bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel case‐based learning approach to integrate basic and clinical sciences
Authors: Averill David B , Yin Aidi , Miller Richard , Engelbert Buxbaum , Wright Bruce E
Published: June 2021

We propose a novel strategy applicable to case‐based learning during the initial years of a medical curriculum. Step 1 presents a clinical vignette that requires students to obtain additional information. Step 2: Students are provided access to an on‐line database containing results of physical exams (PE). Students must pay “PE bucks” to obtain additional findings. The number of “PE bucks” provided to the students is less than the total cost of all possible physical exams. Step 3: The same strategy described above for physical exams is applied to choosing procedures or laboratory tests. The decision to order procedures, laboratory tests, or physical exams depends upon their expense, complexity, and availability. This concept is made more concrete by incorporating “lab bucks” and “PE bucks” as tangible assets. The aforementioned steps help students develop decision‐making processes that address competencies such as relationship‐centered care and systems‐based practice. Step 4: Basic and clinical science faculty serve as content experts to facilitate discussion of multiple choice questions focusing on the basic science or clinical training. This teaching strategy prepares students to integrate basic sciences with clinical situations and should be extremely useful in preparing students for upcoming revision of USMLE Step 1‐Step 2.
From Claude Bernard to the Batcave and beyond: using the life of the Caped Crusader to explore the integrative physiology of stress, exercise and injury
Authors: Zehr E. Paul
Published: June 2021

The pop‐culture icon of Batman was used as an educational vehicle for popularizing physiology. Through his years of rigorous training the fictional human Batman pulled himself to near‐superhuman status. Evaluating the scientific possibility this means examining the Batman mythology from the perspective of integrative physiology. This is the main premise of the book “Becoming Batman: The Possibility of a Superhero” (Johns Hopkins University Press, 2008). The book was created in 4 stages involving: 1) a physiological assessment of occupations that parallel those of the fictitious Dark Knight; 2) articulating the concepts of homeostasis and stress; 3) connecting physiological adaptation in multiple and overlapping systems (e.g. nervous, musculoskeletal, endocrine); & 4) examining the adaptive capacity for these multiple systems to yield peak performance. Thus the approach was about explaining how the human body works and responds to exercise and training using Batman as the metaphor for ultimate performance. Main physiological concepts addressed in the text include the concept of homeostasis, Seyle's general adaptation syndrome, hierarchical organization of the nervous system, neural adaptations to skill training and motor learning, the neuropsychology of martial arts training and combat, pathophysiology of concussion, and the role for repeated trauma in the etiology of neurodegenerative diseases such as Alzheimer's. This approach was meant to bring scientific understanding to non‐specialists and the broader public by using an anchor point that was well understood (that of the physical image and impression everyone has of Batman) and then connecting science to that anchor. The objective is to share with other academics the process and positive and negative outcomes of using such an approach.
Anomalous muscle found between splenius and suboccipital group of posterior cervical muscles
Authors: Gana Karen M , Ball Jennette
Published: June 2021

Variations of musculature of the splenius, semispinalis, and suboccipital muscles have been reported through the years. Recent publications have also identified a connective tissue bridge between the suboccipital fascia and the dura mater. In the present case study, the unilateral presence of a muscle in a layer between the semispinalis capitis and the suboccipital muscles was investigated. It has been shown that this muscle has special attachment to the rectus capitis posterior minor and was enveloped in fascia that attached to all muscles of the region as well as into the ligamentum nuchae levels C5–7 and the inferior nuchal line of the occiput. This variation may provide clinical significance for manifestations of headache and neck pain, as well as for surgical procedures.
Morphology of the thoracic intervertebral disc in the adult spine
Authors: Gillis Heather , Johnson Marjorie , Padfield Beverly , Obright Kathy , Galil Khadry , Hrycyshyn Alan W.
Published: June 2021

Objective

To determine the morphology of the thoracic intervertebral disc (IVD) in the adult spine.

Introduction

There is a paucity of research available regarding the structure of the thoracic intervertebral disc. Despite this, a knowledge and understanding of morphology provides the clinician with a better understanding of regional function and pathology.

Methodology

The morphology of the intervertebral disc of 8 embalmed spines was studied via micro‐dissection of the posterior aspect of the motion segment. As a control of dissection technique one cervical spine and two lumbar segments were micro‐dissected and compared to published findings. Spines were removed from the donor using a posterior approach and were cleaned of superficial soft tissue and sectioned transversely through even numbered vertebral bodies. The posterior neural arch was removed via paracoronal sectioning and the costovertebral joint accessed. The posterior longitudinal ligament was removed in sequential layer dissection. The superior vertebral body was resected down to the vertebral endplate which was then cleared via curette. Results were journal and photo documented.

Results

The thoracic IVD demonstrates a morphological structure that is unique to other regions. Horizontal cleft fissuring as demonstrated in the cervical spine was significantly less developed and limited to the costovertebral region with no posterior midline migration. One spine failed to reveal fissuring. There was a high rate of end plate defects and variable nuclear fibrosis.

Conclusions

The thoracic IVD is a morphologically distinct structure from the adjacent cervical and lumbar spines.

