bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutral sphingomyelinase 3 modulates myotube density and is regulated by microRNA‐133
Authors: Wolf Erin M. , McCarthy John J. , Moylan Jennifer S. , Smith Jeffrey D. , Reid Michael B.
Published: June 2021

The neutral sphingomyelinase (nSMase) family is a group of enzymes that catalyzes the conversion of sphingomyelin to ceramide in a pH dependent manner. Sphingomyelinase activity and sphingolipid signaling play a role in the regulation of cellular processes. Our current data suggest that a novel, muscle‐specific neutral sphingomyelinase, nSMase3, may regulate muscle cell size. Overexpression of nSMase3 in C2C12 myotubes causes a decrease in myotube density, while treatment with an nSMase3 siRNA causes an increase in myotube density. MicroRNA‐133 (miR‐133) is a muscle‐specific small RNA molecule that has roles in proliferation and differentiation of myoblasts. A sequence within the 3′ untranslated region (UTR) of nSMase3 contains a predicted miR‐133 binding site. We hypothesize that miR‐133 inhibits nSMase3 translation and therefore plays a role in its regulation of cell size. In support of our hypothesis, expression of miR‐133 progressively increased while nSMase3 protein levels decreased during myotube differentiation. To test our hypothesis, a luciferase reporter gene containing the 3′ UTR of nSMase3 was cloned and used in a transient transfection assay. Co‐transfection of the reporter gene and miR‐133 decreased luciferase activity. These data suggest that miR‐133 is capable of regulating the expression of nSMase3 and, thus, may have a role in the regulation of muscle size.
Loss of Alpha(E)‐Catenin: Potential Role in the Renal Disrepair Following Injury in the Aging Kidney
Authors: Nichols LaNita A. , Grunz‐Borgmann Elizabeth A. , Parrish Alan R.
Published: June 2021

As the kidney ages, morphological and functional changes lead to renal dysfunction. Clinical data indicates decreased ability to repair following acute insult which leads to increased susceptibility and severity of acute kidney injury (AKI). We have previously shown the aging kidney has a marked loss of α‐catenin expression in the proximal tubular epithelium. We hypothesize that the loss of this protein may be involved in the decreased repair of the aging kidney. To study the effects of reduced α‐catenin, shRNA knock‐down of α‐catenin was generated in NRK‐52E cells. Single cell clones were selected for a non‐targeted (NT3) control and a targeted knock‐down (C2). The 75% reduction of α‐catenin mRNA and protein levels was stable over several passages compared to the NT3 control. Interestingly, the catenin knockdown affected expression of other components of the cadherin/catenin complex. Most notably, mRNA expression of N‐cadherin was reduced >;99% and protein was undetectable in C2. This was paralleled by a decrease in cadherin/catenin function as assessed by aggregate formation, as well as inhibition of wound healing as determined by a scratch assay. NextGen RNA sequencing revealed alterations in several genes which are implicated in renal repair, including proliferation/growth factor signaling, and cytoskeletal organization. This work was supported by NIH AG034154.
Combining case studies and concept maps within a single assignment creates a powerful teaching tool for physiology
Authors: Watson Cheryl L
Published: June 2021

Case studies provide an opportunity for students to apply the principles they learn during a physiology course. Similarly, concept maps are an excellent construct for training students to think causally and mechanistically. Concept maps train students to think as physiologists. While both pedagogical techniques are excellent, they are even more powerful when combined. Course management using these techniques requires that a case study involving normal physiology be provided with each unit. The student assignment is to “solve” the case study using a concept map format. This requires students to 1) identify mechanisms in order of occurrence 2) indicate causality 3) indicate feedback mechanisms 4) identify concurrent physiological mechanisms. Using these techniques students learn physiology and simultaneously learn to ask higher level questions as they create mechanistic maps. Grading these assignments, an instructor can easily identify misunderstandings or confusion. Unlike essays, concept maps do not allow obfuscation, and errors are apparent. After twelve years of using this technique, I've found that students retain their maps and use them for study in health care professional schools.
Converting an Undergraduate Research Program into an Undergraduate Cardiovascular Physiology Class
Authors: Gatson Sarah N. , Quick Christopher
Published: June 2021

Using higher‐order thinking skills (HOTS) in lower‐division classes is hampered by the belief that it is necessary to build upon foundational knowledge. The panacea of implementing high‐impact educational practices (HIEPs) is hampered by the belief that they require extensive resources. The DeBakey Undergraduate Research Program evolving at TAMU since 2003 may offer a novel solution. Complementary cardiovascular research projects are assigned to teams consisting of three multidisciplinary, multilevel undergraduates lead by a graduate student interested in learning to mentor and manage projects. Neophytes learn from more experienced teammates, and teams produce video lectures about their project to enhance inter‐team collaboration. Synergy amongst the HIEPs (learning communities, collaborative projects, research) made it possible to implement them without additional resources. Using mathematical models, students create hypotheses, evaluate results to determine validity, and analyze literature for novelty of predictions. The resulting discovery of knowledge is new either to the scientific community (research) or the student (education). In fact, our research program introduces HOTS (create, evaluate, analyze) by inadvertently flipping Bloom's revised taxonomy of educational outcomes. We thus converted it into a stacked, “flipped”, cornerstone cardiovascular physiology course in 2013.
Emerging changes in medical student procurement in the USA
Authors: Lee Tulp Orien , Einstein George P. , Bailey R. Keith , Robinson Bruce H.
Published: June 2021

