bluuebunny/crossref_metadata_2025 huggingface dataset:
Specificity studies of the aromatic desulfinase, 2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) from Nocardia asteroides A3H1
Authors: Hoang Dylan Minh , Smith Emily , Watkins Linette
Published: June 2021

2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) is a desulfinating enzyme that catalyzes the final carbon‐sulfur bond cleavage reaction in the biodesulfurization (BDS) of dibenzothiophene (DBT), a major organosulfur compound in fossil fuels. Homology studies suggest that Alanine 195 and Alanine 200 are important in determining the specificity of the active site. Point mutations were created to determine the effect of switching A200 to an Arginine (A200R), as well as switching A195 to an Arginine (A195R) and switching both A195 and A200 to Arginine (A195/200R). By altering amino acids in the active site of DszB, the effects of amino acid A195 and A200 on the active site specificity can be determined. Codon optimized DszB was cloned into pTAC‐MAT‐Tag‐2, overexpressed in E. coli, and purified using a Ni2+ column. Enzyme stored in aliquots containing 15% DMSO was stable at −80 °C for several weeks. The natural substrate 2‐(2′‐hydroxyphenyl)benzenesulfinate (HPBS) was tested, along with the smaller ring analogs 2‐(2′‐hydroxyphenyl)enthen‐1‐sulfinate (HPES) and benzenesulfinate (BS), in order to test whether the active site prefers one, two or three rings. The kinetic data of the codon optimized DszB showed a Km of 3.71 ± 0.60 μM and kcat of 52.8 ± 5.49 hr−1. The specificity of DszB‐A3H1‐A200R showed a change in specificity, preferring HPES over BS.

Support or Funding Information

National Science Foundation (CHE 1062629)
Factors that Influence Recombinant Lysine Deacetylase Activity
Authors: Imbraguglio Samantha A , Hylton Brandon J , Toro Tasha B , Watt Terry J
Published: June 2021

Lysine deacetylases (KDACs) are the enzymes that reverse the post translational modification of acetylation, resulting in an unmodified lysine residue and acetate. KDACs play a critical role in the cell and changes to their activity have been linked to diseases. In order to characterize KDAC activity in a biologically relevant fashion, in vitro assay buffer should ideally mimic the in vivo environment. In this project, our research objective is to determine the components of buffer that influence KDAC activity while remaining biologically relevant and cost efficient. The starting buffer used consisted of phosphate, glycerol and potassium chloride. The buffer parameters varied included the viscosity, buffering agent, and metabolite concentrations. The effect of these alterations was measured through the use of fluorescence‐based activity assays. KDAC8 was the primary enzyme used to observe the effects of these variations. Although the variation of most of these parameters either did not affect KDAC8 activity or decreased it, the addition of a metabolite mixture to the reaction slightly increased KDAC8 activity. In order to determine which metabolite caused the activity increase, citrate was selected for further investigation due to its metal chelating properties. KDAC8 and other Class IIa KDACs require a metal cation at their active site to function. In excess, some of these metals can also bind to an inhibitory site on the enzyme. When citrate was added to the reaction in the presence of excess zinc, activity was partially restored. However, the addition of citrate alone had no effect on KDAC8 activity. In conclusion, a minimal phosphate buffer optimizes KDAC8 activity while remaining biologically relevant and cost efficient. Furthermore, the addition of citrate is a biologically relevant method to ensure that excess trace metals are not inhibiting KDAC8.

Support or Funding Information

U.S. Army Research Laboratory and the U.S. Army Research Office DoD W911NF‐13‐1‐0129, Summer Undergraduate Research Fellowship and the Center for Undergraduate Research at Xavier University of Louisiana.
Expression, Purification, and Characterization of Codon Optimized and mutant variations of DszB from N. asteroides
Authors: Gumpf Jacob John , Idrizi Keid , Watkins Linette
Published: June 2021

2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) catalyzes the final carbon‐sulfur bond cleavage reaction in the rate‐limiting step in the biodesulfurization of dibenzothiophene, a recalcitrant organosulfur compound found in fossil fuels. The dszb gene from Nocardia asteroides (A3H1) was codon‐optimized and overexpressed in E. coli. Expression was examined by FT‐IR and activity assays. His‐tagged DszB expressed from a codon‐optimized gene requires co‐expression with GroEL and GroES to fold properly. The DszB enzyme was purified using a Ni2+ column, and a kcat of 52.8 ± 5.49 hr−1 and a Km of 3.71 ± 0.60 μM was determined. Enzyme stored in aliquots containing 15% DMSO was stable at ‐80 °C for several weeks. DszB has optimal activity at 35 °C but is not stable for longer than 30 minutes at room temperature. Three mutated enzymes were expressed, purified and characterized. The A195R, A200R, and A195R/A200R DszB mutant altered kinetic parameters, increased temperature optimum, and increased temperature stability.