Grant Funding Source: NA
LOCATION OF SENTINEL LYMPH NODE IN BREAST CANCER
Authors: MATUSZ PETRU L , PUSZTAI AGNETA MARIA , GRUJIC DACIANA , MASTACANEANU MIHAELA , BRATU TIBERIU
Published: June 2021

Breast lymph drainage is complex. In case of breast cancer, the primary tumour drains into the first regional lymph node, the so called sentinel lymph node (SLN), which captures disseminated tumoral cells. In case of breast cancer, SLN biopsy evaluates node status, axillary stage of the disease. The study lot consisted of 45 female patients with malignant, palpable, mammary tumour. All patients underwent lymphoscintigraphic evaluation: planar study after intradermal and peritumoral injection of 15–30 MBq 99mTc–Nanocoll. In 43 cases (95.55%) we found the SLN; in 2 cases (4.45%), scintigraphic examination revealed no SLN. For the 43 cases, SLN location was as follows: axillary lymph nodes (74.42%), parasternal lymph nodes (4.65%), axillary and parasternal lymph nodes (18.60%), and subclavicular lymph nodes (2.33%). Most frequent location of SLN is at axillary level. In 6.98% cases, SLN is at a distance from the axillary fossa. (Supported by CNMP 41058/2007).
Osseous anatomy of scapula obtained from southwestern Nigerians
Authors: Osuagwu Ferdnand C , Owoeye Olatunde
Published: June 2021

Background

The scapular is an important bony component of the upper limb and detailed knowledge of its anatomy helps when making surgical decisions regarding the scapular and surgical instruments utilized for scapular surgery.

This study was conducted to determine the detailed osseous anatomy and morphometry of scapula obtained from Southwestern Nigerians

Materials and Methods

We examined seventy‐eight dried adult scapula bones. All the scapulas were evaluated and dimensions obtained by a single investigator

Results

The mean maximum distance between the highest point of the superior angle and the lowest point of the inferior angle was 14.6±0.15cm, (mean±standard error of mean, N= 78). The mean maximum scapular width was 10.4±0.1cm. The mean glenoid cavity height was 3.8±0.04cm and the mean glenoid cavity width was 2.6±0.03cm. The mean ratio of the maximum distance between the highest point of the superior angle and the lowest point of the inferior angle versus the mean maximum scapular width was 1.40±0.01. The mean ratio of the glenoid cavity height versus the mean glenoid cavity width was 1.43±0.02. The mean ratio of the maximum distance between the highest point of the superior angle and the lowest point of the inferior angle versus the mean glenoid cavity height was 3.9±0.03 while the mean ratio between the maximum scapular width versus the glenoid cavity width was 4.0±0.04.

Conclusion

The ratio of the maximal length of the scapular versus the scapular width did not differ significantly from the ratio of the glenoid cavity height versus the glenoid cavity width. Also the ratio of the maximal scapular length to the glenoid cavity height did not differ significantly from the ratio of the maximal width of the scapular to the glenoid cavity width.

Grant Funding Source: None
Variation in the Bifurcation of the Radial Nerve: Clinical Implications
Authors: Ross David Aaron , Kershaw Matthew , Marquez Samuel
Published: June 2021

The radial nerve, in the cubital fossa, bifurcates into the deep and superficial branches serving a vital role in providing innervation for the extensor muscles in the forearm and hand. Studies have shown radial nerve injuries are the most common cases associated with fractures of the humeral shaft. While previous studies have focused on the effect of the fracture on the innervation of the radial nerve, few have emphasized the variations of the bifurcation of the radial nerve and if it can be relied upon as a landmark when treating such injuries. This study examines 48 donor body arms. Using a digital caliper, the location of the bifurcation of the radial nerve was measured from the lateral epicondyle of the humerus. In addition, it was noted whether the bifurcation occurred above, at or below a horizontal line drawn between the epicondyles across the cubital fossa. Results show a mean distance from the lateral epicondyle to the bifurcation of 253.4mm, with a standard deviation of 5.39mm. 12.5% of the radial nerves examined bifurcated above the horizontal line and 16.67% bifurcated below the line. Only one sample exhibited a statistically significant deviation from the rest of the samples (p <.05, CI 242.83–263.97). These findings suggest that despite variations in the bifurcation of the radial nerve longitudinally, the bifurcation can be confidently predicted within a certain absolute distance from the lateral epicondyle.

Grant Funding Source: NA
The Frequency and Predictability of the Palmaris Longus and Plantaris Muscles: A Five Year Study
Authors: Abramowitz Jason , Ahamed Tarnima , Weedon Jeremy , Marquez Samuel
Published: June 2021

Due to their length and tensile strength, the homologous palmaris longus (PL) and plantaris (P) muscles (MM) have been frequently chosen by reconstructive surgeons for a variety of craniofacial plastic and cosmetic surgeries of replacement or repair of congenital ptosis, lip augmentation, as well as joint reinforcement. This five year study tracks the frequency (P/A) of these muscles as well as their ability to predict the P/A on the contralateral side. Donor specimens (n=83) from an urban population of the SUNY Downstate anatomy program were used. The presence and absence of the MM was assessed as well as the sex of the donor. 26 (31%) of the donors had an absence of at least one PL and 18 (22%) of the donors had an absence of at least one P, both of which numbers are higher than that noted in the literature. Of those missing a PL and P muscle, 53.8% and 33.3% were bilateral, respectively. Prevalence of an absent muscle on both L & R sides was significantly greater for PL than for P (exact McNemar test p=0.039). Substantial concordance was found for P/A of L & R PL (kappa=0.61) and of L & R P (kappa=0.42). Four (11%) of 38 females were absent all four MMs, compared with none of 45 males (Fisher exact test p=0.040). The high absence of both MM suggests that additional factors may be responsible for their P/A and should be considered by surgeons when selecting the tendons for reconstructive procedures.