In the past century, the world has witnessed the greatest increase in its population and longevity in history due to combined effects of scientific advances in medical care, greater longevity & other factors. Sadly, the number of new physicians being trained in US Medical schools has not kept pace with population trends, resulting in a lower physician density. The emergence of mid level providers[MLPs] (PAs, NPs, and others} MLPs have made many contributions to the delivery of medical care, but to date have not been widely tapped to expand the physician provider base. MLPs are often adult learners or non‐traditional applicants [NTAs], and often have extensive collateral medical education and clinical experience. The results of our experience indicates that NTAs can compete favourably with traditional applicants and play an increasingly greater role in solving the US physician shortage, and ameliorate the strain on traditional academic institutions. Institutional support.
Development of a Game‐Based‐Learning App for exposing students to chemistry and life science as a research endeavor
Authors: Silva Thanuci , Galembeck Eduardo , Takase Rodrigo , Pelaez Nancy , Anderson Trevor Ryan
Published: June 2021

We have developed a gamified Virtual Learning Environment (VLE), called 3DClass, to deliver homework to chemistry and life science students through a user‐friendly and enjoyable interface. Homework from four courses was delivered using the 3DClass, a computer app that acts as an interface between Moodle and the Apple Game Center. This App allows users to play while learning or to learn while playing. The results show that this App can be used in a flexible manner with designs that can be customized to courses with different characteristics, such as with online collaboration or face‐to‐face lectures, and for participant numbers that vary for courses with 20 to 600 students. The design accommodates the delivery of quizzes, and 3DClass also allows students to compete with each other for achievements in the Apple Game Center. It can deliver video, images, and promote student interactions using a forum. The Open Quizzes can be taken without being registered in any of the courses and anyone can download the 3DClass App without cost at the Apple App store. Partial research supported by FAPESP and CNPq, Brazil, IIE, and a College of Science grant from Purdue University.
Does the Embalming Process Induce Pseudo‐Aortic Dissection?
Authors: Rae Guenevere , Husain Mujtaba , Mark Jennifer , Elsayed Galal , Elsayed Mohammed , Kostrubala Anastasia , Schumacher Paul , Swartz William J
Published: June 2021

The purpose of this study is to examine the incidence of aortic dissection and pseudo‐aortic dissection (dissection that has occurred as a result of the embalming process) in the gross anatomy lab. Eighty cadaveric specimens from two separate schools of medicine were screened for aortic dissection. Institution A received anatomical donations that had been initially embalmed by funeral services and embalmed a second time by the institution's director of anatomical facilities (N=31). Anatomical specimens screened at Institution B were subjected to a single embalming process at the institutional site (N=49). The incidence of suspected dissections from Institution A was 9.7%. The incidence of suspected dissections from Institution B was 6.1%. The cadavers suspected of incidence had their aortae removed and sectioned for gross and/or microscopic examination (N=3 from Institution A; N=3 from Institution B). Routine H&E preparations were used to confirm true aortic dissection after gross examination. Tissue changes that occur, as part of the embalming process, are important to identify when using aortic specimens for both teaching and research purposes. Results indicate that pseudo‐aortic dissections may occur during the embalming process and, consequently, resemble pathological disease.

Grant Funding Source: none
The expression of thyroid hormone receptor mRNA and myosin heavy chain isoforms in mouse neonatal masseter muscle growth
Authors: Miwa Yoko , Sato Iwao , Sunohara Masataka , Shimada Kazuyuki
Published: June 2021

Objectives

Thyroid hormone receptors (THRs) are composed of two isoforms, designated alpha and beta. It has been suggested that the thyroid hormone and THRs contribute to the regulation of masseter muscle development by affecting mitochondrial respiratory chain‐related biogenesis. However, the relationship between the mRNA transcript levels of THRs and myosin heavy chain isoforms (Myh1, Myh2, Myh4, Myh3, Myh6) in the mouse masseter muscle is unknown.

Methods

Quantitative real‐time RT‐PCR was used to measure the expression of THR mRNAs in the developing postnatal mouse masseter muscle.

Results

The expression of THR beta RNAs in the developing mouse masseter muscle decreased significantly from 0 to 10 days of age. Of the myosin heavy chains studied, Myh4 and Myh5 were the most highly expressed in the 10‐day‐old mice.

Conclusions

These data, along with the analysis of the RNA transcript levels of these genes, support a role for THRs as regulator of the growth and development of the postnatal mouse masseter muscle.
Mas‐related gene receptor D is expressed in human gut and modulates sensory pathways during intestinal inflammation in mouse models
Authors: Avula Leela , Buckinx Roeland , Pintelon Isabel , Adriaensen Dirk , De Winter Benedicte , Van Nassauw Luc , Timmermans Jean‐Pierre
Published: June 2021

