bluuebunny/crossref_metadata_2025 huggingface dataset:
The Primitive Arterial Connections within the Central Nervous System and their Neurological Implications in Everyday Medical Practice
Authors: Zarzecki Michał P. , Brzegowy Karolina , Pękala Jakub R. , Pękala Przemysław A. , Walocha Jerzy A. , Tomaszewski Krzysztof A.
Published: June 2021

Introduction

The primitive trigeminal artery (PTA) is an anastomosis between the internal carotid artery (ICA) and the basilar artery (BA), that runs close to the trigeminal ganglion (hence its name). Conversely, the primitive trigeminal artery variants (PTAV) connect the ICA with cerebellar arteries. The trigeminal artery is said to regress following the development of the posterior communicating artery during the foetal life. However, when it persists, it might cause neuralgia and nerve palsies due to its proximity and compression of the III, IV, V and VIth cranial nerves.

Aim

The following study aimed to find the pooled prevalence estimate (PPE) of the presence of the PTA and the PTAVs, their types, as well as probe for sources of heterogeneity.

Materials and methods

The authors conducted a thorough search of major medical online databases. The references of the primarily identified studies were screened for any other potentially eligible articles. Data from the studies that met the inclusion criteria was extracted and pooled into the Meta‐analysis. The quality of the included studies was evaluated by the AQUA tool.

Results

The authors deemed eligible 39 studies (110 866 patients). The overall PPE of the PTA and PTAVs was found to be 0.39% (95% CI: 0.31–0.49), slightly more prevalent in females (0.7%, 95% CI: 0.5–0.8) than males (0.4%, 95% CI:0.3–0.6). The PTA was solely found in 0.3% (95% CI: 0.2–0.4), whereas the PTAV only was noted in 0.2% (95% CI: 0.1–0.3). The PTA and the PTAVs originated most commonly from the C4 of the ICA (97.6%, 95% CI:93.7–100.0), had the lateral (parasellar) course in 89.0% (95% CI: 84.2–93.1) and the medial (intrasellar) in 11.0% (95% CI: 6.9–15.8). The anastomosis between the ICA and the anterior inferior cerebellar artery predominated amongst the PTAVs (72.1%, 95% CI: 50.0–95.7). Hypoplasia of the BA was noted in 42.5% (95% CI: 23.0–63.1)of patients with the PTA or the PTAV.

Conclusions

Although this study showed that both the PTA and PTAV are rare variants (0.3% and 0.2% respectively), a neurosurgeon should be vigilant of their presence especially during the transspehnoidal approach to the pituitary neoplasms. The medial (intrasellar) type might pose a higher risk of iatrogenic complications due to its proximity to the pituitary gland. Similarly, the lateral (parasellar) type might complicate the percutaneous gasserian ganglion procedure.

Support or Funding Information

This study was supported by “The Best of the Best! 3.0 (Najlepszy z Najlepszych! 3.0)” Grant of the Polish Ministry of Science and Higher Education.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Variations of the Fibularis Tertius
Authors: Hurley Mary
Published: June 2021

Fibularis tertius, also called peroneus tertius, is a muscle located in the anterior compartment of the leg. It shares a synovial sheath with extensor digitorum longus and attaches at the base of the fifth metatarsal. It is noted in both Moore's Clinically Oriented Anatomy and Gray's Anatomy that the muscle is ‘not always present’. This study examined the dorsum of the feet of 16 cadavers for the presence of fibularis tertius tendons and variations of the tendon. Two cadavers were lacking the fibularis tendon on the right foot. Their left feet and the other 14 cadavers bilaterally had fibularis tertius tendons. Variations seen included insertion of the tendon on the shaft of the fifth metatarsal, tendons branching from the extensor digitorum longus tendon of the fifth digit, and multiple fibularis tertius tendons.

Support or Funding Information

Work was supported by D'Youville College Department of Biology and Math.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Right to Left Crossed Fused Renal Ectopia: An Incidental Finding in Cadaveric Dissection
Authors: Syed Mohsin , Akhter Noor , Phelan Kevin D , Davies David L , Warren William C , Eltahawy Ehab , Whisman Michella K , Scott Hayden , Scott‐Kirchen Logan M , Sellers James T , Sharabura Anna , Shaw Collie M
Published: June 2021

Development of the kidney begins in the sacral region during the 4th week of gestation. As the development progresses, kidneys ascend to reach their adult position in the upper portion of the posterior abdominal wall. During the complex process of ascension, several different congenital anomalies have been shown to develop.

Congenital urinary tract and renal malformations are not uncommon. One such anomaly is the fusion of the kidneys during the early embryonic period called crossed fused renal ectopia (CFRE). CFRE is a rare type of congenital malformation where both kidneys are situated on one side of the midline. In about 90% of cases, the crossed ectopic kidney is fused with the normally placed contralateral kidney. Prevalence of CFRE is estimated to be 1:1300 to 1:7500 (Turkvatan et al 2009, Bauer SB 2002). This anomaly is usually detected as incidental finding as it is often asymptomatic.

