevalence of SIJ anatomical variants using CT.

Materials and methods

A retrospective study was made based on pelvic and/or abdominal CT that allowed the complete visualization of the SIJ. SIJ space was measured, and variants were classified as: Accessory SIJ, ileosacro complex, bipartite bone plate, semicircular defect, iliac bone plate, and ossification center.

Results

Four hundred CT scans were included for analysis. The mean age was 49 years, 180 (45%) were men and 220 (55%) women. More than half (209, 52.2%) of the patients presented an anatomical variant, and were more predominant in women (65.4% vs 36.2%), as well as those older than 40 years of age (60% vs 40%). The mean joint space was similar between sides (right 2.41 ± 0.65mm vs. left 2.37 ± 0.65mm). Incidence was 19.8% accessory SIJ, 6.5% ileosacro complex, 12.3% bipartite bone plate, 8% semicircular defect, 5% iliac bone plate, and 0.8% for ossification center. No statistically significant differences were found comparing the space with the variables of gender, age, or type of variant.

Conclusion

The prevalence of anatomical variants of the SIJ is high in the Latin population, higher in women and those older than 40 years. The most prevalent variant was accessory SIJ.

Support or Funding Information

None

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Unilateral and Bilateral Absence of the Musculocutaneous Nerve
Authors: Hunter Dawn D , Nestor Nicholas S , Bolden Alexys M , Arnold Danielle A , Lambert H. Wayne , Zdilla Matthew J
Published: June 2021

The brachial plexus is responsible for the innervation to the upper limb. The musculocutaneous and median nerves are terminal branches off the brachial plexus. The musculocutaneous nerve supplies motor innervation to anterior flexor muscles of the arm. The median nerve provides motor innervation to most flexor muscles of the forearm, pronator muscles of the forearm and passes through the carpal tunnel to innervate certain muscles of the hand. In the absence of the musculocutaneous nerve, the median nerve typically supplies the musculature of the anterior flexor compartment of the arm, including: biceps brachii muscle, brachialis muscle and at times the corocobrachialis muscle. In the case of a high median nerve injury, a variation of an absent musculocutaneous nerve may not only result in typical palsy of the forearm and hand, but also experience palsy in the arm. Therefore, understanding the prevalence and anatomical characteristics of the absence of the musculocutaneous nerve is clinically important. Among 44 brachial plexuses from 22 cadavers assessed, an absent musculocutaneous nerve was observed in 4 plexuses (9.1%) among 3 (13.6%) cadavers. A single female cadaver revealed a bilateral absence of the musculocutaneous nerves in comparison to one female cadaver showing a right unilateral absence of the musculocutaneous nerve and one male demonstrating a left unilateral absence of the musculocutaneous nerve. In all cases, the median nerve supplied both the biceps brachii and brachialis muscles. In summary, a systematic review of the absence of the musculocutaneous nerve demonstrated that females are under‐represented, relative to males and a unilateral absence of the musculocutaneous nerve in a female should be considered a particularly rare finding.

Support or Funding Information

WV Research Challenge Fund [HEPC.dsr.17.06]

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Correlation of the Common Carotid Artery Morphology with Age of Death
Authors: Bee Mary Tracy , Garmo Alana , Luciow Tavala , DeDolph Sarah , Garmo Atalie , Barton Scott
Published: June 2021

In multi‐campus and multi‐year studies, we investigated the relationship of the size of the common carotid artery versus age of death. A significant and positive correlation was identified bilaterally in the diameter of the common carotid arteries in relationship to age of death in 120 human cadavers ranging from 23 to 102 years old (p=0.017). The diameter of the common carotid artery has been associated with the occurrence and severity of strokes. This may provide insight into circulatory‐related procedures in the cervical region or may be indicative of a related genetic predisposition to a higher vascular flow.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anatomical Features of the Basioccipital Bone including the Transverse Basilar Cleft, Clival Foramen, Clival Canal, Foveae, and Foveolae
Authors: French Evan , Russell Michelle , Pancake Jacob , Koons Aaron , Zdilla Matthew
Published: June 2021