Grant Funding Source: SUNY Downstate Alumni Association
Further characterization of a teratogen in tap water
Authors: Hrubec Terry C , Mallela Murali K , Przbyla Jenny , Etzkorn Felicia A , Blodgett Dennis
Published: June 2021

In May of 2006, we began to observe neural tube defects (NTDs ‐ a common birth defect resulting in spina bifida and anencephaly) in untreated mice. The cause of these defects was eventually traced to the municipal tap water provided to the mice. Many teratogens are routinely found at low concentrations in tap water. To narrow down possible suspect contaminants, F2 mice were exposed to municipal tap water with different water treatment regimes and from different locations within the water treatment plant. Tap water was also fractionated chemically into volatile and not‐volatile fractions and provided to F2 mice. All mice were exposed for 8 weeks before breeding and throughout gestation. Embryos were examined on gestational day 9.5 for NTDs. Municipal tap water derived from well water was not teratogenic while all samples derived from surface waters were teratogenic. Water treatment method did not affect teratogenicity. The teratogen appeared to be volatile, but some teratogenic effect remained in the non‐volatile fraction. Thus either the teratogen is of low volatility and is partitioning into both fractions, or there are two teratogenic compounds acting synergistically to produce the NTDs in mice. This work was funded through the Harvey Peter's Foundation; Institute for Critical Technology and Applied Science (ICTAS); and NIH grant K01RR16241.
Ephs and ephrins control palate fusion
Authors: Khobahy Ida , Miguel Symone San , Serrano Maria , Miller Megan , Svoboda Kathy , Benson M. Douglas
Published: June 2021

Fusion of the palate is accomplished by juxtaposition of the palatal shelves and epithelial to mesenchymal transition (EMT) and eventual apoptosis of the epithelial layers. EMT requires the action of TGF‐ß3. We observed expression of the EphB2 receptor tyrosine kinase and one of its ligands, ephrin‐B2, in palatal epithelial cells. We hypothesized that forward signaling through the Eph, and/or reverse signaling through the ephrin regulate fusion. We cultured E8 chicken palates with TGF‐ß3 and/or recombinant extracellular domains of ephrin‐B2 or EphB2, fused to human Fc. Negative controls received IgG‐Fc protein. Tissues were then processed for histology and scored on a 1 to 4 scale. TGF‐ß3+IgG‐Fc palates showed partial fusion (mean score 3). As expected, IgG‐Fc‐only palates did not fuse (mean score 2). Ephrin‐B2 alone caused partial fusion (mean score 3), and EphB2 caused complete fusion (mean score 4). Curiously, TGF‐ß3+EphB2 displayed reduced fusion compared to EphB2 alone. Our data suggest that Eph/ephrin forward and reverse signaling induce palate fusion downstream of, or in parallel with, TGF‐ß3 signaling. Further studies will address the action of Ephs and ephrins on the signaling pathways that mediate palate EMT and fusion.
MDCT in Detection of Anomalous Coronary Origin
Authors: Miclaus Gratian Dragoslav , Zahoi Delia Elena , Ples Horia
Published: June 2021

MDCT performed in daily practice can detect a large number of coronary anomalies, asymptomatic or unknown to the physician or patient.

Although coronary anomalies are discovered accidentally, they represent an important finding, especially when are of malignant type, because can cause symptoms such as arrhythmias, chest pain in young patients with no cardiovascular risk factors, unexplained syncope or even sudden cardiac death.

In our Diagnostic Centre, we discovered few cases with anomalous origin of coronary arteries, arising from pulmonary trunk, right coronary artery arising from left main with anterior or posterior course, and left circumflex arising from right coronary artery.

All patients were referred for atypical chest pain and some of them for unexplained arrhythmias.

MDCT proved to be a powerful noninvasive technique to detect and evaluate anomalous origin of the coronary arteries, especially using 3D VRT reconstructions, which offer an anatomical view of the course of coronary arteries.
Differential pattern of fMRI signal in patients with Mild Cognitive Impairment compared to healthy controls during working memory tasks involving a task‐irrelevant fearful face distracter
Authors: Tichenoff Emily , McClure Matthew , Burhan Amer
Published: June 2021

Mild cognitive impairment (MCI) represents a transitional stage between normal age‐related cognitive changes and mild dementia. Patients with MCI show neurodegenerative changes that lead to modification of many fundamental neural pathways, including those involved in processing emotion and memory. To date, the literature on these networks in this patient population is very limited. A block‐design, delayed match to sample paradigm with task‐irrelevant emotional distracter was used and presented in a 3‐Tesla MRI scanner. Women, ages 55–85, MCI or healthy controls (HC) were presented with “what” and “where” information at low or high‐item loading with task‐irrelevant fearful or neutral faces. The change in blood oxygen level dependent (BOLD) signal was analyzed using SPM8 and two‐tailed t‐tests were used to compare different combinations of loading and emotional valence factors between groups. Despite comparable behavioral data between groups, MCI patients showed significant differential patterns of activation during working memory (WM) tasks when compared to HC. This pattern was different across information type, loading and emotional valence. Emotional distracters modified this pattern depending on the type of information presented. This work highlights changes in WM and emotional networks in MCI patients and is promising as an early detection tool for Alzheimer disease.