We present a case of a crossed fused renal ectopia (CFRE), discovered by freshman medical students during a cadaveric dissection in a gross anatomy course at the University of Arkansas for Medical Sciences (UAMS). The body donor was a middle‐aged Caucasian female whose death was attributed to natural causes. In this case, the right kidney was positioned inferiorly and transversely fused with the lower pole of the left kidney, resulting in a single fused L‐shaped kidney (MC Donald and MC Clellan, 1957) at the gross level. The renal vascular supply was also unusual. The left renal artery arose from the normal position as a lateral branch of the abdominal aorta. Whereas, the right ectopic kidney was supplied by three arterial branches: a branch from the anterior aspect of the abdominal aorta, a second branch from the left common iliac artery and a third branch from the point of bifurcation of the abdominal aorta. In addition, the inferior mesenteric artery arose from the abdominal aorta at a much higher level than normal. It was at the level of the L1/L2 intervertebral disc. The ureteric openings in the bladder were more inferior than usual. Several small cysts were found on the kidney surface and microscopic examination of a biopsy samples from the ectopic right kidney revealed nephrosclerosis.

CFRE with vascular anomalies can be clinically confusing as it would give rise to symptoms in the lower abdomen rather than in the loin. As such, surgeons who encounter this during abdominal procedures need to be aware of the multiple aberrant arteries that can make vascular control a challenge.

Support or Funding Information

Support

Two UAMS Departments: a) Neurobiology and Developmental Sciences, b) Pathology

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sexual Dimorphism and the Scaling of Intrinsic Thumb Muscle Physiological Cross‐Sectional Area and Trapeziometacarpal Joint Size
Authors: Kashyap Ritesh , Orr Caley
Published: June 2021

The thumb is a common site for the development of osteoarthritis (OA) and studies show that there is greater prevalence of OA of the thumb joint in women. While a number of explanations have been proposed, the mechanism underlying the sex distribution of OA remains unknown. One possible contributing factor is the generally smaller size of females. Given constraints on size at the trapeziometacarpal (TMC) joint (which is locked into the wrist complex), it is possible that stress (force per unit area) tends to be greater in smaller individuals. This may lead to quicker progression of the degenerative processes characteristic of OA. However, increased stress requires a similar muscle force acting on a relatively smaller joint, and this might be the case if females must maintain a minimum level of grip strength for day‐to‐day function. Thus, we hypothesize that females have higher TMC joint stress due to scaling constraints (small joint surfaces with relatively large muscles). To test this hypothesis, cadaveric samples from the University of Colorado School of Medicine were used to measure the physiological cross‐sectional area (PCSA) of intrinsic thumb muscles and scaled against the surface area of the TMC joint. Preliminary data suggest that females tend to have a higher total intrinsic thumb muscle PCSA compared to males, but a clear scaling pattern between the variables was not evident. This suggests a more complicated relationship between joint size and thumb muscle force potential. However, further data and analysis might reveal muscle and joint scaling patterns that could provide an explanatory mechanism for the higher prevalence of OA in women than men. In turn, this could provide a background for future research into methods of preventing OA.

Support or Funding Information

Modern Human Anatomy 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.
A Horseshoe Kidney with Unique and Rare Renal Vasculature—A Case Report
Authors: Zhang Guiyun , Schmidt Richard R
Published: June 2021

Horseshoe kidney with variable vascular supply is of interest to clinicians and anatomists due to its immense surgical significance. A horseshoe kidney encountered during cadaveric dissection is reported here. It is L‐shaped with the left kidney vertical and the right kidney horizontal. The isthmus is narrow, 1.5cm inferior to the inferior mesenteric artery and is deviated away from the midline to the left. The hilum of the right kidney opens anteriorly and the left opens anteromedially. The ureters lie on the anterior surfaces of both kidneys. In addition to renal arteries arising from the aorta described in previous reports, a unique and rare renal artery (Renal Artery 3) was found supplying the right kidney. This RA3 originates from the aorta at L3 vertebral level and takes a long and tortuous course to reach the hilum of the right kidney. Such arterial variation is important for surgical procedures involving the abdominal aorta.

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.
A Cadaveric Study of Skeletal Facial Foramina in the Hispanic/Latino Population
Authors: Hester Kathylin Mei Li , Rahimi Omid , Fry Constance , Nation Haley
Published: June 2021