The basioccipital bone is particularly important with regard to the developing cranium. There is broad anatomical diversity in the anatomy of fetal and infantile basioccipitals. Specifically, some basioccipitals have transverse basilar clefts, clival foramina, and clival canals. Additionally, basioccipitals are diverse with regard to foveae and foveolae patterns. The size and shape of the basioccipital also changes throughout development. Canonical variate analysis revealed shape change from a relatively narrow/long with mild concavity at the foramen magnum in the 5th and 6th intrauterine months to relatively broad/short with more pronounced concavity in the 5th postnatal month. This report highlights the development of the basioccipital bone in addition to detailing the prevalence of the aforementioned anatomical features of the basioccipital and discusses the implications for anthropology, forensic osteology, as well as clinical implications of variant basioccipital anatomy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Presence of a Variant Obturator Artery: The Corona Mortis
Authors: da Fonseca e Silva da Costa Cordeiro Roberto Claudio , Da Silva Denize Augusto , Duro Gabriella Mares , Da Silva Pereira Raiza , Pezzi Lucia Helena Antunes
Published: June 2021

INTRODUCTION

The obturator artery (OA) is a vessel of variable origin with anterior‐lateral direction on the wall of the pelvis. It is followed by the obturator vein and nerve leaving the pelvis through the obturator canal1. This vessel is often a branch of the internal iliac artery, however, it is subject to important anatomical and surgical variations 2,3. The most important variation is referred to corona mortis4 (Crown of Death). The aim of this study is to verify and quantify the OA different origins and to determine the presence of conora mortis.

METHOD

Forty cadavers of Brazilian decent (27males and 13 females) were dissected in order to observe the presence or absence of anatomical variations of OA. The study was conducted in the Laboratory of Anatomy of the Estácio de Sá University (Joao Uchoa Campus), Rio de Janeiro, Brazil. The data analysis was conducted using SPSS software.

RESULTS

The corona mortis (Figures 1 and 2) was found in 22.5% of subjects (55,6% Males and 44,4% females) (Tables 1 and 2). The external iliac artery had a common trunk that originated the obturator and inferior epigastric arteries.

DISCUSSION

The presence of the corona mortis is very relevant for surgery because it is a common variation of the obturator artery. The knowledge of this variation is important because of the possibility of injury during surgical interventions especially when staples are used for endoscopic repair of inguinal and femoral hernias2,3,4,5,6.

CONCLUSION

The presence of corona mortis variation was significant, demonstrating that the knowledge of this variation, is important to surgery.

Support or Funding Information

Own financing and support from Estácio de Sá University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CRISPR‐cas9 gene editing in undergraduate laboratory experience
Authors: Kee H. Lynn , Pieczynski Jay
Published: June 2021

Undergraduate biology students learn that gene editing is revolutionizing basic science and biomedicine. We developed lab experiences for mid‐level undergraduate laboratory that actively engages students in the full process of design, implementation of a gene editing strategy, and interpretation of their results within a 15‐week semester where lab meets once a week for 3 hours. The laboratory experiences were centered around the use and application of Clustered Regularly Interspaced Short Palindromic Repeats‐cas9 (CRISPR‐cas9) gene editing technology in bacterial E. coli cells. In one course, students developed and implemented a strategy to deactivate expression of Green Fluorescent Protein (GFP). In another course, students carried out experiments to try edit a specific gene in bacterial cells to make the cells resistant to a specific antibiotic. Students were immersed in various methods that include: molecular cloning of plasmid construction, bacterial transformation, PCR, DNA sequencing to analyze gene edits, and microbiology techniques. Student assessment revealed many students achieved student learning outcomes, although there were challenges associated with the ambiguity associated with open‐ended research. We conclude that these lab experiences are accessible and effective in engaging students in the scientific practices and principles centered around targeted CRISPR‐cas9 gene editing.