Lawson Health Research Institute, London, ON

Grant Funding Source: Internal Funding
Molecular and genetic studies of histone‐modifying complexes in thyroid hormone action during Xenopus development.
Authors: Shi Yun‐Bo
Published: June 2021

Thyroid hormone (T3) plays a causative role in frog metamorphosis. T3 functions to alter gene expression through T3 receptor (TR). We have analyzed the role of TR‐binding cofactor complexes during frog development. We will show that the histone acetylase/methylase‐containing coactivator complexes SCR/p300//PRMT1 are recruited by liganded TR to endogenous target genes. More importantly, these complexes are necessary for gene regulation by TR and frog metamorphosis and their levels regulate the rate of metamorphosis. On the other hand, during premetamorphic development, unliganded TR recruits histone deacetylase complexes containing TR‐binding corepressors N‐CoR/SMRT. Disrupting the recruitment of corepressor complexes with a dominant negative form of N‐CoR leads to derepression of T3‐response genes in transgenic animals. Interestingly, transgenic tadpoles develop faster than wild type siblings, initiating metamorphosis by as much as 7 days earlier out of the 30‐day experiment. These data thus demonstrate that deacetylase complexes participate in gene repression to control metamorphic timing while acetylase/methylase complexes are involved in gene activation to control the rate of metamorphosis. The similarities between metamorphosis and postembryonic development in mammals suggest that such mechanisms are conserved during evolution.
CDK8 is a positive regulator of transcriptional elongation within the serum response network.
Authors: Espinosa Joaquin M , Donner Aaron J , Galbraith Matthew , Taatjes Dylan , Ebmeier Chris
Published: June 2021

The Mediator complex allows communication between transcription factors and RNA polymerase II (RNAPII). CDK8, the kinase found in some variants of Mediator, has been characterized mostly as a transcriptional repressor. Recently, CDK8 was demonstrated to be a potent oncoprotein. Interestingly, we found that CDK8 is prevalently a positive regulator of gene expression within the serum response network, as it is required for expression of several members of the AP‐1 and EGR family of oncogenic transcription factors (e.g. FOS, JUN, EGR1–3). Mechanistic studies demonstrate that CDK8 is not required for recruitment of RNAPII and promoter escape at these loci. Instead, CDK8 depletion leads to the appearance of slower elongation complexes carrying hypophosphorylated RNAPII. We determined that CDK8‐Mediator regulates precise steps in the assembly of a functional elongation complex, including the recruitment of P‐TEFb and BRD4, but is dispensable for recruitment of SPT5 and FACT. Furthermore, CDK8‐Mediator specifically interacts with P‐TEFb. Thus, we uncovered a novel role for CDK8 in transcriptional regulation that may contribute to its oncogenic effects.
Hepatic eEF2K downregulation is associated with altered nucleosome positioning in neonatal IUGR rats
Authors: Fu Qi , McKnight Robert A. , Yu Xing , Callaway Christopher W. , Lane Robert H.
Published: June 2021

Uteroplacental insufficiency (UPI) alters protein synthesis in intrauterine growth restriction (IUGR). Protein synthesis is regulated in part, by eukaryotic elongation factor 2 kinase (eEF2K). eEF2K, a CpG‐rich gene, is down regulated in IUGR rat liver. Our group has shown that UPI alters epigenetic events on genes that have multiple promoters and splice variants. What is not known is how UPI influences epigenetic regulation in CpG island genes. We therefore analyzed the effects of UPI on eEF2K in terms of DNA methylation, histone covalent modifications, and nucleosome positioning around the transcription start in IUGR rat liver. Bisulfite sequencing, ChIP and SssI MTase footprining were performed. The eEF2K promoter had significantly increased DNA methylation, decreased H3acK9, K14, me1K4, me3K4, and me3K36. In addition, the +1 nucleosome accessibility was significantly restricted, together with increased H2A occupancy and decreased RNA polII loading in IUGR relative to the control. In conclusion, UPI induced changes in DNA and histone covalent modifications, nucleosome position and polII loading. We speculate that these changes contribute to eEF2K downregulation.
MLL histone methylases in estrogen‐mediated regulation of HOX genes involved in hair follicle development and leukemia
Authors: Mandal Subhrangsu S , Ansari Khairul I , Hussain Imran , Kasiri Sahba , Shrestha Bishakha
Published: June 2021

Mixed lineage leukemias (MLLs) are human histone H3 lysine 4 specific methyl‐transferases that play critical roles in gene activation. MLLs are key players in HOX genes regulation. Although, MLL are well recognized as master players in HOX gene regulation and development, the mechanism of endocrine mediated HOX gene regulation and the roles of MLLs in this process is largely unknown. Our studies demonstrate that selected HOX genes such as HOXC13 are transcriptionally regulated by estrogen (E2). HOXC13 is a key player in hair follicle development and leukemia. HOXC13 promoter contains several estrogen response elements (EREs). Estrogen receptors (ERs) bind to these EREs (especially ERE1 and ERE2) in an E2‐depedent manner. Knockdown of ERa and ERb suppressed E2‐mediated activation of HOXC13. Similarly, knockdown of histone methylase MLL3 suppressed E2‐induced activation of HOXC13. Overall, our results demonstrated that MLL‐histone methylases in coordination with ERs, regulate HOXC13 gene expression in presence of steroid hormone estrogen implicating critical roles of HOXC13 as well as MLLs in hair follicle development and related diseases.

