bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of vitamin D level, leg extensor power, and neuromuscular function in mobility‐limited older adults (863.7)
Authors: Kirn Dylan , Laussen Jonathan , Hau Cynthia , Reid Kieran , Fielding Roger
Published: June 2021

Purpose: Low vitamin D level is associated with poor muscle strength in older adults, but little is known about its relationship with muscle power and neuromuscular function in a mobility‐limited population. This study examines the cross‐sectional relationship between vitamin D level, leg power, and neuromuscular activation in mobility‐limited older adults.

Methods: Sixty‐two mobility‐limited older adults completed the study (age= 77.2±4.7 years; Short Physical Performance Battery score = 7.8±1.2). Leg extensor peak power was assessed using bilateral leg press exercise and rate of vastus lateralis neuromuscular activation was assessed using surface electromyography.

Results: As vitamin D level increased, a decrease was seen in muscle power (r= ‐0.27, p= .04) and neuromuscular activation (r= ‐.45, p= .06). Neuromuscular activation was significantly higher in subjects with vitamin D <30 ng/ml compared to those 蠅30 (.24±.09 vs. .11±.14, respectively; p= .02).

Conclusion: This data suggests a negative association between vitamin D level, leg power and neuromuscular function, though further analysis is needed to confirm these findings.

Grant Funding Source: USDA (58‐1950‐0‐014)
Chemosensitive locus coeruleus neurons in the Savannah monitor lizard, Varanus exanthematicus (879.13)
Authors: Zena Lucas , Fonseca Elisa , Santin Joseph , Bícego Kênia , Gargaglioni Luciane , Hartzler Lynn
Published: June 2021

Savannah monitor lizards, Varanus exanthematicus, are unusual among air‐breathing, ectothermic vertebrates by maintaining arterial pH nearly constant during changes in body temperature (‐0.003 U °C‐1) in contrast to the typical α‐stat regulating strategy (‐0.015 to ‐0.017 U °C‐1) of most other ectotherms. Given the importance of pH in the control of ventilation, we examined the CO2/H+ sensitivity of neurons from the locus coeruleus (LC) region of Savannah monitor lizard brainstems. Whole‐cell patch clamp electrophysiology was used to record membrane voltage (Vm) in neurons within the LC region in brainstem slices; changes in firing rate of LC neurons were calculated from action potential recordings. Upon exposure to hypercapnic acidosis (HA) at 20°C, 60% of neurons significantly decreased firing rate (1.8±0.3 vs. 0.6±0.1 Hz; P<0.05). The percentage of LC neurons inhibited by HA is high at 20°C compared with published percentages from bullfrogs and rats. Increasing temperature to 30°C abolished the inhibited response to HA (2.4±0.9 Hz vs. 2.6±1.0 Hz; P=1.0). The large proportion of neurons inhibited by HA at 20°C may provide a mechanism whereby retention of whole body CO2 stores with cooling enables the pH‐stat regulation strategy of Varanus exanthematicus.

Grant Funding Source: Supported by NSF IOS 1257338
Dose dependent effects of intramuscular administered atropine on heart rate in Trachemys scripta (879.16)
Authors: Cruz Carissa Nicole , Williams Cassondra , Hicks James
Published: June 2021

Atropine sulfate is a common anticholingeric drug that is used in human medicine and veterinary medicine, as well as in scientific research. At doses less than 0.1mg/kg, it is used to treat bradycardia, ptyalism, and as an antidote for organophosphate poisoning. Although the effects of atropine have not been thoroughly examined in reptiles, previous studies utilized atropine, 1‐5mg/kg doses, to abolish shunting in reptiles, including snakes, crocodilians, and chelonians . In the present study, we investigated the influence of intramuscular administered atropine on heart rate in freshwater turtles, Trachemys scripta. To achieve this objective, an untethered electrocardiogram device recorded continuously in healthy female adult turtles (n=5, m=1‐2kg) before, during, and after the injections of veterinary grade atropine (0.01‐5mg/kg I.M.) with minimal observer interference. Heart rate was then derived from these measurements. Turtles showed a rapid increase to the peak heart rate less than ten minutes after the injection of atropine, except after the 0.01mg/kg dose, then gradually returned to the resting heart rate. In all doses above 0.01mg/kg, the length of the peak elevated heart rate constitutes about 1/5 of the total time atropine has an effect on heart rate. As a result, the extent of the elevated heart rates in the turtles increased exponentially as the dose of atropine also increased. Thus, we conclude that the lasting consequence of atropine on elevated heart rate is dose‐related. These results suggest that scientists can effectively eliminate shunting in turtles at lower atropine doses without subjecting the animal to prolonged tachycardia and associated cardiovascular adjustments.

Grant Funding Source: Supported by NSF (1121324‐IOS)
Cardiorespiratory and metabolic changes during hypoxic flight in bar‐headed geese (879.20)
Authors: York Julia , Meir Jessica , Willy Jardine , Chua Beverly , Hawkes Lucy , Bishop Charles , Butler Patrick , Frappell Peter , Milsom William
Published: June 2021