Support or Funding Information

Funding was supplied by the Stetson University Biology Department. Molecular reagents from New England Biolabs were provided by the Course Support Program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Infrared Thermography Applied to the Study of Enzyme Kinetics
Authors: Cochran Desiree Ann , Torelli Andrew T
Published: June 2021

Kinetic analysis of enzyme activity typically relies on spectrophotometric detection of changes in product or substrate concentrations. Spectrophotometric detection can directly monitor enzyme activity when a suitable chromophore is involved in the reaction itself, or reaction progress can be tracked via a coupled reaction that involves a chromophore with quantifiable signal changes. An alternative, potentially universal strategy for tracking reaction rates involves monitoring the evolution of heat from the enzyme‐catalyzed reaction. The heat generated by a reaction can then be used to calculate the moles of product formed by taking into account the molar enthalpy change for conversion of substrate into product. As with other enthalpimetric approaches, e.g. isothermal titration calorimetry, analysis of the reaction is not dependent on the use of a specific chromophore, nor is it challenged by optically‐dense or heterogeneous samples. Such methodologies have been in use for decades using near‐adiabatic instrumentation to quantify the total heat change. More recently, infrared imaging technologies such as the 200USD FLIR ONE camera open new opportunities to analyze kinetics on the basis of the heat evolved by the enzyme‐catalyzed reaction. We report our preliminary work to demonstrate the proof‐of‐principle use of infrared thermography to track enzyme reaction rates. In addition, the unique advantages of IR imaging are well suited for applications of this approach in a biochemistry laboratory course setting.

Support or Funding Information

National Science Foundation DUE Award Number:1813313; “Collaborative Research: Visualizing Chemistry with Infrared Imaging”

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gains in Affect and in Scientific Literacy from Peer‐Led Team Learning in a Literature‐Based First‐Semester Biochemistry Course
Authors: Dries Daniel Robert
Published: June 2021

Science is a social endeavor that requires communication and collaboration with scientists of various expertise and background. Traditional lecture, however, can cast learning as a passive, individualized task. To model the scientific community for students, a sophomore‐level biochemistry course was redesigned using the primary literature as the principal content from which biochemical concepts were extracted. Working in groups alongside a peer facilitator, students constructed their own knowledge by interrogating the literature, researching answers to their questions, and sharing with their group, while instructor‐led activities and just‐in‐time mini‐lectures grounded the class in a common experience. Attitudinal post‐surveys showed student affective gains in teamwork, inquisitiveness, and scientific literacy. Moreover, graduates self‐reported greater preparation for post‐graduate work than peers in their program. Together, these results suggest that peer‐led team learning using the primary literature can lead to gains in competencies and skill development while still accruing knowledge of biochemistry content.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of YAP Oncogene in Metastasis and Mechano‐medicine
Authors: Sudol Marius
Published: June 2021

YAP is a WW domain‐containing protein that acts as a potent oncogene and stemness factor. It is one of the two main effectors of the Hippo tumor suppressor pathway. YAP is a transforming gene of the chromosome 11q22 amplicon, and its expression is elevated at high frequency in human cancers, including liver, breast, ovarian and stomach cancer. Our recent work showed that YAP regulates the acto‐myosin network by suppressing Rho‐GTPase via Rho‐GTPase activating protein 29 (ARHGAP29) being a direct transcriptional target of YAP in human gastric cancer. We showed that YAP promoted the expression of ARHGAP29 to suppress the RhoA‐LIMK‐cofilin pathway, thereby destabilizing F‐actin. The overexpression of YAP caused cytoskeletal rearrangement by altering the dynamics of F‐actin/G‐actin turnover, thus promoting migration. In a mouse model, circulating tumor cells (CTCs) exhibited an increase in ARHGAP29 RNA level compared with cells at primary tumor sites. Moreover, increased ARHGAP29 expression correlated with shortened survival of human gastric cancer patients. Importantly, we showed that ARHGAP29 is critical in regulating cancer metastasis in a mouse model of liver cancer metastasizing to lungs.

We have also identified YAP and TAZ specific transcripts and we are characterizing them now as mechano‐responsive transcripts that regulate cell metastasis and mechano‐sensing.

Cancer cells are generally softer than normal cells. By increasing the rigidity of cancer cells to the level of normal cells via novel therapeutic interventions, and by reducing rigidity of extracellular matrix of tumors we could provide a new modality to treat cancer in unison with other standard therapies.