Support or Funding Information

James Madison University, National Science Foundation (CHE 1062629)
Regulation of the Arabidopsis thaliana Ca2+‐dependent protein kinase, CPK28, by autophosphorylation and Calmodulin‐binding
Authors: Bender Kyle W. , Zielinski Raymond E , Huber Steven C.
Published: June 2021

Ca2+dependent protein kinases (CPKs in plants, CDPKs in protists) fulfill central roles as signal transducers downstream of myriad biotic and abiotic stress stimuli in plant systems. Related to Ca2+/CaM‐dependent protein kinases from the animal lineage, CPKs consist of a conserved Ser/Thr protein kinase domain tethered to a Ca2+‐binding regulatory domain known as the CDPK‐activation domain (CAD). CPK activation by Ca2+‐binding has been extensively studied, but the role of autophosphorylation and binding of interacting proteins in CPK regulation is less well understood. Using recombinant proteins, we tested the hypothesis that plant CPKs are regulated by Calmodulin (CaM) binding, and assessed the impact of autophosphorylation on Ca2+‐activation of a CPK involved in plant immunity. CaM specifically bound CPK28 and binding inhibited CPK28 peptide kinase activity. To test the effect of autophosphorylation on Ca2+‐activation of CPK28, we produced dephosphorylated and in situ autophosphorylated forms of the kinase in E. coli. Ca2+‐sensitivity of peptide kinase activity was tested at different Ca2+ concentrations spanning the physiological range (~0.1 to 1 μM in plants). Dephosphorylated and autophosphorylated CPK28 were both responsive to Ca2+ in the physiological range, however, the autophosphorylated kinase was activated more robustly and at lower levels of Ca2+ compared to the dephosphorylated protein. Thus autophosphorylation serves as a mechanism to ‘prime’ Ca2+‐sensitivity of CPK28. Collectively, our analyses reveal subfunctionalization of plant CPKs in terms of CaM‐binding and suggest the possibility for multiple modes of regulation of CPK28 in vivo by Ca2+, autophosphorylation, and CaM‐binding. Importantly, our study provides mechanistic support for the Ca2+‐sensitivity priming hypothesis for Ca2+‐signaling specificity in plant systems. Current work is focused on identifying the phosphosite(s) mediating Ca2+‐sensitivity priming and understanding the in vivo significance of both autophosphorylation and CaM‐binding.

Support or Funding Information

Funding for this work was provided by the National Science Foundation (grant number IOS‐1354094 to REZ and SCH) and the USDA Agricultural Research Service (to SCH).
Identification of Novel Variants of the CB1 Cannabinoid Receptor in Cancer Cells
Authors: Yarbrough Azure , Pyrek Sebastian , Wood Lindsey , Urbaniak Alicja , Bush John , Prather Paul , Radominska‐Pandya Anna
Published: June 2021

Cannabinoid receptors (CBRs) are G‐protein coupled receptors (GPCRs) that are activated by the binding of cannabinoid ligands. It has recently been shown that CB1, found mostly in the brain, and CB2, found mostly in white blood cells and the spleen, have been found to be overexpressed in different cancer models. It has been found that besides the wild‐type CB1, different, shorter versions of the CB1 protein, called CB1 splice variants, are also found in the human brain. However, their function is unknown and requires further investigation. Currently, there is no information available on the expression of these CB1 variants in cancer. Preliminary results using semi‐quantitative reverse transcription polymerase chain reaction (RTPCR) utilizing primers corresponding to CB1 brain variants was used to demonstrate that in several cancer lines of brain, pancreatic, and breast tissue, there were CB1 transcripts of varying sizes were, in fact, being expressed. High, inter‐cancer variations were observed; some cancer lines did not possess any of the constructs while others expressed brain specific variants, and some lines even expressed other potential variants of differing lengths. Based upon our analysis of the results, we hypothesize that in these cancer cells, variants identical to those observed in the brain were being expressed, and that different, cancer‐specific CB1 variants were also being expressed. Currently, we are identifying numerous PCR products obtained by cloning and sequencing. Eventually, the isolated novel variant CBR1 genes will be overexpressed in mammalian cells, and functional analysis of each variant receptor conducted. If successful, the physiological significance of these splice variants and their use as potential novel targets for cannabinoid anti‐cancer drug developments will be determined.

Support or Funding Information

[DoD‐W81XWH1110795, UAMS TRI ABCRP to AR‐P, P20 RR‐16460 INBRE, NIH/NIDA DA039143 to ARP and PLP]
New Delhi Metallo‐Beta‐Lactamase Variants NDM‐4 and NDM‐12 from E. coli Clinical Isolates Exhibit Increased Activity and Stability
Authors: Williams Cameron , VanPelt Jamie , Poth Robert , Cottingim Kelsey , Stewart Alesha , Nix Jay C , Fast Walter , Tierney David L , Crowder Michael W , Page Richard C
Published: June 2021

Metallo‐Beta‐Lactamases (MBLs) confer resistance to carbapenems, cephalosporins, and penicillins in several clinically relevant Gram‐negative bacteria including Acinetobacter, Pseudomonas aeruginosa, and many Enterobacteriaceae. New Delhi Metallo‐Beta‐Lactamases (NDMs) are amongst some of the most worrisome and prevalent MBLs. This family of MBLs is capable of hydrolyzing all generations of bicyclic beta‐lactams. Two new NDM variants, NDM‐4 and ‐12, have recently been identified in E. coli clinical isolates. NDM‐4 contains an M154L substitution while NDM‐12 bears both M154L and G222D mutations. Considering the clinical significance of Gram‐negative species possessing these enzymes, there is a need to characterize the structures and activities of these variants in order to provide a better basis for drug discovery efforts. We have found that NDM‐4 and NDM‐12 possess higher hydrolytic activity compared to NDM‐1. Our differential scanning fluorimetry studies suggest that the M154L mutation confers increased thermal stability and improved affinity for active site catalytic Zn metal ions. The additional G222D mutation of NDM‐12 further improves stability and Zn affinity. M154L, found in the secondary coordination sphere, accounts for changes in stability by acting as a buttress to the loop containing His122, a direct Zn coordinating residue. G222D, on the other hand, is a mutation located in a loop just above the active site approximately 10 Å from the active site zinc ions. Together, our crystal structures of these NDM variants, including the first known structure of NDM‐12, biochemical analyses, and biophysical analyses provide a basis for understanding the roles of key residues surrounding the active site within NDM variants. A better understanding of clinically relevant NDM variants is essential in the fight against MBL‐mediated antibiotic resistance. Our structural, biochemical, and biophysical studies will help to inform drug design efforts to develop new MBL inhibitors.