The bar‐headed goose (Anser indicus) undergoes a biannual migration over the Himalayas, sustaining aerobic flight above 6,000 meters. The goal of our study was to collect cardiorespiratory and metabolic data during flight from geese flying in a wind tunnel in normoxia and two levels of hypoxia simulating altitude. In birds at rest, metabolic rate fell from 26.6 ± 1.3 mL min‐1 kg‐1 to 10.0 ± 0.9 mL min‐1 kg‐1 and 10.7 ± 4.8 mL min‐1 kg‐1 in normoxia, 10.5% O2 and 7% O2 respectively. It rose to 209.8 ± 5.2 mL min‐1 kg‐1 during flight in normoxia but decreased to 181.7 ± 5.0 mL min‐1 kg‐1 in 10.5% O2 and to 131.7 ± 7.1 mL min‐1 kg‐1 in birds during flight in 7% O2. We found no change in heart rate across oxygen levels at rest or in flight. Heart rate at rest averaged 116.3 ± 35.4. While in flight, for any given metabolic rate we found a large range of heart rates in some individuals (184.5 to 457.0) and a very small range of heart rates in other individuals (256.8 to 375.7). There was, however, a strong positive correlation between metabolic rate and O2 pulse (stroke volume x arterial‐venous O2 content difference) at rest (slope=109.71; R2=0.57635) and in flight (slope=187.27; R2=0.65285) independent of the level of inspired O2 in all birds. It appears that the bar‐heads increase metabolic efficiency during hypoxic flight and match O2 supply to O2 demand by a general increase in heart rate and specific increases in O2 pulse.

Grant Funding Source: NSERC of Canada; NSF, USA
Effects of age and lifetime flight behavior on senescence and reproduction in Drosophila (880.2)
Authors: Roberts Stephen , Mancinelli Georgina , Lane Steven , Elekonich Michelle
Published: June 2021

The effects of aerobic activity on physiology and senescence may be profound in variably active species such as flying insects, which attain among the highest metabolic rates ever measured. Flight capacity in insects decreases with age, although limiting flight behavior extends lifespan, slows the age‐related loss of antioxidant capacity and accumulation of oxidative damage, and potentially allows for greater energy investment in non‐locomotor traits such as reproduction. We tested the effects of age and lifetime flight behavior on flight capacity, longevity and reproduction in Drosophila. Specifically, 5 day‐old adult flies were separated into three life‐long treatments: (A) those not allowed to fly (no flight or NF), (B) those allowed ‐ but not forced ‐ to fly (voluntary flight or VF), and (C) those mechanically stimulated to fly (induced flight or IF). Longevity was in the order NF>VF>IF, although the ability to fly and attain high metabolic rates persisted the longest in VF flies. Age‐specific fecundity did not vary among the groups, although NF flies had the greatest lifetime fecundity due to their longer lifespans. These results indicate that aerobic locomotor behavior can accelerate physiological senescence and trade‐off against other traits.

Grant Funding Source: National Science Foundation IOS‐0725030
Epigenetic modification of gene expression during human acclimatization to hypobaric hypoxia (885.4)
Authors: Julian Colleen , Subudhi Andrew , Evero Oghenero , Pedersen Brent , Dvorkin Daniel , Lovering Andrew , Roach Robert
Published: June 2021

Epigenetic processes and hypoxia‐inducible transcription factors work in coordination to regulate transcriptional responses to hypoxia. Our aim was to determine the role of epigenetics and associated changes to gene transcription for human acclimatization to high altitude. We performed genome‐wide methylation (Infinium HumanMethylation450 BeadChip, Illumina) and expression (Affymetrix Human Gene 1.0 ST Array) studies in peripheral blood mononuclear cells obtained from 21 healthy individuals at sea level and on three occasions during acclimatization to 5260m. Acute hypoxia minimally affected DNA methylation status. After 16 days of acclimatization, we identified 183 differentially methylated sites, including several that are associated with genes known to be involved in hypoxic response (e.g., eukaryotic translation initiation factor 2C subunit 2 [EIF2C], heat shock protein [HSP] 27, DNA (cytosine‐5)‐methyltransferase 3A). Indicating the potential functional importance of these epigenetic modifications, using methQTL we found that methylation status altered the transcriptional activity of hypoxia‐related genes considered central to human acclimatization to hypoxia (e.g., HIF1AN, EIFs, HSPs). Our findings support the possibility that epigenetic modification of gene expression contributes to acclimatization to high altitude.

Grant Funding Source: Supported by: W81XWH‐11‐2‐0040 (DoD), NIH‐CCTSI UL1 TR000154 (NIH‐CCTSI), & 5 K12 HD057022‐07 (NIH)
AltitudeOmics: hemoglobin mass and blood volume changes during early acclimatization to and de‐acclimatization from 5260m (885.5)
Authors: Ryan Benjamin , Wachsmuth Nadine , Schmidt Walter , Byrnes William , Lovering Andrew , Subudhi Andrew , Roach Robert
Published: June 2021

The early time course of changes in hemoglobin mass (Hbmass) and red cell volume (RCV) following ascent to and descent from altitudes greater than 5000m has not been previously determined in humans. We examined Hbmass and blood volume (BV) compartments in healthy men (n = 12) and women (n = 9) at sea level (SL) and 5260m following 1, 7, and 16 days of altitude exposure (ALT1/ALT7/ALT16). Subjects were also studied upon return to 5260m following descent to 1525m of either 7 or 21 days. Hbmass was assessed using the optimized CO rebreathing method. Compared to SL, absolute Hbmass was not different at ALT1 but increased by 3.7 ± 5.8% (n = 20; p < 0.01) at ALT7 and 7.6 ± 6.6% (n = 21; p < 0.001) at ALT16. Following descent to 1525m, Hbmass was reduced compared to ALT16 (‐6.0 ± 3.7%; n = 20; p = 0.001) and not different from SL, with no difference in the loss in Hbmass between groups that descended for 7 vs. 21 days. There was a large correlation between the loss in Hbmass and increase in serum ferritin following 7 days at 1525m (r = ‐0.64; n = 13; p < 0.05), suggesting increased red blood cell destruction. We found significant reductions in absolute and relative plasma volume (PV) of ~9‐12% at ALT7 and ALT16 compared to SL; PV returned to SL values following descent to 1525m. Relative BV was not different from SL at any time point due to opposing changes in PV and RCV. Our results demonstrate that large changes in Hbmass can occur within 1‐2 weeks in lowlanders during acclimatization to and de‐acclimatization from altitudes greater than 5000m.