Support or Funding Information

Start‐Up Funds from National University of Singapore (R‐185‐000‐2710‐733) and Mechanobiology Institute (R‐714‐018‐006‐271)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Generation of an Endogenous Dominant Negative Allele of chd5 Using a Novel Homologous Recombination Strategy to Reveal the Role of chd5 in Tumor Suppression and Neural Differentiation
Authors: Denning Ellen , Sorlien Erin , Ogas Joseph
Published: June 2021

CHD5 is a vertebrate specific member of a family of ATP‐dependent chromatin remodeling proteins. Chromodomain/Helicase/DNA‐binding (CHD) proteins play a variety of roles in vertebrate development, and loss of CHD5 causes failure of proper expression in a cohort of genes involved in neural development. CHD5 plays a critical role tumor suppression in humans, and loss of CHD5 contributes to the formation or progression of numerous cancers, in particular neuroblastoma. To understand how CHD5 plays a role in tumor suppression, it is necessary to understand how CHD5 contributes to normal development and how its normal functions are misregulated during tumor progression. Our lab is establishing zebrafish as model system in which to study the role of chd5 dependent processes.

We have engineered chd5 knockout fish using CRISPR‐Cas9 technology. The resulting fish do not exhibit any overt developmental phenotypes, and do not produce tumors during adulthood (N=165, aged >16 months). We hypothesize that the closely related remodelers CHD3/4 function redundantly with CHD5, and propose to use a novel knock‐in approach to generate zebrafish carrying a dominant negative mutation in the ATPase domain that has previously been shown to result in strong mutant phenotypes when generated in closely related remodelers.

To engineer a dominant negative allele of chd5 we have engineered a homologous recombination (HR) strategy that uses piggyBac transposase technology, which has previously been used in Drosophila, mice, and human cells to engineer scarless genomic modifications. This design uses two‐steps to first, introduce a homologous donor carrying the mutation of interest and a fluorescent marker for easy identification. Second, the HR positive fish will be bred with piggyBac expressing fish lines to seamlessly excise the exogenous sequences, thus leaving only the desired genome modification.

Once we have engineered this method into the fish, we will be able to use piggyBac to control when and where we express the dominant negative allele of chd5 so that we can score the resulting fish for developmental and tumor phenotypes. The screening of the dominant negative, knock‐in lines is in progress. The resulting dominant negative lines will give great insight into the role of chd5 in neural development and tumor suppression.

Support or Funding Information

Funding made available by: Purdue University Center for Cancer Research, Purdue University Department of Biochemistry, Purdue University College of Agriculture

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genome Editing of a Secondary Alcohol Dehydrogenase Gene by CRISPR‐Cas9 Technology
Authors: Huang Jenq‐Kuen , Samassekou Kadidia , McClenahan Shawn , Seiver Jacob , Holt Scott , Wen Lisa
Published: June 2021

Nocardia Cholesterolicum NRRL 5767 (NC NRRL5767) is well‐known for its ability to transform ~95% of added oleic acid, an abundant agricultural commodity, to value‐added product of 10‐hydroxystearic acid (10‐HSA). A small amount of unwanted 10‐ketostearic acid (10‐KSA) was also produced. The conversion of oleic acid to 10‐HSA and then to 10‐KSA is catalyzed by oleate hydratase and secondary alcohol dehydrogenase (2°‐ADH), respectively. The objective of this project was to knockout the 2°‐ADH gene in NC NRRL5767 so that the sole biotransformation product from oleic acid would be 10‐HSA. Here, we report construction of CRISPR/Cas9/sgRNA chimeric plasmids that specifically target two different loci in the coding region of the 2°‐ADH gene by golden gate assembly. The constructs were confirmed by DNA sequencing and transformed into NC NRRL 5767 via electroporation. The transformants were selected by apramycin resistance and screened for the presence of the target insert by PCR. The ability of the selected transformants to transform oleic acid to 10‐HSA was examined by TLC. Our results showed that both 10‐HSA and 10‐KSA were produced by the transformants from oleic acid. Apparently, either Cas9 was not expressed or it was expressed but did not target the 2°‐ADH gene. The expression of Cas9 in the cell‐free extracts of selected transformants was examined by Western blotting using Cas9 antibody. However, the Cas9 protein was undetectable in these transformants.