Support or Funding Information

The authors acknowledge financial support from the US National Institutes of Health (Award No. R01 GM111926 to RCP, DLT, MWC, and WF) and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.
The Role of Good Governance and an Enabling Environment in Delivering Good Nutrition
Authors: Kennedy Eileen Theresa
Published: June 2021

It is generally agreed that good governance is essential for effective policy and program development and implementation. There is, however, no formulaic approach to governance that can be applied universally. Using data from Ethiopia this paper provides a analysis of factors contributing to effective governance in the design and implementation of a multi sector nutrition program in Ethiopia.

Support or Funding Information

Funded by the United States Agency for International Development.
Male testicular development is affected by estrogens but not altered in neonatal piglets receiving soy infant formula
Authors: Mercer Kelly E , Chen Celine , Hennings Leah , Dawson Harry , Yeruva Laxmi , Ronis Martin , Badger Thomas
Published: June 2021

Soy infant formula contains many phytochemicals, including phytoestrogens, which are structurally similar to estradiol (E2). As a result of their potentially estrogenic properties, soy infant formulas have been the subject of concern with regards to reproductive toxicity. To address these concerns regarding male reproductive development, we fed male piglets a soy‐based infant formula, a dairy milk‐based infant formula, milk formula supplemented with E2, or milk formula supplemented with putative estrogenic soy phytonutrient genistein (GEN), in comparison with piglets receiving sow's breast milk from postnatal day 2–21. In the E2 supplemented group, serum testosterone and testicular weight were significantly reduced compared to the milk formula‐fed and sow's milk controls. Testis tubule area, Sertoli cell and germ cell numbers were also increased in the E2 supplemented group compared to milk formula controls, P<0.05. When compared to the sow's milk control, RNAseq analysis in testes showed that E2 supplemented milk formula altered expression of 2012 testicular genes (±1.5 fold, P<0.100). Functional annotation analysis indicated up‐regulation of several pathways in the E2 supplemented group, including canonical Wnt signaling, cell differentiation and migration, angiogenesis and spermatogenesis. E2 also down‐regulated cholesterol and androgen synthesis pathways. Real‐time RT‐PCR confirmed the decrease in steroidogenic acute regulatory protein (Star) mRNA in the E2 supplemented group, suggesting a potential mechanism for the observed decrease in serum testosterone in this diet group. In contrast, GEN supplementation of milk formula altered 104 genes, soy formula 49 genes, and the pathways impacted had no clear links to testes function or sex organ development. These data show that early infant diet exposure to phytoestrogens in soy‐based infant formula are not estrogenic in the male reproductive system.

Support or Funding Information

This work was funded by USDA‐ARS Project 6026‐5100‐010‐05S.
DUSP5 Functions in a Feedback Loop to Suppress TNFα‐Induced ERK1/2 Phosphorylation and Inflammation in Adipocytes
Authors: Habibian Justine S , Jefic Mitra , Schepps Abbie , Bagchi Rushita A , McKnight Robert A , McKinsey Timothy A , Morrsion Ron F , Ferguson Bradley S
Published: June 2021

Adipose tissue inflammation is a central pathogenic element that links obesity to insulin resistance and type II diabetes. Evidence demonstrates that extracellular signal‐regulated kinase 1/2 (ERK1/2) acts as a key signaling molecule that can inhibit insulin signaling in the cytosol and promote pro‐inflammatory gene expression in the nucleus. Recent evidence demonstrates that dual specificity phosphatases (DUSPs) dephosphorylate (i.e. inactivate) ERK1/2, thus inhibiting inflammation and insulin resistance. In this report, we observed that the ERK1/2‐specific phosphatase, DUSP5, was induced in response to high fat diet‐induced obesity. Moreover, increased DUSP5 gene expression correlated with increased tumor necrosis factor α (TNFα) gene expression in response to high fat diet‐induced obesity over time. Based on these findings, we examined a role for DUSP5 on ERK1/2 dephosphorylation and pro‐inflammatory gene expression in 3T3‐L1 adipocytes exposed to TNFα. We demonstrate that DUSP5 functions as a negative regulator of TNFα‐mediated ERK1/2 phosphorylation leading to inflammation. We report a rapid phosphorylation and subsequent dephosphorylation of ERK1/2 in 3T3‐L1 adipocytes in response to TNFα stimulation; concomitant to ERK1/2 dephosphorylation we further observed increased DUSP5 expression in 3T3‐L1 adipocytes stimulated with TNFα, suggesting a role for DUSP5 in the regulation of ERK1/2 signaling. Consistent with this hypothesis, genetic knockdown of DUSP5 led to sustained ERK1/2 activation and enhanced inflammatory gene expression in response to TNFα. Mitogen‐activated protein kinases (MAPKs) have been reported to regulate DUSPs, thus creating a feedback inhibitory loop. Indeed, we report that inhibition of both ERK1/2 and c‐Jun N terminal kinase (JNK) attenuated TNFα‐induced DUSP5 expression. Further loss‐of‐function studies are underway to examine a role for DUSP5 on adipose tissue inflammation in vivo using DUSP5 knockout mice in response inflammatory stimuli and/or diet‐induced obesity. Collectively, these data support the postulate that DUSP5 functions in the feedback inhibition of ERK1/2 signaling in response to inflammation and that loss of DUSP5 function exacerbates the inflammatory response in adipocytes.