Clinicians currently follow two surgical guidelines involving skeletal features of the orbit and face to optimize patient care. The first guideline states that along the medial wall of the orbit, the distances between the anterior lacrimal crest, anterior ethmoidal foramen, posterior ethmoidal foramen, and optic canal, follow a “24‐12‐6”mm guideline. The second guideline states that the supraorbital foramen/notch, infraorbital foramen, and mental foramen are equidistant from the midline. Previous studies suggest these two surgical guidelines are not applicable for all ethnicities; however, to our knowledge, no data has been published regarding the accuracy of these guidelines pertaining to the Hispanic/Latino population. A cadaveric study was performed to investigate these skeletal guidelines in the Hispanic/Latino population. Adult male and female cadavers, with no evidence of trauma to the orbit and face, donated to the Human Anatomy Program at UT Health San Antonio were used for this study. To investigate the medial orbital wall “24‐12‐6” guideline, distances between four orbital landmarks (anterior lacrimal crest, anterior and posterior ethmoidal foramina, and optic canal) were measured using a 26‐gauge wire and a Vernier caliper. To investigate whether the supraorbital, infraorbital, and mental foramen, lie within the same sagittal plane, we used a Vernier caliper to measure the distance between each foramen to midline. Our preliminary findings suggest these surgical guidelines may not accurately describe the morphologic characteristics of the Hispanic/Latino bony orbit and face. Preliminary results to date indicate the average distances from the anterior lacrimal crest to the anterior ethmoidal foramen, the anterior ethmoidal foramen to the posterior ethmoidal foramen, and the posterior ethmoidal foramen to the optic canal for Hispanic/Latino orbits (n =5) were 24.4 mm (± 0.686), 16.2 mm (± 2.190), 7.4 mm (± 0.661) respectively. This suggests the anatomy of the Hispanic/Latino boney orbit may differ from the currently accepted “24‐12‐6” surgical guideline. Meanwhile, preliminary data also indicates the average distances from the midline to the supraorbital foramen/notch, infraorbital foramen, and mental foramen for all cadavers studied (94 hemifaces, 47 cadavers) were 26.07 mm (± 3.88), 29.12 mm (± 4.30), 25.46 mm (± 3.15) respectively. This also suggests the currently practiced guideline may not apply to the Hispanic/Latino population. To optimize patient care, it is critical clinicians are aware of ethnic difference between anatomical structures. Our preliminary data is the first to report specific anatomical relationships in the medial orbital wall and facial foramina in the Hispanic/Latino population.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Case Study of Case Studies: Writing Choose‐your‐own‐experiment Cases and Avoiding Common Pitfalls that Decrease Case Study Use
Authors: Hines Justin K
Published: June 2021

Case studies are excellent learning tools and, across multiple online collections, case studies are actually quite numerous. Why then do many instructors who are aware of both the utility and existence of these resources report that they don't use them, or if they do, they write their own? I will present some of the commonly cited barriers to case study implementation as reported by biochemistry faculty in anonymous surveys and discuss methods to avoid creating these barriers to implementation in case study design. Additionally, I will discuss my own experiences creating more than a dozen biochemical case studies to address these issues which often include the use of two important additional design elements. The first is the use of an in‐class iterative cycle which I call “choose‐your‐own‐experiment” in which students choose from a set of experimental options, followed by the immediate acquisition of data, and the subsequent discussion before repeating the cycle with another decision. Cases involving iterative decision‐making and evidence gathering are common in medical education and games, but uncommon in undergraduate science education. Perhaps most notably this game‐like experience is implementable in the context of a regular lecture course by use of a limited set of authentic, but pre‐determined, experiment choices and results. An additional layer of complexity can be added by limiting the ability of students or groups to gather all the data, creating a “jig‐saw” type activity and an opportunity for genuine collaboration toward a common goal. The second notable design element is to avoid a common ‘pre‐ordained destiny’ aspect of many case‐based activities in which every step reveals information that deliberately leads students to the correct answer. Rather, in what I have called an “authentic inquiry approach” to case writing, the data is as real as possible which means it does not always help the student and sometimes investigation options fail. Like a real investigation, this unhelpful data must still be considered. The goal of this session is to aid others in the creation of easily implementable and authentic inquiry‐driven activities in the physical and life sciences.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Iterative Design of 3D Physical Serine Protease Models based on Biometric Data to Optimize Cognitive Load and Decrease Misconceptions in Undergraduate Biochemistry
Authors: Terrell Cassidy Renee , Calvert Jenifer , Randolph Adriane , Cortes Kimberly Linenberger
Published: June 2021

Biochemistry students misconceptions related to enzyme‐substrate interactions can stem from prior knowledge in biology and chemistry where this and related concepts are inadequately represented visually, explained and/or assessed. Moreover, research has shown two‐dimensional representations not only fail to effectively convey biochemical concepts, but also propagate misconceptions. We hypothesize that three‐dimensional (3D) physical models used in conjunction with targeted active learning assessments will increase student understanding of shape, stereochemistry, and electrostatic interactions involved in enzyme‐substrate interactions. However, little research exists on how to design 3D physical models with corresponding assessments that optimize the cognitive load of the student which would enhance learning. This presentation will describe an iterative design process based on biometric, observational and rubric data analysis. The biometric data is collected using electroencephalographic (EEG) and eye tracking tools and voice recordings from simulated learning environments where biochemistry students completed a serine protease activity with corresponding physical models. In addition, observational and rubric analyses of student responses to activities from the simulated learning environment and real classroom sessions provide further data for the iterative activity design process. Here, we will offer suggestions for faculty interested designing 3D physical models with corresponding active learning assessments that optimize the cognitive load and target student misconceptions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Microbe for Ohio: A Service Learning Project That Swept the State
Authors: Bennett Jennifer A , Bowman Bridget , Hout Sokjita , Clinton Tim
Published: June 2021