Grant Funding Source: Supported in part by DOD grants W81XWH‐11‐2‐0040 and W81XWH‐10‐2‐0114
Morphometry and fabric orientation of trabecular bone in human craniofacial regions (918.17)
Authors: Pryor Smith Leslie , Dechow Paul , Yardley Thomas
Published: June 2021

Variation in primate craniofacial form has been attributed to orofacial function. Strain gage experiments show that during orofacial function the zygoma is subjected to high strain relative to the browridge. Trabecular bone morphometry and orientation are reflective of function, but trabecular bone in these regions has been unexplored. This study uses µCT to quantify the morphometric properties of trabecular bone, anisotropy, and fabric orientation in the human browridge and zygoma. We hypothesize that zygomatic trabecular bone will have significantly greater bone volume fraction and anisotropy relative to the browridge as an adaption to larger, more unidirectional loads. Primary orientations of trabecular bone will differ by region consistent with their function.

No significant differences were found between the browridge and zygoma in BVF (0.38 vs. 0.31), and DA (1.65 vs. 1.85), but SMI was significantly lower in the browridge (0.075; honeycomb‐like vs. SMI=1.0752; ideal plate‐like, p<0.034). Orientation was highly significantly different between regions. The zygoma is made up of mediolaterally oriented plates with a slight inferosuperior tilt, while trabecular bone in the browridge is made of denser sagittally oriented plates.

Both regions may be more similar in overall elasticity than predicted, but further analysis needs to test the combined effects of differences in individual features. Orientation of trabeculae in the zygoma appears to be consistent with what would be predicted based on orofacial strain patterns.

Grant Funding Source: NSF BCS 0725141
Can morphological variables be used to predict hip joint function? An examination of the anthropoid proximal femur and range of abduction (919.10)
Authors: Hammond Ashley , Plavcan J. , Ward Carol
Published: June 2021

Femoral morphology is often been used to make inferences about hip mobility in fossil anthropoids, abduction ability in particular. However, no study has shown that variation in hip morphology is actually related to hip mobility. If hip joint mobility is predicted by morphological variation, this would provide a new tool to more specifically evaluate locomotor adaptation in fossils.

We present results of an in silico model of anthropoid hip joint mobility that has been validated on a large sample of extant taxa (hominoids, cercopithecids, Ateles, Cebus). Pelves and femora of the same taxa were laser scanned and polygonal models were digitally articulated. Maximum hip abduction was modeled using PolyWorks software using strict morphological criteria for joint movement. Several morphologies of the femur that are hypothesized to relate to hip mobility were quantified from the laser scan data using PolyWorks. Regression was used to assess the relationship between morphologies and functional abilities.

Neck‐shaft angle and femoral head orientation were significantly related to degree of abduction. However, the majority of morphologies quantified were not significant predictors of hip abduction ability, suggesting that many of the morphologies attributed to high joint mobility may not relate to increased abduction ability. This work has applications to reconstructing locomotor abilities in fossil anthropoids.

Grant Funding Source: Study supported by NSF (BCS 1232393, 0647557, 0716244), Wenner Gren Foundation, LSB Leakey Foundatio
How do the bone elastic properties vary through different layers of the skull? (920.1)
Authors: Zapata Uriel , Wang Qian
Published: June 2021

One of the major roles of the skull is to protect the brain from physical injuries. To accomplish this critical condition, the cranial vault functions as a dome‐shaped structure composed of both inner and outer tables of cortical bone and diploe in between, suggesting independent biomechanical purposes for every stratum. However, questions regarding how the combination of the external and internal mechanical properties of these components in the cranial vault would maximize its capabilities to undertake functional loadings and minimize the employment of bone mass have not been systematically studied. We hypothesized that bone mass and elastic properties are different between two cortical layers. In this study, twelve pairs of the cortical samples from the parietal bone of a female baboon were collected, with each inner‐outer cortical paired sample from the same site. Cortical thicknesses and densities were measured, and elastic properties were assessed using ultrasonic technique. Results demonstrated remarkable difference in both thickness (F= 1.131, p 蠄0.05) and density (F=0.274, p= 0.015) between inner and outer paired samples. Differences in orthotropic elastic behavior (E1 and E3) were found between two cortical layers as well. How these differences are linked to the stress environments of the inner and outer cranial cortical layers awaits further studies.

Grant Funding Source: Supported by NSF BCS‐0725183, and COLCIENCIAS (Bogotá, Colombia).
Fetal developmental anatomy of the human cardiovascular and central nervous systems using Lugol’s iodine staining and micro‐computed tomography (923.6)
Authors: Spaw Alexandra , Witmer Lawrence
Published: June 2021

Iodine staining with Lugol’s solution is a fast‐developing technique that is revolutionizing imaging studies, because it provides the opportunity to distinguish between soft tissues through soaking of the specimen followed by computed tomography (CT scanning). Earlier protocols that resulted in marked tissue shrinkage have been modified by treating specimens with a buffered sucrose solution prior to staining with isotonic Lugol’s, yielding excellent results. This technique was used to compare the cardiovascular and central nervous systems of a series of human fetal specimens ranging from six weeks to 28 weeks (ages approximated by crown‐rump measurements). Anatomical structures were analyzed and segmented using the computer program Avizo, providing the basis for the generation of labeled images, 3D reconstructions, 3D interactive PDFs, movies, and other forms of digital representations of the information provided in the CT scan data. Results include all representations which lead to a catalog of developing anatomy from the late embryonic stage well into the fetal stage of gestation. The catalog created provides students and educators with multiple forms of media to study the fetal anatomy of these two systems and allows professionals to better visualize and interact with the reconstructed versions of key organ systems. The study of both normal and teratological anatomy will lead to greater knowledge of any defects and could potentially lead to improved treatment and surgical plans.