Support or Funding Information

This work is supported by the USDA National Institute of Food and Agriculture (Hatch‐NEV00727). Core facilities used for Research reported in this publication was supported by National Institute of General Medical Sciences of the National Institutes of Health (P20 GM103554).
Effects of voluntary running with defined distances on body adiposity and its associated inflammation in mice fed a high‐fat diet
Authors: Yan Lin , Sundaram Sneha
Published: June 2021

Sedentary lifestyle contributes to obesity. This study examined the effect of quantitative voluntary running on body adiposity and its associated inflammation in mice fed a high‐fat diet. Male C57BL/6 mice were assigned into six groups and fed the AIN93G (sedentary) or a high‐fat diet (sedentary, unrestricted running, or with 25%, 50%, or 75% restriction in running activity) for 12 weeks. The average daily running distance was 8.3, 6.3, 4.2, and 2.1 km for the unrestricted, 25%, 50%, and 75% restricted runners, respectively. The high‐fat diet increased body fat mass by 46% compared to the AIN93G diet in sedentary mice. Running reduced the fat mass in a dose dependent manner; there was no difference between sedentariness and running at 2.1 km/d. The high‐fat diet significantly increased blood glucose, plasma insulin, HOMA‐IR, and plasma and adipose concentrations of leptin and monocyte chemotactic protein‐1 (MCP‐1) in sedentary mice. Running, despite continued consumption of the high‐diet, reduced these variables in dose‐dependent manners. Running at 8.3 (unrestricted) and 6.3 km/d (25% restricted) had the greatest but similar reductions in, whereas running at 2.1 km/d did not affect (except MCP‐1), these measurements. Running, regardless of daily distance, significantly reduced MCP‐1 in both plasma and adipose tissues. In conclusion, voluntary running at 6.3 km/d is optimal to reduce body adiposity and its associated inflammation and metabolic disturbance in C57BL/6 mice. Results that running at 2.1 km/d reduced MCP‐1 without affecting body fat mass show the benefits of moderate amount of exercise in improving low‐grade inflammation. It suggests that different mechanisms, rather than reducing body adiposity, are responsible for this improvement.

Support or Funding Information

The U.S. Department of Agriculture, ARS, research project 5450‐51000‐045‐00D.
Weight loss due to low‐fat feeding in female mice with obesity induced by high‐fat feeding reduces both body fat and bone status
Authors: Sawin Emily A , Stroup Bridget M , Murali Sangita G , Ney Denise M
Published: June 2021

Background

Glycomacropeptide (GMP) is a 64 amino acid (AA) glycophosphopeptide released from κ‐casein during cheese making that promotes satiety, reduces body fat, increases bone mass and infers prebiotic and anti‐inflammatory effects.

Objective

To determine if GMP, when fed as the primary protein source, increases fat oxidation with weight loss and improves body composition and bone status after weight loss compared to casein.

Methods

Weanling female C57Bl6 mice were fed a high fat casein diet (60% en) for 28 wks and then crossed over to high and low fat diets (HF 60% en; LF 13% en) with casein or GMP for another 10 wks. We assessed body weight, food intake and body composition by DXA. Fatty acid oxidation was measured in liver, muscle, and fat tissues using 14C‐palmitate. Plasma concentrations of amino acids, hormones and cytokines were determined. Bone biomechanical performance was assessed with the 3‐point loading test.

Results

Mice that were fed the low fat diets after crossover had significantly reduced body mass, body fat, and energy intake compared with the mice that were fed the high fat diets. After weight loss, female mice fed the low fat diets had significantly smaller femoral cross‐sectional area and femoral bone mineral density compared to mice that were crossed over to the high fat diets.

Conclusion

Crossing over to low fat diets after weight gain on a high fat diet improves body composition by reducing overall body mass and body fat through reduction in energy intake. The reduction in body mass coincides with a reduction in bone status as femoral cross‐sectional area and bone mineral density are reduced.

Support or Funding Information

Supported by USDA Hatch WIS01790 and a Wisconsin Alumni Research Foundation Accelerator Grant.
Minimum Selenium Requirements Based on Muscle and Kidney Glutathione Peroxidase Activity in the Turkey
Authors: Taylor Rachel M , Bourget Victor G , Sunde Roger A
Published: June 2021

Liver and blood glutathione peroxidase (GPX) activities have been used extensively as biomarkers of selenium (Se) status in laboratory and livestock animals. In livestock species used for meat, selenoenzyme activity in the muscle should be considered when setting Se requirements. We recently reported that the Se requirement in rapidly growing young turkey poults is 0.3 μg Se/g diet, based on plasma and liver GPX activity (PLOS ONE 11:e0151665), vs. the published requirement of 0.2 μg Se/g diet (NRC 1994). To examine Se requirements based on breast and thigh muscle, cardiac muscle, and kidney (a tissue with high GPX activity), we fed day‐old male poults (n=50) 10 graded levels of Se (0–1.0 μg/g) for 28 d, and measured GPX1 and GPX4 activity, and transcript expression of all 24 turkey selenoproteins in each tissue. Se response curves and plateau breakpoints were plotted to determine extent of deficiency and minimum Se requirements for each biomarker. The minimum dietary Se requirement for growth was 0.05 μg Se/g. Poults receiving 0 μg Se/g diet had 1 and 8% of plateau GPX1 activity in kidney and heart, respectively, and minimum Se requirements were 0.30 and 0.34 μg Se/g diet, respectively. Breast and thigh GPX1 activity Se response curves were hyperbolic but did not reach well defined plateaus, as GPX1 activity was significantly higher in poults fed 1.0 μg Se/g vs. 0.4 μg Se/g. GPX4 activities in Se‐deficient poults were 9, 22, 8, and 27% of GPX4 plateau activities in kidney, heart, breast and thigh, respectively, and minimum Se requirements were 0.25, 0.31, 0.32 and 0.30 μg Se/g diet, respectively. qPCR of total RNA from each tissue showed that 2–6 transcripts out of 24 total selenoprotein transcripts were significantly downregulated by Se deficiency to 31–73% of Se‐adequate plateaus, with GPX1 and SELH transcripts downregulated in every tissue studied; minimum Se requirements based on transcript expression were 0.08–0.19 μg Se/g diet, and no selenoprotein transcript was up‐ or downregulated significantly by supernutritional Se. This study shows that the turkey minimum dietary Se requirement is 0.3 μg Se/g diet based on heart and kidney GPX activity and on breast and thigh GPX4 activity, just as determined previously based on gizzard, liver and plasma GPX activities. The hyperbolic Se response curves for breast and thigh GPX1 activity suggest that 0.4 μg Se/g diet may be warranted for the 0–4 wk old rapidly growing poult.