It is common for states across America to adopt official symbols that are used for representation. For example, states may have an official state tree, animal, flower, and song. Ohio has at least 25 state symbols, including a state beverage and a state fossil. At this time it does not yet have a state microbe. There is only one state that currently has an official microbe. Oregon has chosen Saccharomyces cerevisiae, commonly referred to as brewer's and baker's yeast, because of the state's brewing industry and many craft breweries. Three other states have selected a microbe and are pursuing legislation to have the microbe officially recognized. After learning of the stories surrounding the microbes selected in other states, I decided to introduce this as a unique service learning project into my upper‐level microbiology course at Otterbein University. This is a writing intensive course that serves as an upper‐level elective for Biology as well as Biochemistry and Molecular Biology majors. Each student was required to choose a microbe that they felt represented Ohio and write a persuasive paper on the topic. Six of these microbes were then selected for a vote by the general public. While the students in the course learned about many new microbes and fulfilled their writing requirements for the course, the major service goal of the project was to educate the public about microbiology, especially the fact that there are beneficial microbes. Microbes and their beneficial roles were presented to the public in a fun, educational way via social media. This multi‐disciplinary project involved many departments at Otterbein, including Biology and Earth Science, Communication, and History and Political Science, as well as the Biochemistry and Molecular Biology Program. The American Society for Microbiology and American Society for Biochemistry and Molecular Biology Student Chapters both played a vital role in relaying the information to the public and organizing events. Currently the “Microbe Term Election” is almost finished, and the next step is to write the winning “candidate microbe” into legislation. The “campaign” has been largely successful in its goals and has gained the support of various organizations, colleges and universities throughout Ohio and allowed the participation of the general public, including K‐12 classrooms in the voting process. The service learning initiative will continue throughout the year with a general education microbiology course in Spring 2019. The selection of an official state microbe is anticipated to continue to spark the public interest in microbiology and biochemistry, as people learn how microbes are beneficial and how they fulfill their important roles.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNA Replication Genes Implicated in Heterochromatin Modulation
Authors: Moghul Tasmiya A. , Sharma Pragati , Reilly Eve , Zaratiegui Miguel A.
Published: June 2021

Cellular DNA is stored compactly in the nucleus in complex with protein factors. The condensed structure or chromatin is categorized as euchromatin ‐ a relatively loose form consisting of frequently transcribed genes, and heterochromatin ‐ a denser, more compact form that covers mostly noncoding genes, transposable elements, and satellite repeats. Some of the functions of heterochromatin are: suppression of recombination of transposable elements, proper segregation of chromosomes and regulation of gene expression. Hence, heterochromatin is vital for maintaining genomic stability. Heterochromatin can be further classified as facultative (assembles and dissembles for gene expression regulation) and constitutive (condensed throughout the cell cycle). Although condensed, heterochromatin has to be unraveled during DNA replication. How does the cell know when to change heterochromatin for replication or when to return to its previous compact state? The overarching goal of this project is to understand the molecular factors that help modulate heterochromatin states to allow replication through silenced regions. Using fission yeast, we conducted a forward genetic screen with Hermes transposon as a random mutagen to obtain Position Effect Variegation (PEV) mutants of heterochromatic ade6 reporter gene. With whole genome sequencing, we identified the mutants that showed loss of silencing at the reporter gene; a number of these mutations were found to be in essential genes. Selection for loss of silencing at the pericentromeric ade6 reporter corresponded with an enrichment in mutants of DNA replication and repair genes. We hypothesize that these mutants can be recreated with CRISPR‐Cas9. The primary aim of my project is to generate mutant alleles of essential genes involved in DNA replication and repair, and validate their heterochromatin mutant phenotype. Using CRISPR ‐ Cas9 genome editing, we targeted five such genes in fission yeast: Vid21, Orc4, Cut1, Cut9, Drc1, and Dcr1 as a method control. The results we found show that we were able to generate viable mutant alleles that may be otherwise difficult to generate using conventional genetic manipulations. This study of the characterization of mutant alleles will enable us to understand their role(s) in the coordination of heterochromatin states and DNA replication and/or repair.