Grant Funding Source: Supported by a grant to AS from the Ohio University Honors Tutorial College and to LMW from NSF
The architecture of Tetrahymena telomerase (926.2)
Authors: Feigon Juli
Published: June 2021

Telomerase is an RNA‐protein complex that extends the ends of linear chromosomes, and is a highly regulated determinant of cellular aging, stem cell renewal, and tumorigenesis. We have obtained the 3D structure of endogenously assembled Tetrahymena thermophila telomerase holoenzyme at 25 Å resolution using electron microscopy (EM). The 7 protein subunits and the stem‐loop 2 region of TER have been localized in the 3D structure by affinity labeling. Fitting with the available high‐resolution structures, including a p65‐TER complex, reveals the organization of TERT, TER, and p65 in the RNP catalytic core. Among the other holoenzyme proteins, p50 has an unanticipated role as a hub between the RNP catalytic core, p75‐p19‐p45 subcomplex, and the DNA‐binding Teb1. A complete in vitro holoenzyme reconstitution correlates activity with structure. This first physical and functional network architecture of a telomerase holoenzyme provides unprecedented detail about the structure of the RNP catalytic core and reveals the organization of holoenzyme subunits that confer processivity and bridge telomerase to telomeres.

J. Jiang, E.J. Miracco, K. Hong, B. Eckert, H. Chan, D.D. Cash, B. Min, Z. H. Zhou, K. Collins, and J. Feigon, “The architecture of Tetrahymena telomerase holoenzyme”, Nature, 496, 187‐182 (2013).

K. Hong, H. Upton, E.J. Miracco, J. Jiang, Z.H. Zhou, J. Feigon, and K. Collins, “Tetrahymena telomerase holoenzyme assembly, activation, and inhibition by domains of the p50 central hub”, Mol Cell Biol. 33, 3962‐3971 (2013).

M. Singh, Z. Wang, B.‐K. Koo, A. Patel, D. Cascio, K. Collins, and J. Feigon: “Structural basis for telomerase RNA recognition and RNP assembly by the holoenzyme La family protein p65”, Mol Cell 47, 16‐26 (2012).

Grant Funding Source: NSF MCB1022379, NIH GM48123
Ubiquitin‐fold protein and JAMM/MPN+ metalloprotease constitute a regulating system for enzyme inhibition and activation (931.1)
Authors: Cao Shiyun , Hepowit Nathaniel
Published: June 2021

Molybdenum cofactor (Moco) is essential for diverse enzymes to form the catalytic center. Moco biosynthesis is a conserved pathway existent in all three life domains, and Moco biosynthesis deficiency in humans causes serious disease. Molybdopterin (MPT) synthase catalyzes the biosynthesis of MPT, a precursor of Moco. Crystal structures of MPT synthase have been reported for over 10 years; however, its post‐translational regulation is poorly known. Here we report a MPT synthase inhibiting and activating mechanism by using the archaeon Haloferax volcanii as a model. We provide strong evidence that the MPT synthase activity is inhibited by SAMP1 (an ubiquitin‐fold protein homolog of the small subunit of MPT synthase, MoaD) covalent linkage to MoaE (a homolog of the large subunit of MPT synthase). Furthermore, we show that the JAMM/MPN+ metalloprotease HvJAMM1 is required to cleave covalently linked inactive SAMP1‐MoaE to free SAMP1 and MoaE, which suggests activation of MPT synthase. Since SAMP1 is an ubiquitin‐fold small protein, our research also indicates a broad idea that ubiquitin‐fold proteins can inhibit an enzyme by linking to its active site and that a protease, like HvJAMM1, can cleave this linkage and reactivate the enzyme.

Grant Funding Source: NIH R01 GM057498 and DOE DE‐FG02‐05ER15650
Probing the architecture of the Mediator complex (939.3)
Authors: Sierecki Emma , Polinkovsky Mark , Giles Nichole , Moustaqil Mehdi , Alexandrov Kirill , Gambin Yann
Published: June 2021

Gene transcription is an exquisitely regulated phenomenon in which multiple layers of protein‐protein interactions participate in the integration of the signal. The mechanism is mediated through interaction of transcription factors with a large array of regulatory proteins. “Activators” and “repressors” binding DNA upstream of the transcription factors provide positive or negative signals. Proteins called “co‐activators” or “adaptors” that cannot bind DNA by themselves, are responsible for tethering the transcription factors to the RNA polymerase and coordinate the influence of activators and repressors. These adaptors often form large protein assemblies. The largest complex, called “Mediator” is comprised of up to 25 subunits that are grouped into four modules called head, middle, CDK and tail, with a total mass of approximately 1 MDa.

To study this adaptor protein complex, a rapid access to numerous recombinant subunits is required. Co‐expression of the subunits is also key to succeed as the level of disorder in subunits of Mediator is far greater than in other multiprotein complexes of similar size. Here, for the first time, we present a model for the architecture of the entire Mediator complex, obtained using a novel combination of protein analysis techniques. All possible subunit pairs were co‐expressed in a Leishmania tarentolae‐based cell‐free expression system and screened for their ability to interact by an AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen). Our results correlate with the published structure of the Mediator head and reveal intricate interactions between the subunits.