Support or Funding Information

USDA NIFA (Hatch 233618 and 1004389), and by the Wisconsin Alumni Foundation Selenium Nutrition Research Fund (No. 2046295).
Oncogenic activity of MAN2A1‐FER fusion in liver cancer and other human malignancies
Authors: Luo Jianhua , Chen Zhanghui , Monga Satdarshan , Michalopoulos George , Yu Yan P
Published: June 2021

Oncogenic fusion gene is one of the fundamental mechanisms driving the progression of human cancers. MAN2A1‐FER, a fusion gene between the mannosidase domain of MAN2A1 and tyrosine kinase domain of FER, was found in 6 different types of human malignancies. MAN2A1‐FER fusion translocated FER domain from cytoplasm to Golgi apparatus, and led to phosphorylation of N‐terminus of EGFR and activation of EGFR signaling pathway. Expression of MAN2A1‐FER generated dramatic increase of growth and invasion of cancers, while removal of MAN2A1‐FER through knockout generated significant lower level of growth and metastasis. The presence of MAN2A1‐FER increased the sensitivity of human cancers to FER kinase inhibitor crizotinib or EGFR kinase inhibitor canertinib. Hydrodynamic tail‐vein injection of MAN2A1‐FER gene resulted in rapid development of liver cancer in mice with somatic Pten deletion. Taken together, we concluded that MAN2A1‐FER fusion gene is one of the key drivers for human cancer development.

Support or Funding Information

NCI, DOD and UPCI
Lipidomic Profiling Identifies Cytochrome P450 as A Therapeutic Target for Colitis‐Associated Colorectal Cancer
Authors: Wang Weicang , Yang Jun , Song Mingyue , Wan Debin , Yang Haixia , Sanidad Katherine , Hammock Bruce D. , Xiao Hang , Zhang Guodong
Published: June 2021

Eicosanoids and associated lipid mediators play central roles in regulating inflammation and carcinogenesis. While most previous studies of lipid mediators in colorectal cancer have only analyzed single or limited number of lipid mediators, few systematic studies have been carried out. Here we used a LC‐MS/MS‐based lipidomics, which can analyze >100 bioactive lipid mediators produced by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes, to profile azoxymethane (AOM)/dextran sulfate sodium (DSS)‐induced colorectal cancer in mice. Lipidomics showed that CYP‐derived fatty acid epoxides were the only lipid mediators which were increased in both plasma and colon tissues of AOM/DSS‐treated mice. RT‐PCR and western blotting validated that the expressions of CYP were increased, while the expression of soluble epoxide hydrolase (the dominant enzyme in degrading fatty acid epoxides) was reduced, in colon tissues of AOM/DSS‐treated mice. To study the functional roles of CYP enzymes, we treated mice with SKF‐525A and clotrimazole, which are two different selective inhibitors of CYP enzymes involved in biosynthesis of fatty acid epoxides, and found that CYP inhibitors significantly suppressed AOM/DSS‐induced colorectal cancer in mice. To elucidate the specific lipid mediator involved in the pro‐colorectal cancer effects of CYP, we treated mice with epoxyoctadecenoic acids (EpOMEs) which are metabolites of linoleic acid produced by CYP, and found that EpOMEs worsened DSS‐induced colitis. Together, these results suggest that the previously unappreciated CYP pathway plays critical roles in promoting progression of colorectal cancer through formation of pro‐inflammatory EpOMEs, and CYP enzymes could be novel therapeutic targets of colitis‐associated colorectal cancer.

Support or Funding Information

USDA NIFA 2016‐67017‐24423, NIEHS R01 ES002710 and Superfund Research Program P42 ES04699
Protein Kinase C Epsilon Peptide Inhibitor Exerts Cardioprotective Effects in Myocardial Ischemia/Reperfusion Injury
Authors: McIntyre Anahi , Adekayode Christine , Heron Joseph , James Ifeanyi , Woodley John , Dittakavi Tejaswi , Benjamin Israel , Liu Stephanie , Chen Qian , Barsotti Robert , Young Lindon
Published: June 2021