Support or Funding Information

This work was supported by RiSE at Rutgers, NIH grant K12GM093854‐07A1 to IRACDA at Rutgers University, NIH‐NIGMS T32 GM8339 New York for Science Partnerships in Research and Education ‐ Rutgers University Biotechnology Training Program and NIH NIGMS RO1 GM105831.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of RNA Modification Enzymes in Trypanosoma brucei
Authors: Schultz Will , Smith Xiane , Taber Cassandra Carol , Coffey Jordan , Militello Kevin
Published: June 2021

RNA methylation is a type of posttranscriptional modification that plays an important role in controlling gene expression. The organism Trypanosoma brucei, the protozoan parasite responsible for Human African Trypanosomiasis, does not seem to have abundant promoter regions or transcriptional regulation machinery. Thus, RNA methylation may play an especially important role in regulating gene expression in this organism. We have identified the presence of 5‐methylcytosine in T. brucei RNA using both mass spectrometry and sodium bisulfite sequencing. Recently, we have identified seven putative cytosine RNA methyltransferase (CRMT) genes in T. brucei. All seven CRMTs are expressed in bloodstream and procyclic form parasites, as detected by qRT‐PCR. Two of the putative CRMTs, termed CRMT4 and CRMT5, are required for maximum parasite growth. Although we suspect these genes to be RNA methyltransferases, we do not have evidence for RNA methyltransferase activity. Both CRMT4 and CRMT5 were expressed in E. coli with N‐terminal 6x‐histidine tags. CRMT4 was produced in E. coli but was difficult to purify. SDS‐PAGE results for an N‐terminal His tagged protein indicate CRMT4 insolubility. Our next step will be to use PCR to create smaller fragments of CRMT4, which may be soluble. CRMT5 was produced in E. coli with an N‐terminal His tag and purified using a His‐affinity column. Purified CRMT5 was used in a series of methyltransferase assays using luciferase activity as a readout. CRMT5 addition results in luciferase activity in the presence of cytosine‐containing RNA (T. brucei total RNA and Poly‐IC RNA). There was little to no luciferase activity observed in the presence of RNA that lacks cytosine or when a mock purification from E. coli without the CRMT5 gene was used. Our next step will be to perform a methyltransferase reaction with CRMT5 and subsequently isolate the RNA for bisulfite sequencing to confirm the methylation of cytosine bases. Evidence for the presence of 5‐methylcytosine and RNA methyltransferases indicates the presence of a process to create an epitranscriptome in T. brucei.

Support or Funding Information

NIH, 1R15AI133428‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Melanoma Antigen Genes (MAGEs): Drivers or Passengers in CARcinogenesis
Authors: Johnson Jodiene
Published: June 2021

Cancer Testis Antigens (CTAs) comprise a group of genes that are ordinarily expressed in the testis but abnormally expressed in a variety of cancers. The ability of CTAs to elicit an immune response makes them attractive targets for cancer immunotherapy. Melanoma Antigen Genes‐A (MAGE‐A) are Type I CTAs whose expression in cancers have been correlated with poor patient prognosis. The biological function of several members of the MAGE‐A gene family in cancer remains unclear. We have shown that MAGE‐A gene expression is regulated by epigenetic mechanisms such as CpG methylation. Our data indicate that several MAGE‐A genes can act as single gene drivers of cell proliferation and anchorage‐independent growth. We will investigate the molecular mechanisms that MAGE‐A5, ‐A8 and ‐A9 use to enhance cell proliferation by performing RT‐qPCR arrays for cancer‐associated genes and determine binding partners of these MAGE proteins using well‐established IP‐Mass/Spectrometry methods.

Support or Funding Information

NSF Research Initiation Award to S.R. #HRD1764201; Department of Education Award # P382G170104 (TITLE‐VII)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A non‐enzymatic function associated with a putative histone demethylase regulates heterochromatin spreading and inheritance
Authors: Raiymbek Gulzhan , An Sojin , Khurana Nidhi , Gopinath Saarang , Biswas Saikat , Larkin Ajay , Trievel Raymond , Cho Uhn‐Soo , Ragunathan Kaushik
Published: June 2021

Heterochromatin is characterized by histone hypoacetylation and an enrichment of methylation marks on histones which are associated with epigenetic silencing. In fission yeast, a single H3 methyltransferase Clr4 (Suv39h) directs all of the methylation of H3K9 i.e., H3K9me2/3 which enforces the establishment of transcriptionally silent regions in the genome referred to as heterochromatin. A putative histone demethylase, Epe1, is recruited to sites of heterochromatin via interaction with a chromo‐domain containing protein Swi6, homolog of HP1. The loss of Epe1 has been associated with the propagation of silent epigenetic states in a DNA or RNA sequence independent manner. How Epe1 carries out its function as an anti‐silencing factor remains unknown. Despite having sequence homology with known histone demethylases, Epe1 exhibits no detectable demethylase activity in vitro. Here, we demonstrate that mutations within the catalytic JmjC domain of Epe1 disrupt its interaction with Swi6. Using a combination of genetic and biochemical assays, we show that the interaction between Epe1 and Swi6 is direct, depends on the active conformation of the JmjC domain and is sensitive to mutations that affect co‐factor binding. Surprisingly, in cells the interaction between Epe1 and Swi6 requires functional heterochromatin. We reconstituted this dependence in vitro and demonstrate that the presence of an H3K9 tri‐methyl peptide enhances the interaction between the two proteins. We attribute this stimulatory effect of H3K9 methylation to an auto‐inhibitory role for the C‐terminal region of Epe1. Deleting this segment resulted in a dramatic increase in Swi6 binding and no further stimulation in the interaction between Epe1 and Swi6 upon addition of an H3K9me3 peptide. Finally, we demonstrate that this non‐enzymatic role of Epe1 regulates the binding of a histone deacetylase. Our work demonstrates that Epe1 selectively binds and inhibits heterochromatin bound Swi6 molecules which in turn impedes downstream heterochromatin assembly.