Grant Funding Source: Supported by the ARC and NHMRC
The role of bcl‐2 proteins during chronic glucose exposure coupled with oxidative stress (960.3)
Authors: Van Dalfsen Kelsey , Del Gaizo Moore Victoria
Published: June 2021

Diabetic cardiomyopathy (DCM) is a complication of diabetes that affects heart function and contributes to mortality. Chronic, elevated glucose levels negatively effects heart tissue and has been identified as a major contributor to DCM’s development. Given that BCL‐2 family proteins are integral in controlling apoptosis, the effects of hyperglycemia on BCL‐2 proteins are being studied. While hyperglycemia itself doesn’t cause heart cells apoptosis, it does alter BCL‐2 protein expression levels. When hyperglycemic heart cells are then subjected to oxidative stress, apoptosis does indeed ensue. Therefore, BCL‐2 protein alterations during chronic hyperglycemia may play a pivotal role in heart cell damage caused by secondary pathologic events such as ischemia‐reperfusion injury.

Grant Funding Source: Supported by NSF MRI: CHE‐ 1229562
Identification of KASIII enzymes with novel substrate specificities: demonstration of in vivo production of novel ω‐1 hydroxylated fatty acids using a novel KASIII (967.7)
Authors: Garg Shivani , Jin Huanan , Stewart Charles , Yandeau‐Nelson Marna , Noel Joseph , Nikolau Basil
Published: June 2021

Most bacteria and plants typically synthesize straight and/or branched chain fatty acids. The proportion of these two classes of fatty acids is determined by availability of acyl‐CoA precursor pools and by substrate specificity of the enzyme that initiates fatty acid synthesis, 3‐Ketoacyl ACP Synthase III (KASIII). Presence of unusual fatty acids in certain organisms, such as ω‐cyclic or ω‐phenylic fatty acids, suggests that KASIII in such organisms have specificities for unusual acyl‐CoA substrates, such as cyclic or aromatic acyl‐CoAs. In this study, we have biochemically and biophysically characterized unique KASIII enzymes from four diverse bacterial sources, namely C. gingivalis, B. subtilis, T. aquaticus and A. acidocaldarius. From this set, we have identified several KASIIIs that possess novel substrate specificities towards atypical fatty acid synthesis primers, including hydroxylated (3‐hydroxybutyryl‐CoA), unsaturated (crotonyl‐CoA) and aromatic (benzoyl‐CoA) primers. Additionally, we have demonstrated in‐vivo production of ω‐1 hydroxy‐fatty acids in a modified strain of R. rubrum, by recombinantly co‐expressing A. acidocaldarius KASIII which utilizes 3‐hydroxybutyryl‐CoA. Identification of such unique KASIII enzymes introduces the opportunity to synthesize diverse ω‐functionalized fatty acids which can serve as chemical intermediates for production of bio‐based chemicals.

Grant Funding Source: NSF
Characterization of Fas receptor peptides with calmodulin and its derivatives (969.2)
Authors: Jensen Drake , Wei Chin‐Chuan
Published: June 2021

The Fas receptor (FasR)‐mediated apoptotic pathway has been known to be regulated by Ca2+ fluctuations in the cell, specifically through Calmodulin (CaM). CaM contains four EF‐hand motifs, and upon Ca2+ binding, a conformational change is elicited, allowing FasR binding. Here we sought out to perform a characterization using synthetic Fas peptides and recombinant CaM and Troponin C chimera proteins (1TnC, 2TnC, 3TnC, and 4TnC) in which the EF‐hands have been interchanged (i.e. 1TnC contains the first EF‐hand motif of TnC and the 2nd, 3rd and 4th EF‐hands of CaM). Using isothermal titration calorimetry, we determined that a peptide with sequence TLSQVKGFVRKNGVNE binds weakly to CaM (Kd =1.8 x 10‐5 M) whereas a longer peptide (KYITTIAGVMTLSQVKGFVRKNGV) binds moderately to CaM (Kd = 2.6 x 10‐6 M). The replacement of the 1st or 2nd EF‐hands did not significantly alter the peptide binding affinity in comparison to CaM, being driven by both positive entropy and negative enthalpy changes, whereas the replacement of the 3rd and 4th EF‐hands exhibited a weaker, highly enthalpic binding. To understand the binding mechanism related to the conformational change of CaM and chimeras upon ligand binding, we are currently determining their heat capacity changes by ITC and thermostability by differential scanning calorimetry.

Grant Funding Source: Supported by National Science Foundation
The vitamin D receptor is associated with inflammation in human skeletal muscle (LB423)
Authors: Pojednic Rachele , Ceglia Lisa , Lichtenstein Alice , Dawson‐Hughes Bess , Fielding Roger
Published: June 2021

Introduction: Emerging evidence suggests that vitamin D may play a direct role in inflammation mediated by skeletal muscle, possibly through the vitamin D receptor (VDR). Purpose: We examined two models, both a cross sectional and a longitudinal study of vitamin D3 supplementation to determine 1) the association between VDR protein expression and serum 25‐hydroxyvitaminD3 (25OHD) on intramuscular interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNFα) gene expression and protein concentration and 2) the effect of 16‐week supplementation with vitamin D3 on the association between VDR gene expression and IL‐6 and TNFα gene expression. Methods: Muscle biopsies of thirty older, mobility‐limited adults were obtained at baseline. A subset of 12 mobility limited women underwent 16 weeks of 4,000 IU vitamin D3 supplementation and biopsies were obtained at follow‐up. VDR, IL‐6 and TNFα gene expression gene expression and protein concentration were examined. Results: In cross‐sectional analysis intramuscular VDR protein concentration was positively associated with intramuscular IL‐6 gene expression (p=0.04), but serum 25OHD was not (p=0.8). Neither VDR protein or 25OHD was associated with intramuscular TNFα gene expression (p= 0.58; p=0.74). VDR protein concentration negatively predicted intramuscular IL‐6 protein concentration (p=0.03) but serum 25OHD did not (p=0.9). Neither VDR protein nor 25OHD was predictive of intramuscular TNFα protein (p=0.4; p=0.2). VDR gene expression was not associated with IL‐6 or TNFα gene expression or protein concentration (p>0.05). In the longitudinal study, VDR gene expression approached significant positive association with IL‐6 gene expression (p=0.06), but 25OHD did not (p=0.31) after vitamin D3 supplementation. Neither VDR nor 25OHD was associated with TNFα with vitamin D supplementation at baseline (p=0.89; p=0.84) or at follow‐up (p=0.09; p=0.32).