During myocardial ischemia/reperfusion (I/R), the generation of reactive oxygen species (ROS) contributes to post‐reperfusion cardiac injury and contractile dysfunction. Activation of protein kinase C epsilon (PKC ɛ) during I/R has been shown to increase ROS release, in part, by its stimulation of increased uncoupled endothelial nitric oxide synthase activity. We hypothesize that using a cell permeable PKC ɛ peptide inhibitor (PKC ɛ‐) (N‐myr‐EAVSLKPT, MW=1054 g/mol, 10μM or 20μM) will improve post‐reperfused cardiac function and attenuate infarct size compared to untreated controls in isolated perfused rat hearts subjected to I(30 min)/R(90 min). Male Sprague‐Dawley rats (275–325 g) were anesthetized with sodium pentobarbital (60 mg/kg) and anticoagulated with heparin 1000 units IP. PKC ɛ‐was dissolved in Krebs' buffer and infused during the first 10 min of reperfusion. PKC ɛ‐treated hearts exhibited significant improvement in post‐reperfused cardiac function at 90 min in the maximal rate of left ventricular developed pressure (+dP/dtmax): 56±5%; n=6 (10μM) and 46±3%; n=4 (20μM) compared to untreated controls (n=6) which only recovered to 32±5% of baseline values for +dP/dtmax respectively (p<0.05). Furthermore, PKC ɛ‐treated hearts showed significant reduction in infarct size of 27±2% (10μM) and 28 ±2% (20μM) compared to untreated control I/R hearts, 40±3% (p<0.05). The results suggest that PKC ɛ‐is effective in improving cardiac function and reducing infarct size and is a putative treatment that could aid in clinical myocardial infarction/organ transplantation patient recovery.

Support or Funding Information

This study was supported by the Center for Chronic Disorders of Aging, the Division of Research and the Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.
Chronic Dehydration Induced Changes in Mouse Supraoptic Nucleus (SON): Contribution to Chronic Kidney Diseases (CKD)?
Authors: Song Zhilin , Roncal Carlos , Milagres Tamara , Lanaspa‐Garcia Miguel , Andres‐Hernando Ana , Johnson Richard
Published: June 2021

Chronic recurrent dehydration (WAN) can cause CKD, maybe through a fructokinase (KHK) dependent way. VP is a peptide hormone that is synthesized in the hypothalamic SON and paraventricular nucleus (PVN), and is subsequently transported to and stored in posterior pituitary (PP), from where it is released into systemic circulation. VP may mediate some types of CKD through currently unclear mechanisms. Previous studies have demonstrated that acute dehydration (24 hr water deprivation) may activate a local polyol pathway: aldose reductase (AR)‐fructose‐KHK, leading to elevated VP synthesis and secretion, thus worsening CKD. Current studies evaluated the effects of chronic dehydration on the AR‐KHK axis in mouse SON. Mice were subject to heat stress for 30 min at every hour for seven hours during day with limited water access at night only, for five weeks. Mice were sacrificed and SONs and PPs were extracted out and assayed by rtPCR and ELISA for mRNA expression and hormone content, respectively. Chronic dehydration induced significant increase in mRNA expression of the following markers: AR, KHK‐A, KHK‐C, VP, transient receptor potential vanilloid I (TRPV1, the osmo‐sensor), and oxytocin, indicating activation of the AR‐KHK axis and increased VP synthesis /secretion. Further analysis revealed that VP storage in PP was slightly reduced in WT mice by chronic dehydration but was significantly reduced in KHK A/C double knockout mice. KHK‐KO did not significantly affect VP storage in PP of control mice. These results suggest that chronic dehydration may lead to CKD, at least partially, through elevated VP synthesis and secretion driven by activation of the AR‐fructose‐KHK pathway. Future studies will further clarify the underlying mechanisms.

Support or Funding Information

DOD PR130106;

NIDDK DK048520;

AHA

12GRNT11940024
Sex Differences in the Utilization of Essential and Non‐Essential Amino Acids in Lepidoptera
Authors: Davidowitz Goggy , McCue Marshall , Levin Eran
Published: June 2021

The different reproductive strategies of males and females have selected for differences in function and behavior that may translate into differences in resource use. Here we studied sexual differences in the utilization of essential and non‐essential amino acids by the Carolina sphinx moth (Manduca sexta: Sphingidae). We labeled two essential amino acids (leucine and phenylalanine) and one non‐essential amino acid (glycine) with an isotopically enriched first carbon (13C). These isotopically enriched amino acids were added either to the larval diet or to the adult nectar. We determined the allocation of these amino acids using a stable carbon isotope analyzer of the 13CO2 of the moth's breath as well as allocation to flight muscle and to the spermatophore. On day one after eclosion from the pupae, unfed male moths oxidized larval‐derived amino acids in higher ratios than unfed females, but this trend reversed after four days of starvation: females oxidized more amino acids than males. Males oxidized nectar‐derived essential amino acids more than females, whereas there was no difference between males and females in the oxidation of non‐essential amino acids. Males allocated significantly more nectar‐derived essential and non‐essential amino acids to flight muscle than did females. Last, males allocated relatively few nectar‐derived amino acids to their first spermataphore compared to a four‐fold increase to their second and third spermatophores. We conclude that males and females have fundamentally different allocation strategies of amino acids and that these allocation strategies differ for essential, compared to non‐essential, amino acids. We suggest that these differences in amino acid allocation strategies reflect the need of the males to maintain their ability to fly and find mates, a lower priority for females.