We have previously shown that the Mas‐related gene receptor D (MrgD) is expressed de novo in the mouse intestine during inflammation, specifically in intrinsic sensory neurons and mucosal mast cells (MMCs). We have also shown that MrgD deletion leads to increased MMC recruitment and sensory neuropeptide expression. These findings indicate role(s) for MrgD in the regulation of mastocytosis and (mast cell mediated)‐sensory neuromodulation. To further examine the effect of MrgD on intestinal function, we compared the motility of the ileum of healthy and Schistosoma mansoni‐infected wild‐type and MrgD−/− mice. Furthermore, to determine the mechanism of activation of MrgD in MMCs, calcium live cell imaging was performed on mouse bone marrow‐derived mast cells (BMMCs). MrgD deletion did not alter intestinal motility indicating that MrgD‐induced changes only influence the sensory pathways during inflammation. The MrgD ligand â‐alanine evoked a Ca2+ influx in a subset of BMMCs expressing MrgD, suggesting that â‐alanine can mediate MrgD activation during intestinal inflammation. As in mouse, MrgD was expressed in neurons and mast cells in the human intestine, suggesting translational potential. The findings justify the further exploration of MrgD as an experimental target in intestinal inflammatory pathologies, focussing on sensory perception and neuro‐immune interactions. Supported by FWO grant G.0179.08

Grant Funding Source: N/A
Unilateral superficial ulnar artery arising from the axillary artery: a case report
Authors: Hartmann Carol , Hutchinson Erin
Published: June 2021

Knowledge of arterial variations within a clinical setting is essential. During routine dissection of the left upper limb of a 69 year‐old white, male cadaver, the ulnar artery was found to arise from the anterior aspect of the second part of the axillary artery. In its course the artery passed superficial to the medial and lateral roots of the median nerve and continued distally within the subcutaneous tissue on the medial aspect of the upper limb. No branches were observed in the arm or forearm before the artery passed through the ulnar canal to form the superficial palmar arch. The superficial ulnar artery positioned within the subcutaneous tissue of the upper limb is a notable variation due to the risk of intra‐arterial injection. This case further emphasizes the importance of all health care professionals being aware of clinically important variations.
An investigation of the dorsal scapular artery
Authors: Lichter Andrew , Raya Nischal , Saltzman Barbara S , Hankin Mark H
Published: June 2021

The dorsal scapular artery (DSA) arises in the root of the neck and courses along the medial scapular border. It may arise either from the subclavian (SCA) ‐ a direct DSA, or indirectly from SCA via the transverse cervical artery (TCA) ‐ an indirect DSA. This cadaveric study (N=56) assessed the frequency of each variant, including any side and sex differences. We found a direct DSA in 74% of cases and an indirect DSA in 26% of cases; slightly more direct DSAs were observed on the left than on the right (80% vs. 68%; P<0.05). Regardless of side, the direct DSA almost always originated from the 2nd or 3rd part of the SCA. Within an individual, the origins of the DSAs were symmetrical in 70% of cases (P=0.005). The pathway of the direct DSA was also stereotyped: 67% of direct DSAs passed between the superior and middle trunks of the brachial plexus; 29% passed between the middle and inferior trunks; 4% passed inferior to the plexus. The indirect DSA arose from the TCA, which coursed posteriorly across the superior trunk; the indirect DSA never went through or inferior to the plexus. Improved understanding of the DSA will aid in clinical practice (e.g., in trapezius flap procedures, brachial plexus blocks). The proposed nomenclature will not only improve communication among practitioners, but also offers a simple resolution to the nomenclature discrepancies in current anatomical and surgical literature.

Grant Funding Source: None
Asymmetry and vestigial structures in extant birds
Authors: Sartin Catherine E.
Published: June 2021

It is commonly asserted that vestigial structures have a greater degree of fluctuating asymmetry than functional structures. The reason given for this link between vestigial structures and fluctuating asymmetry is relaxed selection pressure, as well as fitness benefits associated with redirection of energy and other resources to functional structures. Perhaps surprisingly, few studies have examined and quantified the amount of fluctuating asymmetry in paired vestigial structures, especially limbs.

The present study is an attempt to fill this void and to test the following hypotheses: (1) Skeletal elements of limbs not used for locomotion will be more asymmetric than the skeletal elements of limbs used for locomotion (both within specimens and between groups), and (2) limb asymmetry will increase with time since the limb became vestigial. Four locomotor categories were examined: Tropicbirds (regularly use forelimb, but not hindlimb for locomotion), Flighted rails (regularly use both fore‐and hindlimb for locomotion), Grounded Rails (only use hindlimb for locomotion; lost flight recently), Ratites (only use hindlimb for locomotion; did not lose flight recently). Within each category, attempts were made to obtain a statistically‐significant sample (n ≥ 30) while still controlling for phylogeny and body size.

Eight bilateral measures were taken, including maximum lengths and condylar breadths. Dimenstions were converted into percentage directional (%DA) and absolute (%AA) asymmetries. Results reveal significant fluctuating asymmetry in the forelimb elements of flightless taxa; however, tropicbirds do no show significant asymmetry in their hindlimb elements. Despite a small sample size of ratites, results also suggest a greater degree of fluctuating asymmetry in the forelimbs with time since the limb became vestigial.

