bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationNational 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 InformationNational 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 InformationGrant 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 InformationFunding provided by the Australian Research Council.