Conclusion: This study provides support for the hypothesis that intramuscular vitamin D receptor, rather than serum 25‐hydroxyvitaminD3 status, affects intramuscular IL‐6 expression and concentration, but not TNFα, in human skeletal muscle.

Grant Funding Source: Supported by USDA Agricultural Research Service, under agreements No. 58‐1950‐7‐707 (to BDH and LC) and No. 58‐1950‐0‐014 (to RAF); The Dairy Research Institute (R.A.F.), NHLBI pre‐doctoral training grant (T32HL69772 to RMP)
Young Cambodian Women’s Beliefs, Attitudes, and Perspectives of a Healthy Diet (LB474)
Authors: Boudreau Emily , Sibeko Lindiwe , Cordeiro Lorraine , Peterman Jerusha
Published: June 2021

Cambodians were resettled in the U.S. as a consequence of domestic civil strife in the 1970s. Refugees in the U.S. experience poorer health outcomes than U.S. born residents or other immigrants. Trauma from war, displacement, and loss of family may be associated with higher incidences of chronic disease. Intergenerational health risks can predispose children of survivors to poor health. For many, dietary practices may contribute to poor health outcomes. Implementation of culturally sensitive nutrition education can be an important strategy to motivate dietary behavioral change. The purpose of this study is to better understand young Cambodian women’s beliefs, attitudes and perspectives of what constitutes a healthy diet. Semi‐structured focus groups (n=7) with 38 Cambodian women 15 to 30 years old were conducted in Lowell, Massachusetts in 2013 and 2014. Five groups were held in English, and two in Khmer. Content analysis identified three broad categories from transcribed data: healthy food, unhealthy food and motivators for food choices, with discussions including both traditional and participant identified American foods. Within these categories, emergent themes were food safety and sugar, which were categorized as both healthy and unhealthy, while appearance and behavior were the most prevalent motivators impacting food choices. These findings contribute important culturally informed knowledge that can be used to help develop meaningful nutrition interventions for young Cambodian women.

Grant Funding Source: Supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, the Massachusetts Agricultural Experiment Station and the Nutrition Department of the University of Massachusetts Amherst.
Ontogeny and microanatomy of the nasal turbinals in four genera of lemuriformes
Authors: Smith Tim D , Martell Molly C , Rossie James B , DeLeon Valerie B
Published: June 2021

The nasal cavity of strepsirrhine primates (lemurs and lorises) has the most primitive arrangement of extant primates. The numerous turbinals of the ethmoid bear a large surface area of olfactory mucosa (OM), although to date this has only been carefully studied in smaller nocturnal species. In the present study, we examine turbinal development in four genera of diurnal or cathemeral lemuriformes. In addition, we examined an age series of each genus in order to detect whether structures bearing OM as opposed to respiratory mucosa (RM) develop differently, as has been observed in nocturnal strepsirrhines. In adults, throughout its entire length the maxilloturbinal is covered by highly vascular respiratory mucosa with large sinusoidal vessels in the lamina propria; any turbinal that closely borders the maxilloturbinal has a similar vascular RM. Posteriorly, the most vascular RM is restricted in the nasopharyngeal duct, which becomes partitioned from the dorsal olfactory region. A comparison of newborns to adults reveals that the first ethmoturbinal increases more in length in the parts that are covered with RM than OM, which supports the idea that ethmoturbinals can specialize in more than olfactory function. Finally, we observe that the regions of turbinals that are ultimately covered with RM develop more accessory laminae or additional surface area of existing scrolls compared to the regions covered with OM. Because such outgrowths of bone develop postnatally and without cartilaginous precursors, we hypothesize that the complexity of olfactory lamellae within the ethmoturbinal complex is primarily established at birth, while respiratory lamellae become elaborated due to the epigenetic influence of respiratory physiology.

Support or Funding Information

This study was supported by NSF # BCS‐1231350; NSF # BCS‐1231717; NSF # BCS‐0959438
Using Threshold Concepts To Improve BMB Instruction – An Evidence Based Approach
Authors: Loertscher Jennifer , Lewis Jennifer , Minderhout Vicky , Xu Xiaoying
Published: June 2021

Threshold concepts (TCs) as a theoretical framework was proposed by Meyer and Land as a means to improve instruction by focusing on those concepts that would move students from novice to expert understanding of a discipline. TCs are concepts that, when mastered, represent a transformed understanding of a discipline, without which the learner cannot progress. We identified five TCs for biochemistry through a collaborative effort involving over 70 faculty members and 50 undergraduate students at five diverse institutions. The TCs are steady state, biochemical pathway dynamics and regulation, the physical basis of interactions, thermodynamics of macromolecular structure formation, and free energy. Experts deemed these concepts important for understanding biochemistry and students exhibited limited understanding of the concepts. Efforts are underway to develop instructional materials and assessment instruments for these TCs. Specifically, we have analyzed student responses from assessment instruments related to steady state and the thermodynamics of structure formation piloted at diverse institutions in the United States in order to inform development of instructional materials. Ultimately data collected from these implementations will be used in a feedback loop to revise instructional materials and improve teaching and learning in undergraduate biochemistry courses.