Grant Funding Source: Center for Functional Anatomy and Evolution, Johns Hopkins
The Effect of Posture on the Neck and Shoulder Suspension Apparatus of Humans: A 3D Biomechanical Analysis
Authors: Osborn Michelle L , Ge Jinghua , Melancon Keith P , Rau A Ravi P , Homberger Dominique G
Published: June 2021

Clinical issues arising from bad posture and misalignment of the neck can significantly affect the quality of life of individuals. An understanding of the physical configuration and biomechanics of the neck and its associated parts in various postures is fundamental for successful treatments. We have re‐conceptualized the human head, neck and shoulder complex as a shoulder suspension apparatus, and we hypothesize that habitual bad postures lead to changes in the balance of forces acting on this apparatus. We tested our hypothesis by comparing the forces acting on the shoulder suspension apparatus of persons with different postures as ascertained from clinical x‐ray images. We applied the free‐body diagram force analysis to 3D models of the shoulder suspension apparatus in good and poor postures, which were reconstructed from data of x‐ray CT scans of a patient. Our biomechanical model suggests that in a good posture the head and neck are stabilized by the core muscles, the nuchal ligament is relaxed, and the shoulders are suspended from the head by fascia and the connective tissue of the clavotrapezius and cleidomastoid muscles. In poor postures, various muscles must be recruited to re‐balance the shoulder suspension apparatus, which, in turn, may cause tightened muscles, myo‐fascial pain, poor circulation to the head, head‐aches, and inflammation of the joints.

Grant Funding Source: LSU Foundation to DGH
Radiofrequency preserves tendinous stiffness after experimental injury
Authors: Akamatsu Flávia Emi , Teodoro Walcy Paganelli Rosolia , Saleh Samir Omar , Martinez Carlos Augusto Real , Ribeiro Marcelo Lima , Andrade Mauro , Jacomo Alfredo Luiz
Published: June 2021

BACKGROUND

Calcaneous tendon injuries are commonly seen as consequences of sports trauma. Ideal management should provide rapid healing so early mobilization can avoid tissue adhesions. Radiofrequency (RF) produces heat by molecular friction caused by polarity changes and its therapeutic use aims reduction of the local inflammatory response. We investigated the action of the RF on the calcaneous tendon healing process after experimental lesions in rats.

METHODS AND MATERIAL

Thirty adult male Wistar rats weighing 250 to 300 g were surgically submitted to bilateral partial transverse section of the calcaneous tendon. The right tendon was treated with RF whereas the left tendon served as control. On the third postoperative day, the rats were divided into three experimental groups consisting of ten rats each which were treated with monopolar RF (Tonederm™) adjusted to 650 kHz and 1–2w, for two minutes twice a week, until they were sacrificed on the 7th, 14th and 28th days, respectively. We studied collagen I gene expression and immunofluorescent staining to evaluate collagen Type I and III.

RESULTS

A larger amount of collagen I without correspondent collagen I gene expression was observed within 7 days.

CONCLUSION

RF treatment preserves stiffness and accelerates the healing process of the calcaneous tendon and may be a useful tool in tendinous lesion management.
Human Achilles tendon: Absence of renewal during adult life revealed by nuclear bomb 14C
Authors: Heinemeier Katja Maria , Schjerling Peter , Heinemeier Jan , Magnusson Stig Peter
Published: June 2021

Tendons connect muscle to bone and transmit enormous, repetitive forces during movement. The prevalence of tendon overuse‐injuries is high, and these conditions are often prolonged due to inadequate tissue regeneration. Whether this relates to a slow tissue turnover is unknown since current methods provide diverging estimates of tendon protein half‐life that range from two months to 200 years. With use of the bomb pulse method we have studied life‐long Achilles tendon turnover and can show that human core tendon tissue is practically inert during adulthood. The extremely slow turnover of adult tendon is demonstrated by tissue concentrations of radiocarbon (14C), corresponding to the elevated atmospheric levels left over from nuclear bomb tests in 1955–1963, decades before the tendon tissue was sampled. We find that tendons contain a concentration of 14C approximately reflecting the atmospheric levels during the first 17 years of life, indicating that tendons are formed during height growth, but are essentially inert thereafter. Our observations resolve a basic question in tendon physiology and firmly disprove previous suggestions that tendon tissue is turned over at a rate comparable to the fast turnover of skeletal muscle. The absence of tendon tissue renewal during adulthood is a fundamental premise for understanding tendon function and for future research in tendon pathology and treatment of tendon injury.
Remodeling and Regeneration in the Goldfish Heart
Authors: Haag Maria T , Grivas Jamie A , Lafontant Pascal J
Published: June 2021

A remarkable ability for heart regeneration has been demonstrated in the zebrafish and giant danio, two members of the cyprinids family. While regenerative capacities appear widely distributed within genera, they can vary significantly in closely related species. Here we describe using light and electron microscopy the injury response and repair mechanisms in the heart of another cyprinid, the goldfish, following cautery injury to a small portion of the ventricular myocardium. We observed a robust inflammatory response in the first week consisting of infiltrating macrophages and heterophils. These inflammatory cells were identified in the lumen of spongy heart and in the compact layer of myocardium both adjacent and remote to the injury. Marked accumulation of collagen was observed during the second week in a border zone between healthy and injured myocardium, in adjacent sub‐epicardial regions, and was concurrent with a robust angiogenic response. The replacement of the ablated region of the ventricle was achieved by six weeks post‐cautery. PCNA‐positive cardiac myocytes (CM), as well as apparent de‐differentiated cardiac CM were present during repair. This data suggest that effective regeneration mechanisms operate in the injured goldfish heart, similar to that demonstrated in zebrafish and giant danio, and that cardiac regeneration may be widespread among cyprinids.