Support or Funding Information

Western Illinois University Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential nicking patterns of Cas12a variants
Authors: Murugan Karthik , Sashital Dipali G
Published: June 2021

Cas12a is an endonuclease from the bacterial CRISPR‐Cas immune system that can be easily programmed for use in various biotechnological applications. Despite its prevalent use in genome editing, the cleavage specificity and activity of Cas12a remain to be fully determined. We employed high‐throughput in vitro cleavage assays to determine and compare the native specificities of different Cas12a variants. Surprisingly, we observe pervasive nicking of randomized target libraries. Cas12a has distinct nicking activity against targets with multiple mismatches, where it can linearize certain mismatch targets, but only nick others. Our results also demonstrate that Cas12a variants have differential in vitro cleavage activity and mismatch tolerance. Francisella novicida (Fn)Cas12a is less tolerant to mismatches than Lachnospiraceae bacterium (Lb)Cas12a and Acidaminococcus sp (As)Cas12a. The mismatch position plays a vital role in determining target specificity of Cas12a. Like Streptococcus pyogenes (Sp)Cas9, the mismatches in the PAM‐proximal “seed” region are especially deleterious to Cas12a target cleavage, however, the seed region is shorter (~ 6bp) when compared to SpCas9 (~ 8–10bp). Overall, we observe that Cas12a variants have sequence‐ and seed‐specific nicking and cleavage activities, with variable sensitivities to mismatch position.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Divulging the Enigma of Epitranscriptome in Plasmodium falciparum
Authors: Govindaraju Gayathri , Jabeena CA , sethumadavan Devadathan , Raja Nivethika , Rajavelu Arumugam
Published: June 2021

In spite of prime posttranscriptional modifications, eukaryotic RNA is subjected to internal epitranscriptomic marks. Thus, these changes influence the rate of transcription which regulates the translational plasticity. However, RNA methylome and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, for the foremost a piece of our work demonstrated the cytosine methylation in the tRNA Asp at the C38 position of Plasmodium falciparum, which is accomplished by the tRNA aspartic acid methyl transferase1 (TRDMT1), a conserved homologue of DNA methyltransferase DNMT2. Through RNA bisulfite sequencing, we mapped the 5‐methylcytosines in endogenous tRNA Asp, followed by in‐vitro characterisation of TRDMT1. Accordingly, P. falciparum proteome divulged the significant role of polyaspartic acid repeat‐containing proteins and its abundance. Those proteins may play astonishing roles in host‐pathogen interactions. Further studies focus on the generation of TRDMT1 knock out parasites which may aid us to find out the critical role of TRDMT1 mediated C38 methylation of aspartic acid tRNA. In particular, translational regulation of essential proteins which could modulate the malaria parasite pathogenesis. On the other hand, the significant genes in P. falciparum show discrepancy in mRNA expression and its cognate protein, through which ‘time shift’ is displayed. Moreover, P. falciparum exhibits decelerated ring stage as a distinctive policy for drug resistance. In recent years, it is evidently proven that the epigenetic modifications are existed in eukaryotic mRNA, especially methylation at the nucleotide level and is the reason for time shift.

In support, our preliminary data's on P. falciparum RNA implied the methylated nucleotides. We anticipate that parasites may modulate the translation process at the transcription level through methylation that could be installed by a multicomponent methyltransferase complex and follow‐up by the RNA binding proteins. Thus so, parasite might adopt the ‘just‐in‐time’ transcription and translation strategy for its emergence that are controlled by epigenetic machinery. Consequently, further mining of RNA methylome in parasite could be insightful about the host pathogen interaction. Hence, by cracking the epitrancriptome of P. falciparum, the underlying mechanisms of dynamic gene regulation of the parasite pathogenesis could be explored.

Support or Funding Information

Department of science and technology, Government of India.