This work is supported in parts by Texas Advanced Research program (ARP) and American Heart Association
Structural basis for the recognition of Set1 family Win motifs by WDR5
Authors: Dharmarajan Venkatasubramanian , Patel Anamika , Vought Valarie E , Cosgrove Michael S
Published: June 2021

The Mixed Lineage Leukemia protein‐1 (MLL1) associates with WDR5, RbBP5 and Ash2L to form the MLL1 core complex that catalyzes mono‐ and dimethylation of histone H3 Lysine 4 (H3K4). We have recently established that WDR5 interacts with MLL1 through a highly conserved WDR5 interaction (Win) motif in MLL1 and a peptide derived from the Win motif in MLL1 has been shown to disrupt this interaction and specifically inhibit the H3K4 dimethylation activity of the MLL1 core complex. The Win motif is highly conserved among the metazoan Set1 family members, however, little is known about how the different Set1 family Win motifs are recognized by WDR5. To understand the molecular details of these interactions, here we compare 3‐dimensional structures and thermodynamic binding properties of different human Set1 family Win motif based peptides bound to WDR5. We show that Win motif peptides derived from human Set1 family members (MLL2, MLL3, MLL4, SET1a and SET1b) interact with WDR5 with dissociation constants (Kd) ranging from 50–470 nM. These binding affinities are 4 to 30‐fold tighter than the interaction between MLL1 Win motif and WDR5. The crystal structure of WDR5 bound to the human SET1a Win motif peptide reveals more favorable interactions outside the Arginine binding pocket in WDR5 that contributes to the increased affinity as compared to that of MLL1 Win motif peptide. We also demonstrate that the human SET1a Win motif peptide is a four‐fold better inhibitor of the H3K4 dimethylation activity of the MLL1 core complex with an IC50 value of 70 μM. These studies establish the structural basis for the differential interaction affinities between WDR5 and Set1 family Win motifs and suggest a framework for the design of Win motif based peptide inhibitors of Set1 family complexes.
Order Of Phosphorylation Events In Activation Of Ribosomal Protein S6 Kinase 1 (S6K1)
Authors: Keshwani Malik M , Taylor Susan S
Published: June 2021

Protein phosphorylation is key trigger of signal transduction and AGC subfamily of serine/threonine protein kinases are pivotal in regulating various aspects of cellular processes via phosphorylating itself and its downstream affectors. Most AGC kinases have three conserved phosphorylation sites namely in the activation loop, turn motif and hydrophobic motif (HM) that must be phosphorylated for kinase activity. The 70 kD ribosomal protein S6 Kinase 1 (S6K1), an AGC kinase plays an important role in protein synthesis and is regulated by mitogen, insulin and nutrient signaling pathways. Activation of S6K1 is understood to be driven by only two of the three conserved phosphorylation sites and the role of turn motif site has been completely ignored. Also, there has been no order of phosphorylation established in any of the AGC kinases. We have found that S6K1 when expressed in insect cells, has turn motif constitutively phosphorylated and when we transfect insect cells with S6K1 and PDK1 (activation loop kinase), we get all three sites phosphorylated. We carried out a suite of in‐vitro phosphorylation and dephosphorylation studies with S6K1 followed by western analysis and establish the order of phosphorylation of S6K1. We have also found that turn motif phosphorylation is the priming step in S6K1 activation followed by the activation loop and HM phosphorylation.
Mechanism of Calcium‐Induced Electron Transfer in NADPH Oxidase 5
Authors: Wei Chin‐Chuan , Motl Nicole , Wetherell Kristen , Levek Kelli , Reynolds Nicole , Williams Tiffany
Published: June 2021

Superoxide generated by non‐phagocyte NADPH oxidase 5 (NOX5) is of growing importance for vascular physiology and pathology. NOX5 consists of a transmembrane heme domain that is linked to a flavoprotein domain that contains FAD and binds NADPH. Its activity appears to be regulated by a self‐contained Ca2+ binding domain (CaBD). Here, we generated recombinant CaBD and E99Q/E143Q mutant, and characterized them using fluorescence spectroscopy. Ca2+ binding to CaBD induces a conformational change that exposes hydrophobic patches and increases the quenching accessibilities of its Trp residues and AEDANS at cys107. The circular dichroism spectra indicated no significant change in the secondary structures of CaBD upon metal binding. We concluded that the C‐terminal half of CaBD has a higher calcium binding affinity, a higher chemical stability, and a slower calcium dissociation. The Mg2+‐bound CaBD was also investigated, and the results indicate that its structure is similar to the apo form, but the rate of Mg2+ dissociation is close to that of calcium dissociation. Our data suggest that N‐ and C‐terminal halves of CaBD are not completely structurally independent. It has been suggested that the interaction between the CaBD and flavoprotein domains is essential for superoxide production. However, the interaction sites are still unclear. Here, we have generated several flavoprotein fragments and are investigating the domain‐domain interactions by fluorescence spectrometry. We also are determining the metal binding affinities of CaBD's individual halves using isotherm titration calorimetry. This work is supported in part by Cottrell College Science Award (CCSA) from Research Corporation.
Identification and characterization of a 3‐deoxy‐D‐manno‐oct‐2‐ulosonic acid (Kdo) oxidase; kdoO
Authors: Chung Hak Suk , Raetz Christian R. H.
Published: June 2021