Grant Funding Source: none
Coordinated processing of 3′ slipped (CAG)n/(CTG)n hairpins by DNA polymerases preferentially induces repeat expansions
Authors: Guo jinzhen , Chan Nelson , Zhang Tianyi Zhang , Hou Caixia , Wu Jianxin , Gu Liya , Li Guo‐Min
Published: June 2021

Expansion of CAG/CTG trinucleotide repeats causes certain familial neurological disorders. Hairpin formation in the nascent strand during DNA synthesis is considered a majorpath for (CAG)n/(CTG)nexpansion. However, the underlying mechanism is unclear. We show here that removal or retention of a nascent strand hairpin depends on hairpin structures and DNA polymerases involved. Pol delta alone removesthe 3′slipped hairpin using its proofreading activity when it contains no immediate 3′complementary sequences. However, in the presence of pol beta, pol delta preferentially facilitates hairpin retention regardless of hairpin structures. In this reaction, pol beta incorporates several nucleotides to the hairpin 3′end, which serves as an effective primer for the continuous DNA synthesis by pol delta, thereby leading to repeat expansion. These findings strongly support a mechanism that involves polymerase switching between pol delta and pol beta in coping with 3′slipped (CAG)n/(CTG)nhairpins during DNA synthesis.
TDG excision of fC may be a predominant element of pathways for active DNA demethylation
Authors: Maiti Atanu , Hwang Bor‐Jang , Armwood Cherece J. , Lu‐Chang A‐Lien , Drohat Alexander C.
Published: June 2021

Base excision repair (BER) is essential for maintaining genetic integrity and for active DNA demethylation, a central element of epigenetic gene regulation. A key player in both processes is thymine DNA glycosylase (TDG), which excises modified forms of 5‐methylcytosine (mC). TDG excises thymine from mutagenic G/T mispairs arising from mC deamination. TDG also excises 5‐formylcytosine (fC) and 5‐carboxylcytosine (caC), oxidized forms of mC generated by Tet enzymes. TDG is essential for embryonic development, reflecting a crucial function in regulating gene expression that likely involves a key step in active DNA demethylation. We used structural, biochemical, and cell‐based methods to understand how TDG attains specificity for excising select forms of modified mC (i.e., T, fC, and caC). We identified a TDG variant that retains fC activity, but lacks detectible caC activity, indicating a fundamental difference in the excision mechanism for these related bases. We used this variant to study the role of TDG in active DNA demethylation in human cells. The results indicate that TDG excision of fC can account for findings that caC is depleted in cells expressing TDG, consistent with the fact that fC is a precursor for Tet‐mediated formation of caC. The results suggest that TDG excision of fC could be a predominant element in a pathway for active DNA demethylation. Supported by the NIH (R01‐GM072711).
Rational design of an aptamer‐based biosensor for potassium ions
Authors: Diegelman‐Parente Amy
Published: June 2021

Biosensors provide a unique platform for analyte detection, generating a dose‐dependent response through a binding‐specific conformational change. While most ligand‐binding biomolecules are not biosensing by default, many can be engineered to be so through the introduction of an alternative nonbinding (and thus nonsignaling) conformation. While this strategy has been met with limited success with proteins, the sequence‐structure‐function relationship inherent with oligonucleotides has led to the creation of structure‐switching aptamer‐based biosensors for many target ligands. G‐quartet sequences (GQS) generate a characteristic structural motif in the presence of potassium ions. While previous work has been done to engineer this motif into structure‐switching biosensors for potassium, no work has been done to edit their dynamic ranges. Here, we report the rational design of six sequences to bind potassium with different apparent binding affinities. We demonstrate a correlation between the experimentally determined binding affinity for potassium (via circular dichroism) and the theoretically determined free energy of the alternative structure (determined computationally by nearest‐neighbor parameters). This work demonstrates the ability to rationally design an aptamer‐based biosensor to operate in any desired range of analyte concentration weaker than the intrinsic binding constant.
Triiodothyronine indirectly increases amphiregulin gene expression levels via estrogen receptor activation
Authors: De Sibio Maria Teresa , Luvizon Aline Carbonera , Conde Sandro Jose , de Oliveira Miriane , Olimpio Regiane Marques Castro , Moretto Fernanda Cristina Fontes , de Melo Azevedo Luvizotto Renata , Fecchio Denise , Nogueira Celia Regina
Published: June 2021

The objective of this study was to determine whether triiodothyronine hormone (T3) exerts direct or indirect modulation in amphiregulin (AREG) gene expression. To elucidate the modulation type, MCF‐7 cells were treated with T3 (10−8M) for 60 minutes in the presence or absence of fulvestran (estrogen receptor inhibitor ‐ ICI) or cycloheximide (protein translation inhibitor – CHX). Gene expression was assessed by RT‐qPCR, considering statistical significance level at 5%. Treatment with T3 increased amphiregulin 1 gene expression (± 0.16, control group) to 13.93 (± 2.09, P <0.001). When treating cells with T3 and ICI simultaneously, amphiregulin mRNA expression decreased to 9.74 (± 1.44, P <0.05) and when treating cells with T3 and CHX, amphiregulin mRNA expression decreased to 0.88 (± 0.07, P <0.001). These results demonstrate that T3 increases amphiregulin gene expression via estrogen receptor. The decrease in amphiregulin expression by CHX in the group treated with T3 demonstrates that such modulation occurs indirectly, i.e., there is a need of protein synthesis prior to gene modulation. Financial support: FAPESP: 2009/15607–2
Duration of anatomy courses and performance on the USMLE Step 1 and Step 2 Examinations: Is there a relationship?
Authors: Pawlina Wojciech , Cuddy Monica M. , Swanson David B. , Drake Richard L.
Published: June 2021