Department of Biotechnology, Government of India.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cypermethrin Induced Liver Toxicity: Altered Gene Expression and DNA Methylation
Authors: Mahna Dolly , Puri Sanjeev , Sharma Shweta
Published: June 2021

Cypermethrin, a type II pyrethroid, is a widely used insecticide worldwide. It is considered a safe insecticide to be used in pet shampoos and household cleaning agents thus ignoring the possible health hazards caused by it. Cypermethrin may contaminate food, water, fruits or get deposited on furniture, mats or floors leading to acute or chronic disorders in non‐target organisms. Very limited studies with low and non‐frequent dose mimicking the unintentional cypermethrin intake in non‐target organisms including human beings have been carried out. The present study was aimed at evaluating the toxic effect of cypermethrin in the liver of Balb/c mice exposed to a low and once in a week oral dose (1/10 LD50) for 28 days. Serum was extracted from blood after the dosing period and utilized for Liver Function Tests. The gene regulatory studies were performed by amplifying the cDNA, employing specific primers for p21 and p53 and other cell cycle regulatory genes through PCR (Polymerase Chain Reaction). DNA methylation studies were carried out by bisulfite treatment of DNA followed by Bisulphite specific PCR. The methylation status was found out by analyzing the gene promoter regions through sequencing.

The changes in liver enzyme activities and histo‐architecture of liver cells show that sub‐chronic exposure to cypermethrin causes liver damage. It also induces a significant increase in hepatic markers enzymes (ALT, AST, ALP) along with necrosis, vacuolar degeneration, increased kupffer cells and a decrease in the number of hepatocytes. A significant change in the gene expression of p21 and p53 is seen in the test animals pointing towards the relation of liver injury by cypermethrin with cell cycle regulatory mechanisms. The gene promoter region of p53 gene show alteration in methylation status in the test animal sample as compared to the control.

Sub‐chronic exposure to cypermethrin causes significant changes in liver enzyme activities, cell architecture, cell cycle regulatory gene expression and methylation status of the concerned genes. These genes along with the other cell cycle regulatory genes can be used as biomarkers for liver injury caused by low doses of cypermethrin.

Support or Funding Information

1. University Grant Commission, New Delhi, India.2. DST‐PURSE grant, New Delhi, India.3. Institute of Forensic Science and Criminology, Panjab University, India.4. Centre for Stem cell and Tissue Engineering, Panjab University, India.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Function and Mechanism of Hepatitis C Virus Non‐structural Protein 3 (HCV NS3) Unfolding of Viral G‐quadruplex RNA Structures
Authors: Belachew Binyam Ateka , Byrd Alicia , Gao Jun , Raney Kevin
Published: June 2021

Hepatitis C virus (HCV) is the major cause of chronic liver disease and hepatocellular carcinoma. It is currently estimated that over 71 million people in the world are chronically infected with HCV. The current HCV drugs are extremely expensive, and thus they are not available for the majority of patients infected with the virus. Moreover, due to high rate of mutation of the HCV genome, it is likely that the virus develops resistance to these drugs. Therefore, it is important to develop a cost‐effective anti‐viral compound that targets conserved regions within the HCV genome and prevent replication of various HCV genotypes and subtypes.

The HCV genome encodes for ten different viral proteins, one of which is Non‐structural protein 3 (NS3). NS3 has both protease and helicase domain. The helicase domain of NS3 is essential for viral replication. However, its mechanism of action is not well understood. Previous works have shown the presence of conserved guanine‐rich consensus sequences within the HCV genome. These guanine rich sequences form G‐quadruplex (G4) secondary structures through Hoogesteen hydrogen bonding. It has been reported that both replication and translation of HCV genome could be inhibited by the formation of G4 structures. However, it is still unknown how these HCV G4RNA structures are regulated. We hypothesized that the Helicase domain of NS3 facilitates HCV replication by unfolding conserved G4 structures within the HCV genome.

To investigate whether NS3 helicase resolves HCV G4RNA structures, we purchased synthetic HCV guanine rich RNA sequences and performed circular dichroism spectroscopy to confirm G4 formation. In addition, fluorescence anisotropy NS3 binding and trap unfolding assays were conducted. We showed that NS3 binds and resolves HCV G4RNA structure in an ATP dependent manner. Understanding the mechanism by which NS3 resolves HCV G4RNA might allow us to find processes and factors that could be targeted to prevent HCV replication in a host cell.