Several important Gram‐negative pathogens, including strains Yersinia pestis, Burkholderia pseudomallei, and Acinetobacter, synthesize an analog of Kdo, known as Ko, in which one of the hydrogen atoms at the Kdo 3‐position is replaced with an hydroxyl group. The origin of Ko has not been explored, but studies with Burkholderia cepacia have shown that Kdo is synthesized and transferred to lipid A by KdtA, exactly as in E. coli. The function of Ko is unknown. We have recently explored the idea that Ko might be derived from Kdo by an enzymatic reaction with a mechanism similar to LpxO. Taking advantage of bioinformatics methods, we cloned and expressed potential genes from Y. pestis and B. ambifaria into E. coli WBB06. Remarkably, 50 % to 90 % of the Kdo2‐lipid A was recovered as Ko‐Kdo‐lipid A in both constructs, as judged by TLC and ESI/MS analysis. Therefore, we designated these genes as kdoO. The formation of Ko requires the presence of O2 during bacterial growth and the oxygen atom in Ko is derived from molecular oxygen. Furthermore we have overexpressed KdoO and reconstituted enzymatic activity in vitro.
A kinetic analysis of each step of protein splicing of the Pyrococcus abyssi PolII intein
Authors: Duffee Lauren R. , York Daniel J. , Dorval Deirdre M. , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

Protein splicing is a post‐translational event by which an intervening polypeptide, called an intein, facilitates its own excision from the flanking polypeptides, called the exteins, and the ligation of the exteins. The intein that interrupts the Pyrococcus abyssi PolII protein can be purified as part of a precursor fusion protein. Splicing can be initiated by an increase in temperature, which allows for detailed kinetic analysis of the overall splicing process as well as the individual steps. We have studied the influence of the residue immediately upstream of the intein on the rate of the first step of splicing, an amide to thioester rearrangement. Modifications leading to changes of that rate can result in splicing side reactions. We also have examined the influence of residues constituting a putative oxyanion hole on the rate of the first step of the reaction and the extent of splicing. Finally, we present data on the influence of two conserved downstream His residues on the rate of step three of splicing, Asn cyclization coupled to peptide bond cleavage.

This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.
Monitoring the cellular dynamic distribution of mammalian Ste20‐like protein kinase 3 by peptide aptamer from phage display
Authors: Chiu Yi‐Rong , Yuan Chiun‐Jye
Published: June 2021

Mammalian Ste20‐like protein kinase 3 (Mst3), a member of the GC‐III subfamily protein kinases, is found to regulate various cellular affairs in response to environmental cues. The localization of endogenous Mst3 is in cytoplasm and may translocate to nucleus upon stimulation. We are interested in screening Mst3‐binding peptide for future drug development. Two phage display libraries, hepta‐peptide and dodeca‐peptide phage libraries had been subjected to an in vitro selection process for Mst3‐binding ligand identification. Following six biopanning process of Mst3‐binding selection and DNA sequencing of selected phages, five Mst3‐binding phages were selected. The five types of Mst3‐binding phages had been confirmed that all could bind with Mst3 but not Mst4 by ELISA assay and Western blotting. The peptides were constructed into EGFP vector to form EGFP fusion proteins in vivo. The peptide‐EGFP fusion proteins will be used to real time monitor the subcellular redistribution of endogenous Mst3 in response to various environmental stimuli.
Roles of interfacial interactions of D249 and R167 in the oligomerization of 3α‐hydroxysteroid dehydrogenase/carbonyl reductase
Authors: Hwang Chi‐Ching , Huang Tzu‐Jung , Hsu Chao‐Nan , Wang Yuan‐Liang
Published: June 2021

The dimerization of 3α‐hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. In this study, we explored the mechanisms of dimerization and stabilization in the 3α‐HSD/CR. Mutants of D249A, D249K, D249S, R167A, R167D, and R167Q 3α‐HSD/CRs were constructed, and their oligomeric forms were characterized through gel filtration chromatography. Enzyme catalysis was studied by kinetic analysis. Protein stability was studied through thermal and urea unfolding by CD spectroscopy. Mutation on D249 and R167 causes different effects on the loss of enzymatic activity and shows a concentration‐dependent dimerization of wide‐type and mutant enzymes. Thermal and urea‐induced unfolding profile for wild‐type and mutant enzymes appeared as a two‐state transition and three‐state transition, respectively. The study from the thermal and urea unfolding indicates the mutation of the residue D249 and R167 destabilize the protein structure. Therefore, the salt‐bridge interaction between D249 and R167 of each subunit is important in stabilizing dimeric formation of 3α‐HSD/CR.
Translation Regulators Mediate the Interaction between Eukaryotic Elongation Factor 1A and the Actin Cytoskeleton in Saccharomyces cerevisiae
Authors: Lewis Marcus Eric , Kinzy Terri Goss
Published: June 2021

Eukaryotic Elongation Factor 1A (eEF1A) is an abundant G‐protein whose primary function is to transport aminoacyl‐tRNA to elongating ribosomes during eukaryotic translation. Additionally, eEF1A has also been identified as an actin‐binding protein. This second property of eEF1A allows it to bind and subsequently bundle filamentous actin into two specific structures: patches and cables. These structures collectively referred to as the actin cytoskeleton, provide the network for intracellular transport and endocytosis in yeast. Evidence has substantiated the eEF1A‐actin interaction; however, regulation of this association has yet to be determined. Two major regulators of translation in yeast are the TOR kinases and Gcn2p. These kinases are activated in response to nutrient and amino acid starvation, respectively, and ultimately lead to decreased translation via blocked initiation. Using strains expressing either an F308L or S405P mutant form of eEF1A, both of which exhibit disorganized actin cytoskeletons and blocked translation initiation, we analyzed the affects of TOR and Gcn2p on the eEF1A‐actin interaction. Our current findings suggest that while eEF1A and TOR appear to promote actin cytoskeleton integrity through two distinct pathways, Gcn2p may be involved in eEF1A's ability to sustain actin organization. This research was supported by the National Institutes of Health.
R‐ to T‐ Transition in Fructose‐1,6‐bisphosphatase: Loop Displacement is the Penultimate Step to Inactivation
Authors: Gao Yang , Iancu Cristina V , Mukund Susmith , Honzatko Richard B
Published: June 2021