During the past two decades, anatomy instruction has changed due to curricular reforms in medical schools. Reduction in total hours of anatomy course and cadaver dissection, as well as shifts towards an integrated curricula has raised questions whether changes have had a negative impact on the students’ knowledge of anatomy. Our purpose was to investigate the effect of changes in course hours, curriculum type, and laboratory experience on USMLE Step 1 and Step 2 CK scores. Results of 2009 AAA online survey of anatomy course directors from 54 LCME‐accredited schools were matched with USMLE scores for 6,411 examinees taking Step 1 for the first time in 2009. The USMLE scores included Step 1 total test scores, Step 1 gross anatomy sub‐scores, and Step 2 CK total test scores. Analyses of data indicate that for all three USMLE scores, examinees from schools with stand‐alone gross anatomy courses slightly outperformed examinees from schools with integrated curricula. Conversely, the number of course hours had no effect on how students performed on Step 1 or Step 2 CK. Examinees that were exposed to both dissection and prosection slightly outperformed on Step 2 examinees that had dissection only. While these relationships are statistically significant, they may not be practically meaningful and more research is needed to fully explore different aspects of anatomy curricula and retention of anatomy knowledge.
SWI/SNF remodels SIR3 heterochromatin during transcription
Authors: Rege Mayuri M , Peterson Craig L.
Published: June 2021

Heterochromatin in S. cerevisiae is assembled and maintained by the Sir proteins, primarily Sir2p, Sir3p, and Sir4p, at telomeres and the silent mating type loci. In addition to these canonically silenced loci, Sir3p is also found within euchromatin, where it appears to hinder gene transcription. Recent work from our lab has shown that the chromatin remodeling enzyme SWI/SNF, can evict Sir3p from nucleosomal arrays in vitro, and this activity can promote homologous recombination in vitro and in vivo. We have asked if SWI/SNF could also alleviate potential blocks to transcription caused by Sir3p. Consistent with this possibility, we find that the growth defects of a swi2Δ are partially suppressed by deletion of SIR3. In contrast, inactivation of either SIR2 or SIR4 does not suppress the phenotypes of a swi2Δ. Gene expression analysis of swi2Δ and swi2Δ sir3Δ mutants have revealed a group of genes involved in cell‐cycle progression and cell wall organization in which SWI/SNF appears to control their expression by antagonizing Sir3p. Taken together, our studies suggest a new role for SWI/SNF to promote transcription via remodeling Sir3p in euchromatin.
Pan‐omic Approaches to Study the Effect of Microgravity on Skin Endothelial Cells Submitted to Endotoxic Insult
Authors: Santos William J , Waddy Edward , Chakraborty Nabarun , Gautam Aarti , Hoke Allison , Jett Marti , Hammamieh Rasha
Published: June 2021

Microgravity enhances bacterial virulence but depresses host immune response. Presumably, pathogenic insults occurring in space may trigger altered immunological pathways in host cells. Understanding this paradigm is essential to establishing proper wound care management. In this study, human microvascular endothelial cells were exposed to lipopolysaccharide (LPS), an endotoxin commonly found in gram‐negative bacteria, for two different time periods (4 h and 8 h). At the end of each time point the cells were fixed in RLT buffer and maintained in a fixed state until after the Space Transport System (STS) ‐135 landed.

Genomics and transcriptomics analysis using high density mRNA and miRNA arrays and coherent functional analysis revealed the arrest of certain key immunological networks, elevated risk of tumorogenesis and neurological degeneration as a result of microgravity. Eliciting more disease footprints like osteo‐atrophy and adenocarcinoma, LPS assault at reduced gravity appears more lethal than at terrestrial gravity. Reduced gravity further altered angeogenesis and vasulogenesis promoting rheumatoid arthritis, tumor growth, and wound repair, some of the common ailments noticed in astronauts. The results were validated using a proteomics panel investigated by Luminex platform, a classic ELISA assay.
Expression of mRNAs for two novel isoforms of mouse heat shock factor 1 in mouse cells and tissues
Authors: Bachman Nancy Jane , LaPilusa Tami , Gong Tze‐Wen , Lomax Margaret I
Published: June 2021

Heat shock transcription factor 1 (HSF1) mediates the response to heat in all cellular organisms. On exposure to heat, the HSF1 in cells refolds, enters the nucleus and switches on genes encoding protective heat shock proteins (hsps). Two isoforms of mammalian HSF1, denoted α and β, were previously known to be derived by alternate splicing of an additional coding exon. These forms differ by the presence of an additional 22 amino acid segment in the HSF1α protein as compared to β. Our previous studies show that these isoforms switch on different subsets of hsp genes and to different extents; HSF1α is generally a better activator of hsp genes. This study provides evidence for expression of the mouse mRNAs for two additional isoforms, also derived by alternate splicing, containing an additional 28 amino acid segment, denoted HSF1γ and δ. Existence of these new isoforms was predicted by bioinformatic analysis of HSF1 expressed sequence tags using the basic local alignment search tool (BLAST). Comparative bioinformatic analysis suggests many vertebrate species, including zebrafish, clawed frogs, mice, and humans possess these isoforms. Based on reverse‐transcriptase PCR analysis, the HSF1γ isoform mRNAs from mouse fibroblast cells and various mouse tissues may be widely expressed, both before and after heat shock, while HSF1δ is expressed only in certain tissues. Experiments are underway to construct mouse expression plasmids that can be used to test the ability of HSF1γ and δ isoforms to activate hsp target genes in mouse fibroblasts to begin to identify their functions. Supported by NIH NIGMS grant 1 R15 GM096231–01.
Induced pluripotent stem cells as tools for disease modelling and drug discovery in Alzheimer's disease
Authors: Ooi Lezanne
Published: June 2021