Support or Funding Information

University of Michigan

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Open complex stability regulates transcription initiation by E. coli RNA polymerase from the rrnB P1 promoter
Authors: Liao Guanyu , Henderson Kate , Wang Hao‐Che , Record M. Thomas
Published: June 2021

Initiation of transcription by E. coli RNA polymerase (RNAP) begins with specific binding to promoter DNA and ends with promoter escape. For the four T7A1 and λPR promoter/discriminator combinations, we found that the discriminator determined the lifetime and stability of the open complex and the escape point of RNAP in initiation from productive promoter complexes (1). Quantitative initiation studies with these promoter/discriminator combinations reveal that the RNA‐DNA hybrid length for promoter escape and the length distribution of short (abortive) RNA released before escape both increase with the lifetime or stability of the initiation‐competent open complex (OC). The escape point for productive complexes increases from the 7‐mer to 8‐mer step for T7A1 discriminators to the 10‐mer to 11‐mer step for λPR discriminators. Nonproductive complexes make a short RNA smaller than the escape length and stall, slowly releasing it and reinitiating. These findings led us to predict that escape from a promoter like the ribosomal rrnB P1 promoter, with an unstable OC, should occur at a very short RNA‐DNA hybrid length and without release of short RNAs (1). Currently, to obtain the experimental data to test this prediction, we are performing single round transcription assays for the rrnB P1 promoter and other promoters with the rrnB P1 discriminator. In preliminary studies, we have found NTP concentrations where a significant fraction of promoters initiate rapidly. For these NTP concentrations, we find that only the shortest RNAs (e.g. 3‐mer) are made by nonproductive complexes during the time period required for productive complexes to escape and begin elongation. In addition, initiation of λPR promoter with rrnB P1 discriminator shows similar pattern comparing to initiation of rrnB P1 promoter with its own discriminator. We conclude that the current results are consistent with the proposal of early escape of RNAP from promoters with unstable OC.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thermodynamic Analysis of a Bulged‐G Motif
Authors: Moore Ethan , Aman Zach , Grover Neena
Published: June 2021

The bulged‐G motif has been recognized in the crystal structure of ribosomal RNA as it contains an S‐turn in the phosphodiester backbone. The S‐turn is formed by a G•U‐A base triple that has a non‐canonical A‐G base pair above the base triple, and an adenine bound to either a guanine or uracil below the base triple. The base triple contains the bulged G nucleotide and is a binding site for elongation factors which ensure that proper translation occurs; this same RNA also binds to sarcin and ricin that inhibit ribosomal function. A similar S‐shape in an RNA backbone was also found to occur in the bacterial c‐di‐GMP‐II riboswitch that contains a A•C‐G base triple. In this study, thermal denaturation and isothermal titration calorimetry (ITC) experiments were performed to delineate the role of specific nucleotides in the formation of the S‐turn and its interaction with magnesium ions. A wild type RNA construct was derived from the bulged‐G motif that contained the G•U‐A base triple flanked by the non‐canonical base pairs, and an RNA triple substitution construct was designed that replaced the base triple with a A•C‐G. The wild type RNA and the base‐triple‐substituted constructs had similar stabilities in 1 M KCl buffer at pH 7.0 and 5.5. RNA constructs are additionally stabilized in 10 mM magnesium, with 2.63 kcal/mol gain in stability for the wild type and 1.45 kcal/mol for A•G‐C constructs. ITC experiments were performed to measure the enthalpy, entropy, and the binding affinity of magnesium ions to RNA. Results for wild type and modified constructs will be presented.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐Function Analysis of the 3′ CITE from the Blackcurrant Reversion Nepovirus Genomic RNA 2 (BRV2)
Authors: Bean Scott‐Wesley M , Baquero Galvis Laura D. , Morrison Evan J. , Filbin‐Wong Megan E.
Published: June 2021