AMP transforms fructose‐1, 6‐bisphosphatase (FBPase) from its active R‐state to its inactive T‐state. During this quaternary transition, a catalytically essential loop (residues 52–72) leaves the active site (engaged conformation) and moves to its inactive (disengaged) conformation. Structures of FBPase and molecular dynamic simulations reveal the mechanism of loop displacement. The AMP complex of Asp10 FBPase provides the first example of an engaged dynamic loop in a near‐T quaternary state. Subunit rotation forces loop 190 (from subunit C1) into contact with the hinge (residues 52–57) of the dynamic loop of subunit C2. Additional conformational changes in residues 264–274 of subunit C2 reduce space available to the engaged conformation of loop 52–72. Target Molecular Dynamics indicates the absence of steric barriers in subunit rotations but requires the displacement of the loop 50–72 from its engaged conformation in order to achieve the T‐state. Principal Component Analysis reveals anti‐correlated motions, which cause steric clashes consistent with the displacement of loop 52–72 from its engaged conformation. This research is supported by NIH grant NS 10546.
Structural genomics of cancer‐preventive phenylpropanoid biosynthetic pathway enzymes
Authors: Kim Wanyeon , Yang Hee Jung , Yun Young Ju , Kim Young Ha , Kwon TaeWoo , Kang ChulHee , Youn BuHyun
Published: June 2021

Lignans and isoflavonoids are major constituents of vascular plants and play important roles in plant defense, helping hosts discriminate between biological pathogens and symbiotic non‐pathogens. In addition to their physiological roles in plants, these compounds have pronounced antimicrobial, antifungal, antiviral, antioxidant and anticancer properties. It is generally accepted that low cancer incidence rates in people with specific plant‐based diets is due to the so‐called chemopreventive effects of particular lignans and isoflavonoids. For these reasons, there is considerable interest in making these compounds more generally available by defining and exploiting the lignan and isoflavonoid biosynthetic pathways. The goal of this project is thus to define the structure‐function relationships of these key proteins and enzymes in lignan and isoflavonoid biosynthesis through high‐resolution X‐ray structural analyses. The information gained from these studies will be very useful for exploring the regulation of lignan and isoflavonoid biosynthesis for ecological plant protection, and for the industrial‐scale regiospecific and stereospecific synthesis of these pharmacologically active substances.
Regulation of cholesterol biosynthetic gene expression by the Ah receptor
Authors: Tanos Rachel , Patel Rushang D. , Perdew Gary H.
Published: June 2021

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor known to drive expression of target genes. Activation of AHR by xenobiotic agonists such as dioxin or TCDD (2,3,7,8‐tetrachlorodibenzo‐p‐dioxin) is known to have toxic consequences. Interestingly, ahr‐null mice exhibit a defined set of physiological phenotypes that indicate an endogenous role for AHR. Recent microarray studies performed in mice and rats treated with TCDD have shown a decrease in the expression of lipid metabolism genes. In our effort to understand the mechanisms of this regulation, we conducted a microarray analysis on liver samples from ligand‐treated transgenic mice expressing a DNA‐binding mutant AHR and showed that AHR DNA‐binding is not essential for the observed repression. In addition, real‐time qPCR performed on mice and human hepatocytes confirmed repression of cholesterol biosynthesis genes namely HMGCR, SQLE and LSS following receptor activation. Using real‐time qPCR and western blot, we showed that absence of the receptor in liver‐specific AHR Cre‐Flox mice leads to upregulation of those genes in the absence of an exogenous ligand. These data firmly establish the role of AHR as regulator of cholesterol biosynthesis independently of its DNA‐binding ability and suggest that AHR may be a previously unrecognized therapeutic target. Funding: NIH ES04869 and Bristol Myers Squibb
The C‐Terminus of α Subunit Determines The Catalytic Activity of AMP‐activated Protein Kinase
Authors: Cui Xiaopei , Gao Haiqing , Hwang Jenn‐Kang , Shyy John Y‐J
Published: June 2021

Comprising a catalytic α subunit and two regulatory β and γ subunits, AMP‐activated protein kinase (AMPK) regulates cellular energy homeostasis as well as cell proliferation and polarity. To date, the mechanism underlying the autoregulation of AMPK kinase activity remains elusive. Using tissues collected from mice ablated with AMPKα, we showed that the stability of β subunit depended on the presence of α. In the absence of α1 or α2 subunit, the decrease in β was due to an accelerated protein degradation, which could be rescued by the α complementation. Similarly, siRNA knocking down of β decreased α expression in cultured cells. Experiments with α2 truncations revealed that residues 412–426 of murine α2 were critical for the binding of β subunit. Exhibited little effect on αβ interaction, a 12‐a.a truncation at the C‐terminal (aa. 541–552) of α2 resulted in decreased kinase activity. This truncation could act as a domain negative mutant to abate endogenous AMPK activity in phosphorylating the downstream targets such as acetyl‐CoA carboxylase and endothelial nitric oxide synthase. Computational simulation suggested a fluctuate structure of β in the newly formed αβ complex which would be responsible for the decreased kinase activity. Taken together, the interaction of αβ is important in maintaining the AMPK stability, whereas the C‐terminal tail of the α is necessary for the kinase activity of AMPK.
Effects of Omega‐3 Fatty Acids on Lipid Metabolism and Signaling Using Lipidomic Analyses
Authors: Brooks Joshua D , Norris Paul C , Dennis Edward A
Published: June 2021