The use of induced pluripotent stem cells (iPSCs), whereby a patient's somatic cells can be reprogrammed to a pluripotent state by the forced expression of a defined set of transcription factors, has the potential to enable in vitro disease modelling and be used for drug discovery programs. Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder that leads to a decline in memory and cognition. Fibroblasts were taken from AD patients (or non‐AD controls) and cultured under specific conditions to generate iPSCs which were then provided with growth factors to allow differentiation into neurons. While AD‐iPSCs were morphologically indistinguishable from control‐iPSCs, differentiated cells showed differing responses to both cellular stresses and neuroprotective drugs. Since these cells are derived from individual patients, the use of iPSCs has provided novel insights into disease pathogenesis, providing information on an individual's variations in the disease process and their cellular response to drugs.
RNAi Screen Reveals Candidate miRNAs Associated with the NF‐κB Pathway
Authors: Redan Benjamin W. , Motobar Omid , Tuzmen Pinar , Guha Rajarshi , Caplen Natasha , Martin Scott E.
Published: June 2021

Gene silencing through RNAi has emerged as a powerful tool for genetic loss‐of‐function studies. This study screened the druggable genome (≈7,000 genes) to illuminate novel components of NF‐kB signaling. A cell line with a β‐lactamase reporter gene was used, which was assayed after stimulation with TNF‐α. Downregulation of IKKα and proteasome constituents were identified as top NF‐κB antagonists. Follow‐up of hits helped us identify novel components of NF‐κB signaling, namely microRNAs (miRNA). miRNAs are about 22 nucleotides in length and function as endogenous post‐transcriptional regulators. Of primary importance is the seed sequence—positions 2–8 of miRNA—because it can bind to the 3’ UTR of a mRNA transcript in order to repress translation and/or facilitate sequestration and degradation of targets. Approximately 900 human miRNA mimics were screened and candidate target genes were identified. One such target is RELA. TargetScan (targetscan.org) predicts that RELA is targeted by the miR‐302 family and miR‐372, supporting our screening data. There is both a conserved and a poorly conserved possible heptamer binding site on the RELA 3′ UTR (5’ AGCACUU 3’). Thus far, the putative properties of two of the miRNAs were confirmed by showing downregulation of RELA by RT‐PCR. Also, an immunofluorescence method has been developed that will investigate downregulation of RELA protein.
MicroRNA‐101 is a Master Regulator of Epigenetic Machinery
Authors: Huang Yi , Chen Hua‐Chien , Chen Shu‐Jen , Chou Chen‐Kung
Published: June 2021

MicroRNAs (miRNAs) are small non‐coding RNA molecules that play important role in cellular functions by silencing hundreds to thousands of protein‐coding genes. Considering the function of their target genes, miRNAs have been implicated as key regulators in various physiological and pathological processes. Besides, epigenetic modulations induce heritable changes on gene expression by altering the conformation of chromatin to regulate biological functions as well. Our previous study revealed that several epigenetic modulators were present in a two‐ layered network of microRNA‐101 for regulating HepG2 cell growth. Although cumulating evidence has recently indicated an intricate interplay between miRNAs and epigenetic machinery, the studies that systematically investigate these mechanisms are still lacking. In this study, we incorporated computational and biochemical approaches to delineate the impact of miR‐101 on epigenetic machinery. In miR‐101 over‐expressing HepG2 cells, a significantly altered gene signature of epigenetic machinery was identified by gene‐set enrichment analysis (GSEA). A similar miR‐101‐induced gene‐signature of epigenetic machinery was also identified in human bladder cancer cell line, suggesting that this altered gene signature was not liver‐specific. Comparing to this signature in the cells manipulated with other miRNAs indicated that miR‐101 regulates epigenetic machinery in a specific manner. Furthermore, differential histone post‐translational modifications (PTMs) caused by miR‐101 putative targeting or non‐ targeting epigenetic modifying enzymes were detected by western blotting. Our findings suggest that miR‐101 is a master regulator of epigenetic machinery.
Photosynthetic Enzymes and Carbon Partitioning Studies in Different Switchgrass Populations
Authors: Soundararajan Madhavan , Wahlmeier Steve
Published: June 2021

Switchgrass (Panicum virgatum L.) is a C4 perennial lignocellulosic biomass producing biofuel plant. Several populations of this plant have been already established and their potential use in biofuel production has become a topic of significant discussion. The large variation across various populations of switchgrass warranted an examination of carbon partitioning as well as primary photosynthetic enzymes. Using 13C as a tracer, we were able to examine the assimilation and partitioning of carbon into various carbohydrates, both structural and non‐structural in switchgrass over its growing season. Levels and activities of the primary photosynthetic enzymes (Ribulose 1,5‐bisphosphate carboxylase/oxygenase, Phosphoenolpyruvate carboxylase, NADP – Malic enzyme, Pyruvate Pi Dikinase) involved in the assimilation and fixation of carbon, in a few switchgrass populations were determined. Results from these studies suggest that not only is there a large amount of variation across populations, but also within them.
Novel Approach to the Biosynthesis of a Potentially Therapeutic Immunomodulatory Bacterial Polysaccharide
Authors: Mostafavi Anahita Z , Sharma Sunita , Troutman Jerry M
Published: June 2021