Canonical eukaryotic translation initiation is enhanced by interactions between the 5′ 7‐methylguanosine cap (m7G) and the 3′ poly(A) tail, which exist on opposite ends of the mRNA molecule in the untranslated regions (UTRs). Viruses, however, hijack translation components using non‐canonical methods; typically differing in the structure of their 5′ and/or 3′ UTRs. Blackcurrant reversion nepovirus (BRV), a virus that infects the blackcurrant plant, has a genome consisting of two positive‐sense RNAs (RNA1 and RNA2). Neither RNA has a 5′ m7G cap, yet they both have a poly(A) tail. Structures within the 3′ UTRs of RNA1 and RNA2 called cap‐independent translation enhancers (3′ CITEs), are hypothesized to interact with a region of their 5′ UTRs. These distinct structural interactions facilitate the recruitment of essential initiation factors needed for translation, but this exact mechanism is not well understood. It is thought that the 5′ and 3′ UTRs possess conserved and complementary sequences which facilitate interaction. This study seeks to elucidate the specific secondary structure of the RNA2 3′ CITE and to correlate such to its role in translation initiation. Using in silico modeling of folded RNA2 and in vitro RNA structural probing, we determined a preliminary secondary structure for the 3′ CITE. We also identified structural changes when the putative 5′ ‐ 3′ UTR complementarity is disrupted. Using a luciferase reporter gene flanked by the RNA2 3′ CITE and wild‐type BRV2 5′ UTR, we show how the putative 5′ ‐ 3′ UTR complementarity is required for the quantity of and the rate at which luciferase protein is made. We conclude that the 3′ CITE is important for promoting translation initiation and the putative 5′ ‐ 3′ UTR complementarity between the ends of the viral RNA is necessary but not sufficient for protein production. The structure‐function relationship of the BRV RNA2 3′ CITE will lead to a clearer understanding of how BRV can be controlled to prevent Blackcurrant Reversion Disease (BRD) and shed light on vital processes that govern this unique class of nepoviruses.

Support or Funding Information

Colorado‐Wyoming Alliance for Minority Participation in STEM

MSU Denver College of Letters, Arts and Sciences Dean's Seed Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring Clustered Regularly Interspaced Short Palindromic Repeats Diversity in Soil Bacteria
Authors: Hassan Sherouk , Shah Rameen , Wei Yufeng
Published: June 2021

Clustered regularly interspaced short palindromic repeats (CRISPR) is a defense system found in bacteria (and archaea) against bacteriophages, viruses that infect bacteria. CRISPR DNA sequence consists of short, identical nucleotide repeats that are interspaced by spacers. The spacer sequences are records of viral infections experienced by the bacteria and play the role of an “immunization card”: if the spacer sequences match the infecting viral DNA, the bacteria will cut the viral DNA with CRISPR‐associated (Cas) proteins, eliminating the threat of viral infection. We speculate that the spacer sequences reflect the history of bacteriophage infections, which are unique to the environment bacteria reside in. Environmental factors, such as location and time, weather, local ecosystems, and pollutions, will alter the spacer sequences found in bacteria. This research focuses on investigating and discovering new spacer sequences from soil samples collected around NY/NJ metropolitan area, a historically heavily polluted urban ecosystem, at various locations, timepoints, and weather conditions. To accomplish this goal, the genomic DNA was extracted and purified from the collected soil samples. The CRISPR locus was amplified by using polymerase chain reaction (PCR) and then purified via gel electrophoresis. The purified CRISPR DNA was then ligated into a p‐Drive vector and transformed into M15 competent cells. The cells were cultured to amply the vector containing ligated CRISPR sequence, which was subsequently analyzed by aligning against CRISPR spacer database to identify new space sequence. We expect that the newly discovered spacer sequences reflect the history of viral infections unique to the regions investigated, which can potentially inform the understanding of health risks related to environmental bacteria and viruses.

Support or Funding Information

‐ U.S. Education Department Minority Science & Engineering Improvement Program grant # P120A160084‐ NIH AREA Grant # CA179410‐ USED Title III Part F, HSI‐STEM & Articulation Grant #P03C160155

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Genetic Interactions Associated with Splicing Factor Prp5
Authors: Merkhofer Evan C. , Moresco Philip , Copeland Jacqueline
Published: June 2021

Regulation of gene expression occurs at multiple levels, including the removal of non‐coding sequences from protein‐coding pre‐messenger RNA transcripts, commonly known as pre‐mRNA splicing. Correct gene expression requires high fidelity of pre‐mRNA splicing, as up to 15% of mutations leading to disease may be a result of aberrant pre‐mRNA splicing, including cystic fibrosis, spinal muscular atrophy (SMA) and some leukemias. Pre‐mRNA splicing is a multistep process that requires several proteins and RNAs for ATP hydrolysis, including multiple essential DEAD‐box proteins such as Prp5. Here we investigate the role of the DEAD‐box protein Prp5 in splicing using the yeast Saccharomyces cerevisiae as a model. Prp5 has previously been shown to be required for ATP‐dependent RNA remodeling in splicing, facilitating binding of the U2 snRNA to the branchpoint sequence in the pre‐mRNA intron. In addition, unpublished research has revealed genetic interactions between Prp5 and non‐splicing genes such as Gcn5 and Spt8. In this study, additional potential genetic interactions involving Prp5 were investigated. To identify these interactions, a transposon mutagenesis screen was employed. Approximately 20,000 mutations were screened, 61 of which showed putative genetic suppression of the temperature sensitive prp5‐1 mutation. Subsequent growth assays confirmed that many of these mutations exhibited genetic suppression, indicating important genetic interactions between these genes and Prp5. hiTAIL PCR was utilized to identify a number of these genetic interactions, including an interaction between Prp5 and the MMO1 gene, a verified protein in S. cerevisiae with unknown function. Additional experiments to characterize this interaction with MMO1 are currently being performed. Furthermore, we have used high‐throughput sequencing to identify an additional subset of the interactions from the transposon screen. This study will help shed light upon the role of the DEAD‐box protein Prp5 in pre‐mRNA splicing.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Searching for the Function of Ecm14, a Pseudopeptidase, Using a Synthetic Lethal Assay
Authors: McDonald R Christian , Lyons Peter J
Published: June 2021