While arachidonic acid (an omega‐6 fatty acid) and its metabolites play a role in many inflammatory diseases, the omega‐3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) possess potent anti‐inflammatory properties and exert therapeutic benefits. Increased omega‐3 fatty acid intake has been shown to alter the balance of pro‐inflammatory omega‐6‐derived eicosanoid production, but research to date has focused on only a few select lipid metabolites rather than on changes in the cellular‐wide eicosadome. Herein we report overall effects of EPA and DHA on both the cellular lipidome and eicosadome occurring in RAW264.7 macrophages using novel mass spectrometry techniques (JBC, 282, 22834). Our studies have demonstrated that while small variations are seen in the quantity of omega‐6 fatty acids found in the cell, major decreases are seen in some (but not all) of the known omega‐6 fatty acid‐derived eicosanoids forming in macrophages along with a concomitant increase in a few specific omega‐3 fatty acid metabolites. Thus, for the first time, the overall variation in cellular lipid metabolites caused by the presence of EPA and DHA has been examined. Taken together, these studies will help to elucidate the likely signaling molecules responsible for and the molecular mechanisms underlying the beneficial effects of EPA and DHA in inflammation. This work was supported by NIH Grant GM069338.
Production of chlorinated lipids in neutrophil mediated inflammation
Authors: Anbukumar Dhanalakshmi S. , Shornick Laurie P. , Albert Carolyn J. , Ford David A.
Published: June 2021

Neutrophils are critical cellular mediators of the innate immune system. One defense mechanism employed by neutrophils involves the production of myeloperoxidase (MPO)‐derived HOCl, which chlorinates critical functional residues of lipids, proteins and nucleic acids. 2‐Chlorohexadecanal (2‐ClHDA) is a lipid product of HOCl attack of plasmalogens. The present study shows for the first time that novel chlorinated metabolites of 2‐ClHDA (2‐chlorohexadecanoic acid, 2‐ClHA; 2‐chlorohexadecanol, 2‐ClHOH) are produced endogenously in isolated, activated neutrophils and as a result of neutrophil mediated inflammation. Both 2‐ClHA and 2‐ClHOH are produced in activated neutrophils in an MPO dependent manner, and are released by neutrophils into media. In contrast to 2‐ClHDA, 2‐ClHA and 2‐ClHOH do not cause neutrophil chemoattraction and partially inhibit chemoattraction by fMLP. Lastly, a rodent model of virally‐induced lung inflammation resulted in elevated levels of 2‐ClHA in bronchoalveolar lavage fluid and plasma compared to control uninfected counterparts. These data demonstrate that novel chlorinated lipid species are produced during inflammation, and these lipids may potentially serve as mediators and biomarkers of inflammation.

This work was supported in part by NIH grants HL088072 and HL074214 (DAF), and pre‐doctoral fellowship grant 0710163Z (DSA) from the American Heart Association.
Trypanosoma cruzi and its gp83 ligand modulate host thrombospondin 1 and laminin γ‐1 gene networks to recruit trypanosomes in the early infection process
Authors: Nde Pius , Cardenas Tatiana C , Johnson Candice A , Pratap Siddharth , Kleshchenko Yulia Y , Furtak Vyachslav A , Lima Maria F , Villalta Fernando
Published: June 2021

Trypanosoma cruzi, the protozoan that causes Chagas heart disease, uses the extracellular matrix to facilitate infection of human cells. However, the mechanisms underlying this process are unknown. Here we show that T. cruzi gp83 ligand, a GPI‐anchored molecule that the parasite uses to attach to host cells, is cleaved by the trypanosome PLC. The cleaved gp83 signals the host cell to increase laminin γ‐1 expression to facilitate the initial recruitment of trypanosomes to the extracellular matrix. Then, the recruited trypanosomes up‐regulate the expression of thrombospondin 1 (TSP1), which also enhances the initial infection process. The over‐expression of laminin γ‐1 and TSP1 in cells enhances trypanosome infection whereas silencing of laminin γ‐1 and TSP1 significantly reduces the trypanosome infection. We have determined the laminin γ‐1 and TSP1 specific gene networks that interact within the human protein interactome during the early infection process. We conclude that T. cruzi uses different strategies to manipulate the extracellular matrix to facilitate the initial infection process (Supported by NIH grants AI080580‐03, AI007281‐21 and AI083925‐01).
Effect of Fucus evanescens Fucoidan on Expression of Matrix Metalloproteinase‐1 Promoter, mRNA, Protein and Signal Pathway
Authors: Lee Yong Hwan , Ku Mi Jung
Published: June 2021

Fucoidan, a sulfated polysaccharide from the brown algae Fucus evanescens belong to the order Fucales. There are no studies which report effects of F. evanescens fucoidan on skin. In this study, we assessed the effects of F. evanescens fucoidan on UVB‐induced MMP‐1 protein, mRNA, promoter and phosphorylation of MAPKs activities in vitro using the immortalized human keratinocyte (HaCaT) cell line. Pretreatment with fucoidan significantly inhibited MMP‐1 protein, mRNA, promoter activity in a dose‐dependent manner compared to the control. We also found that fucoidan markedly reduced the UVB‐induced activation of ERK. We suggest that F. evanescens fucoidan may be a potential therapeutic agent to prevent and treat skin photoaging.