Capsular Polysaccharide A (CPSA) is found on the surface of Bacteroides fragilis, a symbiotic gut microbe. CPSA is made up of repeats of a tetrasaccharide subunit. Our laboratory is applying an in vitro method to enzymatically synthesize CPSA, which is assembled on a bactoprenyl diphosphate scaffold one sugar at a time. There is evidence suggesting that the first sugar incorporated into this scaffold is acetamido‐4‐amino‐6‐deoxy‐glactopryanose (AADGal). I isolated the presumed dehydratase, UngD2, aminotransferase, WcfR, and the initiating glycosyltransferase, WcfS, proposed to be responsible for bactoprenyl‐dipohsophatelinked AADGal biosynthesis. Utilizing capillary electrophoresis, ion exchange high performance liquid chromatography (HPLC), reverse phase HPLC, and ultraviolet light active bactoprenyl monophosphate analogues we were able to characterize the function of WcfR and WcfS. The role of UngD2, however, appeared to be ambiguous in the conditions tested thus far.
Describing the dual orientation of vaccine candidate P6
Authors: Snyder Joy , Pichichero Michael , Michel Lea Vacca
Published: June 2021

Nontypable Haemophilus influenzae (NTHi) are pathogenic bacteria that cause several illnesses, including otitis media (ear infections). The outer membrane protein P6 is currently a top vaccine candidate for NTHi. Past studies have demonstrated that P6 can interact with molecules both inside AND outside of the cell, but its sequence and structure suggests that it does not cross the outer cellular membrane. P6 is a peptidoglycan associated lipoprotein and therefore known to interact with peptidoglycan inside the cell. To demonstrate surface exposure of P6, we utilized flow cytometry and confocal microscopy. In both experiments, whole NTHi cells were incubated with P6 monoclonal antibody and a secondary antibody conjugated to a fluorophore. Fluorescently labeled cells were detected using flow cytometry and visualized via confocal microscopy. Results from our flow cytometry experiments demonstrate that ~17% of NTHi cells express surface exposed P6. Confocal microscopy corroborates that P6 is indeed on the cell surface of intact cells. We conclude that P6 is expressed in two orientations in NTHi: one in which it is surface exposed and able to interact with extracellular antibodies, and one in which it is internally localized and able to interact with peptidoglycan. This study was funded by the Rochester Institute of Technology, NIH NIDCD RO1 08671 (to MEP), and an ASBMB UAN Undergraduate Research Award to Joy Snyder.
Green Ice in My Freezer! Is Our Drinking Water Safe? A Qualitative and Quantitative Study of Copper Contaminants in NYC Public Water Supply
Authors: Patruno Anna , Benitez Benedetta Sampoli
Published: June 2021

Alzheimer's disease is a progressive and incurable form of dementia that slowly terminates memory and cognitive skills. Though no cause for the disease has been found, recent studies speculate that copper consumption through diet is implicated in the cognitive decline of the aging population and of those suffering from Alzheimer's disease. According to standards set by the Environmental Protection Agency (EPA) under the Lead and Copper Rule, if more than 10 percent of tap water samples exceed the copper action level of 1.3 mg/L or 1.3 ppm, additional steps must be taken to reduce that value through corrosion control and public awareness. Despite the EPA's claim that it is not possible to see copper in water supply, a batch of freezer‐made ice cubes presented a green tinge. It was hypothesized that this was due to an insoluble copper salt since a green solid precipitate was observed when the ice was dissolved. The purpose of this investigation is to determine which compounds were present in the “green ice” samples and to quantify their content. Through qualitative methods, the presence of a copper salt was confirmed. Further tests revealed the salt to be copper carbonate, or CuCO3. Through absorption spectroscopy, the amount of free copper in solution was quantified and found to exceed the EPA Maximum Contaminant Level Goal. Possible reasons for the presence of such high amounts of copper in the drinking water will be presented.
Defying protein isomerization in aging: Baker's yeast limit a common protein damage with novel mechanisms
Authors: Patananan Alexander Nikolich , Clarke Steven Gerard
Published: June 2021

The spontaneous degradation of aspartyl and asparaginyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Although the protein isoaspartyl methyltransferase (PIMT) repairs this modification and its genetic loss results in reduced functions in several organisms, no gene homolog or enzymatic activity is present in Saccharomyces cerevisiae. Therefore, we used biochemical approaches to elucidate how isoaspartyls are metabolized in this organism. Surprisingly, cytosolic isoaspartyl damage quantified by base labile volatility assays was strikingly low compared to cells with PIMT, suggesting a novel regulatory pathway. Although mutating or inhibiting the proteasome and autophagy pathways did not increase isoaspartyls compared to wild type yeast, depleting cells of ATP increased isoaspartyls 5‐fold. Yeast lacking V‐ATPases, enzymes that heavily consume ATP to regulate vacuolar pH, also significantly accumulated isoaspartyls and implicated the vacuole in damage regulation. A screen of genes associated with vacuolar morphology identified a kinase that deacidified the vacuole and drastically elevated isoaspartyls when mutated. Future investigations will elucidate the significance of this enzyme and the vacuole on this novel isoaspartyl regulatory pathway in yeast and higher organisms. Ruth L. Kirschstein National Research Service Award GM007185 and GM026020.