Support or Funding Information

NSF grant IOS‐1053318 to GD
Diet Composition Modulates Intestinal Hydrolytic Enzymes in White‐Footed Mice (Peromyscus leucopus)
Authors: Wang Luping , Karasov William
Published: June 2021

Animals can digestively accommodate to diets with varying substrate levels by routinely possessing plenty of spare digestive capacity or by modulating digestive capacity to match changes in dietary substrate level. There are few studies of this in wild mammals, though in laboratory mice and rats levels of intestinal disaccharidases and peptidases are known to change in correlation with dietary carbohydrate and protein levels, respectively. We maintained white‐footed mice for 12–81 days on diets enriched in either starch (diet C), protein (diet P) or lipid (diet L). There was no effect of either diet, or time on the diet, on their body mass when tissues were harvested. There were no significant effects of body mass on small intestinal mass or length, and no significant effects of diet on small intestinal mass or length. Diet treatment affected the summed activities of maltase, sucrase and aminopeptidase‐N. Post‐hoc Tukey comparisons showed that in all cases summed activity was lowest for diet L, with no differences between diets C and P. Activities of all three enzymes tended to be highest most often in midintestine, and whatever factor(s) reduced enzyme activities on the L diet apparently did so in all intestinal positions. This is only the second study in a wild small mammal, to our knowledge, testing for digestive modulation to diet composition. Like the other study, we found evidence of modulation, but the pattern differed from that observed in laboratory mice and rats in that the expected correlations with dietary carbohydrate and protein were not apparent. Maltasisc activity was relatively high in white‐footed mice, which suggests that they may routinely possess plenty of spare digestive capacity.

Support or Funding Information

Supported by NSF 1354893 to WHK.
Genetic Background Influences Patterning and Implementation of Stereotypy in the Early Postnatal Mammary Ductal Tree
Authors: Hadsell Darryl , Firozi Urvashi , Hadsell Louise , Cox Timother C , Lee Adrian
Published: June 2021

The extent to which ductal branch pattern is controlled by hard‐wired stereotypical mechanisms has been a long standing question in biology. In the mammary gland, prevailing wisdom has held that ductal patterning is stochastic. However, a detailed analysis of mammary ductal patterning using appropriate 3‐dimensional approaches has not been done. Prior published work from our group demonstrates that genetic background has a profound effect on mammary ductal patterning. The hypothesis for these experiments was that ductal patterning and the implementation of stereotypical branching behaviors during early postnatal development differs with genetic background. To test this hypothesis, 12 measures of ductal geometry and two of stereotypy were compared among 3D reconstructions derived from whole‐mounted mammary glands that were stained with the epithelial marker E‐cadherin and imaged by optical projection tomography. Samples were taken on postnatal day (PN) 17 from females of the C57BL/6J (N=5), PWK/PhJ (N=3), and KK/HlJ (N=3) strains. Comparison of graph size, defined as total number of branches, and total ductal length (mm) revealed that overall development was the greatest (P<0.05) in KK/HlJ (56.33±1.76, and 31.57±0.357, respectively), intermediate in C57BL/6J (50.33±6.16, and 23.67±.798, respectively), and least in the PWK/PhJ (41.60±3.12, and 20.51±1.39, respectively). The average local ductal bifurcation angles were also larger (P<0.05) in KK/HlJ than PWK/PhJ (107.01±0.67° and 95.45±3.39°, respectively). Lastly, as a measure of stereotypy, graph discordance was lowest (P<0.05) in KK/HlJ, intermediate in C67BL/6J, and highest in PWK/PhJ (0.47±0.013, 0.54±0.03, and 0.61±0.02, respectively). These results highlight the utility of 3D imaging methodologies in understanding mammary ductal development and support the conclusion that differences in ductal development among differing genetic backgrounds may also be linked with differential implementation of stereotypical patterning.

Support or Funding Information

This work supported by a grant from the DOD number DAMD‐0017.
Studies on HIV‐1 RNA‐protein interactions.
Authors: Hadler Nicole , Stockdale Eryn , Wise Kenneth , O'Carroll Ina
Published: June 2021

In the absence of a cure and a vaccine, the human immunodeficiency virus (HIV‐1) continues to affect millions of people worldwide. HIV is a retrovirus, a unique family of viruses that replicate via a DNA intermediate. The newly replicated HIV RNA must be spliced in order for it to exit the nucleus of the host cell and be packaged into new viral particles. Normally, unspliced or partially spliced HIV RNA's are trapped within the nucleus and degraded. To combat this, HIV has evolved an accessory protein, Rev. The protein Rev binds to the RRE (located on the unspliced or partially spliced RNA) in order to create a complex that allows for the unspliced HIV RNA genome to be exported out of the nucleus.

The secondary structure of the HIV‐1 Rev‐Response Element contains five unique stems. The protein Rev binds to sites situated on stems I and II. We have used electrophoretic mobility shift assays (EMSAs) to assess the significance of the various stems, Stems I, III, IV and V, in the functionality of the Rev‐RRE complex. In this assay, electrophoresis was used to resolve the Rev‐RRE complexes after titrating the mutants with Rev protein. RRE mutant RNAs were obtained in vitro and purified. Free RNAs and protein‐bound RNAs were resolved on a gel; the effectiveness of RNA mutants to bind Rev provide insight into the role of the RNA structure in Rev binding.

Support or Funding Information

This project is supported by the Defense Threat Reduction Agency and the US Naval Academy.
Extraction of Protein Thermodynamic Parameters from High‐Throughput Differential Scanning Fluorimetry Assays
Authors: Wright Thai , Stewart Jamie , Konkolewicz Dominik , Page Richard C
Published: June 2021

Thermodynamic properties of protein unfolding have been extensively studied; however, the methods used have typically required significant preparation time and high protein concentrations. Here, we present a facile, simple, and high‐throughput differential scanning fluorimetry (DSF) method that enables a complete set of thermodynamic parameters of protein unfolding to be extracted. This method assumes a two‐state, reversible protein unfolding mechanism and provides the capacity to quickly analyze the biophysical mechanisms of changes in protein stability and to more thoroughly characterize the effect of additives, inhibitors, or pH. We show the utility of the DSF method by analyzing the thermal denaturation of lysozyme, carbonic anhydrase, chymotrypsin, horseradish peroxidase, and cellulase. Compared to similar biophysical analyses by circular dichroism, DSF provides greater than 24‐fold reduction in experimental time. This study opens to door to rapid characterization of protein stability on low concentration protein samples.