Support or Funding Information

National Science Foundation DUE‐1224868
DNA Distortions Imposed by the Transcriptional Regulator PecS from Pectobacterium atrosepticum are Attenuated on Protein Oxidation
Authors: Deochand Dinesh K , Grove Anne
Published: June 2021

The phytopathogen Pectobacterium atrosepticum is responsible for causing soft rot and blackleg disease in potato. Soft rot is characterized by maceration of plant cell wall and middle lamella by enzymes such as pectate lyases, cellulases, and proteases. Pathogenicity of the bacterium depends on responding to environmental cues for efficient colonization of host plants. MarR family transcriptional regulators modulate virulence and adaptation to hostile environments. Previous studies have shown that in the related bacterium Dickeya dadantii, the MarR transcription factor PecS negatively controls expression of the pectinase gene and secretion of the antioxidant indigoidine. PecS downregulates the expression of both pecS and pecM genes; PecM is an efflux pump, responsible for secretion of indigoidine, which is involved in neutralization of reaction oxygen species. Here, we report that P. atrosepticum PecS binds pecS‐pecM intergenic DNA with very high affinity (Kd = 1.0 ± 0.1 nM) and specificity at pH 7.4. DNase I footprinting analysis reveals that PecS binds two palindromic sites in the pecS‐pecM intergenic region and significantly distorts the DNA upon binding, as evidenced by presence of hypersensitive sites. Structure based modeling shows cysteine at the dimer interface, raising the possibility that oxidizing agents can modulate DNA binding by disulfide bond formation between redox‐active cysteines. PecS forms dimers and multimers in presence of organic and inorganic peroxides, as evidenced by SDS‐PAGE analysis. Thermal shift assays suggest that oxidants change the protein conformation since thermal stability of reduced PecS (Tm ~ 50 °C) is significantly lower in presence of oxidants. DNase I footprinting of oxidized PecS shows identical protection pattern as reduced PecS, however hypersensitive sites are no longer present, indicating that the DNA distortion imposed by reduced PecS is absent on binding oxidized protein. This is reminiscent of our previous observation that an increase in pH from 7.4 to 8.3 is associated with PecS failing to induce hypersensitive DNase I cleavage and in protein‐DNA complexes dissociating during electrophoresis, conditions under which PecS is unable to repress the pecS promoter in vivo. Likewise, we observed reduced DNA binding by oxidized PecS by electrophoretic mobility shift assay. We propose that cysteine oxidation induces conformational changes in the DNA binding domain, which modulate binding to cognate DNA. We further propose that PecS controls gene expression by sensing oxidants.

Support or Funding Information

National Science Foundation(MCB‐1515349)
Characterization of Maize Necrotic Streak Virus (MNeSV) mRNA 3′ Cap Independent Translation Enhancers (3′ CITE) Binding with Eukaryotic Initiation Factors
Authors: Liu Qiao , Goss Dixie J.
Published: June 2021

5′ m7GpppN cap and the 3′ poly adenosine (A) tail of eukaryotic mRNAs are key elements for recruiting translation initiation machinery. Unlike host mRNAs, many viruses lack those elements and yet they are translated efficiently. Plant viruses, in particular, have complex structures within their untranslated regions (UTR) that allow them to bypass some cellular translation control steps. In Maize necrotic streak virus (MNeSV) 3′ UTR, an I‐Shaped RNA Structure (ISS) has been reported to mediate the virus translation initiation progress. The details of how these structures mediate translation are not well understood. Biophysical methods were applied to study the binding of 3′ ISS with eIFs. With fluorescence quenching and anisotropy techniques, it was found that eIF4A‐eIF4B complex increased binding affinity of eIF4F with 3′ISS four fold (from Kd =~173±34 nM to Kd =~48±11 nM). The enhanced binding affinity was not caused by the helicase activity of the eIF4A‐eIF4B complex. Using mutants of the ISS, translational efficiency was found not to correlate with binding affinity to eIF4F. ISS‐iA/B is a single nucleotide mutant of ISS but has a different predicted secondary structure with ISS. ISS‐iA/B′s binding affinity to eIF4F was similar with wild‐type, whereas translation efficiency was lower. Binding of wild‐type ISS with eIF4F was both enthalpically and entropically favorable, while ISS‐iA/B binding with eIF4F was only enthalpically favorable. Kinetics association rate, k1, for ISS binding to eIF4F was found to be 30.08 s−1μm−1 and the dissociation rate, k−1, was found to be 5.08 s−1. These rates are comparable to binding of m7GTP‐capped RNA with eIF4F. Both thermodynamics and kinetics indicate that factors in addition to eIF4F are necessary for MNeSV to outcompete host cell mRNA for translation.

Support or Funding Information

Grant Fund NSF MCB1513737
Bioengineering of Human Heparin: A Next Generation Anti‐coagulant
Authors: Whitelock John , Cheng Bill , Tang Fengying , Lyons Guy , Lord Megan
Published: June 2021

Heparin used clinically is manufactured by extraction from porcine and bovine tissues. Recently, contaminating oversulfated chondroitin sulfates found in batches of porcine heparin led to the deaths of approximately 100 people in the US, which was caused by an uncontrolled anaphylactoid‐type response. This has led researchers to explore alternative methods of producing heparin including chemical synthesis, chemoenzymatic synthesis and metabolic engineering of cells to express high levels of the enzymes involved in heparin biosynthesis. However, these approaches are yet to produce heparins with anticoagulant activity similar to heparin used in the clinic. The aim of this research was to recombinantly express serglycin in human cells as an alternative approach to producing anticoagulant heparin.