Support or Funding Information

This work is supported by MIRA grant R35GM122601

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of Tissue Preparation, Handling and Dissociation Methods for Total RNA
Authors: Easparro Brandon , Soldat Shelby , Proctor Caleb , Nash Rodney J , Atwood James
Published: June 2021

The extraction and isolation of RNA is an integral part of downstream analyses such as RT‐PCR, RT‐qPCR, Northern blotting, and cDNA library construction. The importance of using pure, intact RNA for these processes is well documented and a critical part of downstream analysis success. It is well known that RNA is sensitive to degradation due to mechanical shear, temperature, storage conditions and freeze‐thawing. Furthermore, RNA is highly susceptible to RNAse degradation following the release of nucleases during the tissue disaggregation process. Thus, proper sample handling during the tissue preparation and homogenization processes is crucial when performing any RNA based assay.

Herein, we evaluate the impact of multiple methods for tissue harvesting, storage, storage buffer and mechanical dissociation processes on purified RNA yield and integrity. Multiple, freshly harvested murine tissues, including kidney, liver, heart, and lung were snap frozen in liquid nitrogen and stored without buffer or in an RNA stabilizing reagent at −80°C. Tissues were then dissociated via cryomilling or ambient bead milling and RNA was purified using a silica spin column method. The process above was repeated following one, two and three freeze‐thaw cycles. After each purification, RNA was analyzed on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano kit chip, as per the manufacturers' protocol. Gel images, electrophoretograms and RNA integrity numbers (RIN's) were visualized and analyzed on the 2100 Bioanalyzer Expert software (Agilent).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying a Novel Role for Circular Tau RNAs in Neurodegenerative Diseases
Authors: Welden Justin Ralph , Dockins Stephen , Blackburn Jessica , Nelson Peter , Stamm Stefan
Published: June 2021

The Tau protein, encoded by the Microtubule‐Associated Protein Tau (MAPT) gene, is the major component of the intracellular filamentous deposits that define many neurodegenerative diseases (Tauopathies) and is a critical component in the etiology of Alzheimer's disease. However, the essential regulation of the MAPT gene expression is still poorly understood. It is unclear what causes the formation of neurofibrillary tangles, but tauopathy conditions show changes in mRNA isoforms, generated through alternative pre‐mRNA processing. Furthermore, mutations changing alternative splicing of MAPT cause frontotemporal dementia, underscoring the importance of tau pre‐mRNA processing.

We have identified human‐specific circular RNAs (circRNAs) formed by the tau‐encoding mRNA from the MAPT locus generated by backsplicing of exon 12 (Welden et al. PMID: 29729314) and are investigating a role these circular RNAs may have in neurodegeneration. CircRNAs formed by backsplicing events occurs when pre‐mRNA forms loops, which is aided by primate‐specific Alu‐elements in humans.

The tau circRNAs containing exons 10–12 consist of 281 nucleotides (nt), which is divisible by 3, that would give rise to 96 amino acids and does not include a stop codon. Another tau circRNA containing exons 7–12, with the exclusion of the alternatively spliced exon 8, is also divisible by 3 comprising 681 nt and does not have a stop codon; however, it does have a start codon in exon 7 and would give rise to 227 amino acids. Thus, if translation initiates, they could form high molecular weight multimers containing parts of the tau protein. Using minigenes, we are testing the role of human‐specific Alu‐elements in deciphering which cis‐factors are crucial for circRNA formation.

Transgenic zebrafish expressing the tau circRNAs suggest a physiological relevance of tau circRNAs. We generated transgenic fish expressing only the human tau circRNAs under a neuron‐specific promoter. Importantly, the introduction of an in‐frame start codon that causes frontotemporal dementia in humans in the tau circRNAs causes early neurodegeneration in fish, suggesting that possible translation of the tau circRNAs may be occurring.

Since circRNAs do not contain a canonical ribosomal entry site, we are testing whether tau circRNAs are translated after N‐6 methyl adenosine methylation of the circular RNA in biochemical studies.

In summary, we found evidence of human‐specific tau circular RNAs, suggesting that not all RNAs made from this gene have been discovered. Our finding of a phenotype in fish indicate that the circRNAs could contribute to tauopathies, using a new disease mechanism.

Support or Funding Information

NIA ‐ 5P30AG028383‐13, University of Kentucky College of Medicine Excellence in Graduate Research Fellowship

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.