Many enzyme families contain both catalytically active and inactive members. Although these inactive enzyme‐homologs (pseudoenzymes) share significant similarity with their active counterparts, key active site modifications seem to suggest a loss of traditional catalytic activity. The human M14 metallocarboxypeptidase (MCP) family is one such enzyme family that contains several active and inactive members. The baker's yeast Saccharomyces cerevisiae contains only one M14 MCP member, Ecm14, which is produced by a non‐essential gene in the yeast genome. We show through a phylogenetic analysis that this gene is present throughout the ascomycete lineage of fungi. Although Ecm14 is very similar to human CPA1 (a catalytically active member of the M14 MCP family), multiple key modifications in Ecm14 seem to indicate a loss of traditional peptidase‐like activity. A key technique for elucidating the function of non‐essential genes, including those producing pseudoenzyme products, is the genome‐wide synthetic lethal screen. Biochemical pathways typically involve multiple gene components and many of these gene players provide redundancy in the event that other genes experience deleterious mutations. The synthetic lethal screen is used to identify novel mutant yeast strains whose survival is dependent on a particular gene of interest. Colonies that contain a putative synthetically lethal gene pair are kept alive and identified by harboring a color‐reporting plasmid that complements the deletion of the endogenous gene of interest. Here we demonstrate the insertion of the wild‐type ECM14 gene into this integral pSLS1 plasmid and the determination of gene insert orientation using restriction endonucleases. Additionally, we demonstrate the preparation of knockout yeast strains using drug resistant deletion cassettes, the transformation of these yeast with the above plasmid, and finally, genome‐wide mutagenesis attempts using the chemical mutagen ethyl methanesulfonate. Identification of synthetic lethal genes will be presented, as a step toward a better understanding of the biological role of Ecm14.

Support or Funding Information

Graduate Student Grants in Aid of Research to RCM and Faculty Research Grants to PJL from the Office of Research & Creative Scholarship, Andrews 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.
Unveiling the Impact of the Core Circadian Clock Protein FREQUENCY on Output in Neurospora crassa
Authors: Mosier Alexander Eric , Hurley Jennifer Marie
Published: June 2021

Circadian rhythms are cellular oscillations that tune an organism's biology to the 24‐hour light/dark cycle on Earth. This cellular time‐keeping mechanism aids in the anticipation of environmental changes and increases organismal survival. These rhythms are maintained by a transcriptional/translational negative feedback loop, called the core circadian clock, that is evolutionarily conserved throughout most branches of life. In this core clock, positive arm proteins act to transcriptionally activate negative arm proteins. The negative arms proteins act back on the positive arm, in a time specific manner, to halt the negative arm's activation before degradation and re‐initiation, thus setting the period of the clock. Beyond this core clock, positive arm proteins also act to transcriptionally activate a variety of other genes that do not participate in the core clock mechanism in a time dependent manor, referred to as clock‐controlled genes (ccgs.) Most research into the mechanisms of circadian regulation on output has focused on transcriptional activation by the positive arm proteins. However, recently published work has demonstrated that there are many oscillating proteins for which the corresponding mRNA is not rhythmic. This has led to the theory that the regulation of circadian output is also accomplished via post‐translational mechanisms, which we hypothesize are imparted by the negative arm proteins.

In the model organism Neurospora crassa, the negative arm comprises FREQUENCY (FRQ), Frequency‐Interacting RNA Helicase (FRH), and Casein Kinase 1 (CK‐1). Importantly, FRQ has been computationally and experimentally proven to be an intrinsically disordered protein (IDP). Aside from lacking a single, predetermined three‐dimensional structure, IDPs play key roles in cellular signaling and regulation due, in part, to the transient but highly specific and varied interacting partners they have. These attributes make FRQ a prime candidate to impart post‐translational circadian regulation. The aim of this study is to characterize protein complexes, centered around FRQ, that change in a time specific manner, to determine the impact these interactions could impart post‐translationally on circadian regulatory pathways. To this end, Neurospora samples where crosslinked and purified through co‐immunoprecipitation before protein identification through liquid chromatography mass spectrometry. Initial results suggest that FRQ plays a role in post‐translational regulation on metabolism, the cell cycle, and a variety of other functions.

Support or Funding Information

This work was funded through the support of the NIGMS/NIH R35GM128687 grant and the Rensselaer Polytechnic Institue Start Up Fund.

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