The serglycin recombinantly expressed in human embryonic kidney (HEK‐293) cells was decorated with heparin, heparan sulfate and chondroitin sulfate. The disaccharide composition of the recombinantly expressed heparin was analysed by fluorophore assisted carbohydrate electrophoresis and contained 15% tri‐sulfated disaccharides as well as 40% di‐sulfated, 25% monosulfated and 20% unsulfated disaccharides. The disaccharide composition of low molecular weight heparin was also analysed and contained a higher proportion of sulfated disaccharides than the recombinantly expressed heparin being 40% trisulfated, 45% disulfated, 10% monosulfated and 5% unsulfated disaccharides.

The activated partial clotting time assay is used clinically to monitor heparin levels in patients after its administration and was used in this study to determine the anticoagulant activity of the recombinant human heparin and to compare this to the activity of low molecular weight porcine heparin. In this assay porcine heparin was approximately 20 times more potent in its anticoagulant activity than the recombinant human heparin on a weight basis.

These data demonstrate for the first time the successful production of human heparin from cells transfected with the human serglycin gene. This is a significant step towards alternative sources of heparin that has distinct advantages for patient safety due to the use of production methods with a lower inherent risk of contamination. Human cell‐derived heparin will also provide an alternative source to animal‐derived heparin that will enable its use by patients who, due to religious beliefs and/or dietary choices, have so far been excluded from heparin treatment.

Support or Funding Information

Funding provided by the Australian Research Council.
Purification and Biophysical Characterization of the Photosystem I Complex from Botryococcus braunii
Authors: Joaquín Ovalle Freisa M. , Guihurt Grace , Torres‐Martínez Zally , Morales‐Lozada Yermary , Griebenow Kai
Published: June 2021

Botryococcus braunii (B. braunii) is a green photosynthetic microalga that is highly investigated due to its capacity of hydrocarbon accumulation, secretion, and synthesis. However, its photosynthetic apparatus has not been elucidated. Photosystem I (PSI) is a membrane‐bound pigment‐protein complex that mediates the electron transfer from plastocyanin on the lumenal side to ferredoxin on the cytoplasmic side. In this study, our purpose was to isolate, purify and characterize PSI core subunits and light harvesting proteins. After the initial isolation via differential ultracentrifugation and solubilization of thylakoid membranes with 1% of n‐dodecyl β‐D‐maltoside, the resulting supernatant was loaded onto a Toyopearl DEAE‐650 ion exchange chromatography column. The purified PSI fraction obtained was pooled and concentrated using an Amicon centrifugal filter unit. The resulting protein concentration was 1 mg/ml, and the PSI characteristic 677 nm absorption maximum was attained. The biophysical characterization was performed via UV/Vis and fluorescence spectroscopy, native and SDS‐polyacrylamide gel electrophoresis, and MALDI‐MS, with the characteristic peaks and molecular weight results of the proteins and subunits found in PSI. Our characterization results show that PSI has been successfully purified, which may provide the fundamental knowledge of the function and possible applications of this integral membrane protein complex in B. braunii.

Support or Funding Information

This research was funded by NIH Research Initiative for Scientific Enhancement (RISE) Program Grant No. 5R25GM061151‐14 and DOD Contract No. W911NF‐11‐1‐0218.
Effects of Mutation at Position 557 on the Catalytic Properties of Soybean Lipoxygenase‐1
Authors: Bassett Natasha F. , Clapp Charles H.
Published: June 2021

Soybean lipoxygenase‐1 (SBLO‐1) catalyzes the oxygenation of linoleic acid to give primarily 13‐hydroperoxy‐9,11‐octadecadienoic acid (13‐HPOD) plus a small amount (10% at pH 7.5) of 9‐hydroperoxy‐10,12‐octadecadienoic acid (9‐HPOD). Replacement of phenylalanine‐557 with valine increases the amount of 9‐HPOD formed to 30–40%. Following earlier suggestions by Hornung et al. (Proc. Natl. Acad. Sci. USA 96 (1999) 4192–4197), our working hypothesis is that 13‐HPOD arises from tail‐first binding with the methyl terminus of linoleic acid near phe‐557, and that replacement of phe‐557, with smaller residues enables linoleic acid to bind in a head‐first manner leading to 9‐HPOD. To test this model, the F557S mutant was prepared, and its activity was determined with linoleic acid and 20‐hydroxy‐5,8,11,14‐eicosatetraenoic acid (20‐HETE), which has a hydroxyl group on the terminal carbon that might form a hydrogen bond with the hydroxyl group of serine‐557. Kinetics studies on the mutant were complicated by pronounced substrate inhibition at high substrate concentration. At low substrate concentration, the activity of the F557S mutant is 25% of wild‐type with linoleic acid as substrate and 65% of wild‐type with 20‐HETE as substrate. Oxygenation of linoleic acid by the F557S mutant produced only 8% of 9‐HPOD, considerably less than the percentage of 9‐HPOD produced by the F557V mutant. This result indicates that the size of the residue at position 557 is not the only factor that affects the regiochemistry of the reaction. To test the hypothesis that 9‐oxygenation results from head‐first binding, linoleoyl‐L‐valine (LLV) and linoleoyl‐D‐valine (LDV) have been investigated as substrates of the F557V mutant. The percentage of 9‐oxygenation with these substrates is comparable to what is observed with linoleic acid and shows little or no dependence on the stereochemistry of the valine moiety. These findings suggest that the 9‐oxygenation of LLV and LDV does not involve head‐first binding.

Support or Funding Information

NSF CHE‐1213262