Support or Funding Information

The authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP) and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP).
Ras Residue Y71 Promotes Flexibility of Switch I and Switch II
Authors: Guterres Hugo , Mattos Carla
Published: June 2021

Ras is a small GTPase of 21 KDa that switches between GTP‐bound active state and GDP‐bound inactive state. Structural studies based on X‐ray crystallography and NMR have shown that in its active GTP‐bound state, Ras can adopt multiple conformations, with the switch II R‐(reactive) state and T‐(tardy) state, and switch I state 1, and state 2 being among the most well studied. Ras must be in state 2 to interact with effector proteins and in the R‐state to be catalytically competent. Through accelerated MD (molecular dynamics) simulation analysis of Ras, residue Y71 on switch II stands out as one of the most flexible side chains, spanning a conformational range sufficiently large to interact with Y96 on helix 3 on one side of switch II, and with E37 on switch I. In addition, this dynamic behavior of Y71 tends to accompany R and T state conformational transition. These observations lead to further study of the role of residue Y71 in Ras. Here we present a crystal structure of H‐RasY71A solved to 1.81 Å resolution and intrinsic hydrolysis rates for H‐RasY71A and K‐RasY71F, relative to their wild type counterparts. In addition, we study the differences in dynamics of these proteins using classical MD simulation. The T‐state conformation is stabilized in the crystal structure of H‐RasY71A, with fewer H‐bond networks than the wild type connecting helix 3 to switch II. MD simulations show that switch I and switch II are more rigid in both H‐RasY71A and K‐RasY71F mutants relative to the wild type H‐Ras. Interestingly, intrinsic hydrolysis assays gives a higher rate constant for H‐RasY71A than for wild type, while for K‐RasY71F the rate constant is smaller. Overall, our results suggest that residue Y71 plays a significant role in promoting flexibility of switches I and II and that it plays an important role in the conformational transition between T and R states important in promoting intrinsic hydrolysis of GTP on Ras.

Support or Funding Information

NSF MCB‐1517295
Engineering a Tunnel to a More Versatile Lipoic Acid Ligase
Authors: Schmitz Benjamin , Konkolewicz Dominik , Page Richard C
Published: June 2021

Protein‐polymer conjugates have become a rapidly expanding area of research. As a result of broad utility in a range of disciplines, protein‐polymer conjugates can now be applied to therapeutics, biofuels, and even protein tags. Grafting‐to and Grafting‐from methods have been successful in creating these conjugates but have limitations on the specificity of attachment (Lele et al. 2005). Lipoic acid ligase (LplA) has been demonstrated to be convenient tool for attaching small molecule precursors that can enable subsequent attachment of a polymer to specific sites of target proteins containing a lipoic acid ligase acceptor peptide (LAP) sequence. Studies on LplA have demonstrated that mutation of tryptophan 37 to valine allows LplA to accommodate 10‐azidodecanoic acid (Plaks et al., 2015). Paired with an alkyne‐bearing polymer this enables the use of click chemistry to attach a polymer to the protein‐ligated 10‐azidodecanoic acid. While useful, attachment of a pre‐clicked polymer would enable for more rapid exploration of protein‐polymer conjugates with a wide library of polymers. In this study, we describe structure‐guided, activity‐based efforts to engineer a tunnel within LplA to further open the binding pocket and allow LplA to accept pre‐clicked polymers.

Support or Funding Information

The authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP), the Burroughs Wellcome Fund (Award No. 1014031 to RCP), and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP).
Regulation of Mitotic Microtubule Dynamic Instability in Monopolar Spindles by Bundling and Kinetochore Attachment
Authors: Gergely Zachary R , Flynn Patrick J , Montes Salvador , McIntosh J. Richard , Betterton M. D.
Published: June 2021

Objective

The dynamics of mitotic microtubules are important for mitotic spindle assembly and chromosome segregation, but descriptive parameters have proved challenging to measure directly. We are studying the dynamic instability of single and bundled mitotic microtubules (MTs) that originate from the spindle pole bodies in fission yeasts. We marked the α‐tubulin with an m‐Cherry tag expressed at low levels to minimally perturb MT dynamics and labeled the kinetochores with a GFP tag. Using a temperature sensitive kinesin‐5 allele (cut7–24) at restrictive temperature (37°C) in which bipolar spindle assembly is blocked, we observe monopolar spindles whose dynamic MTs can be imaged directly.

Results and Conclusions

This persistent monopolar spindle allows the measurement of growth, shrinkage, catastrophe, and rescue rates for both single and bundled MTs. The kinetochores are dynamic and appear to move along bundled MTs allowing us to study correlated dynamics of the MTs and kinetochores. We have compared MT dynamics and bundling in this strain with others that contain additional perturbations to proteins that affect MT dynamics and bundling. To quantify these parameters, we have authored software to automate detection and measurement of MT and kinetochore dynamics and their changes over time in monopolar spindles. To quantify the rate of tubulin turnover in this system, we have performed photobleaching experiments in which we detect the substitution of unbleached tubulin dimers for bleached ones using fluorescence recovery (FRAP).

Support or Funding Information

This work was supported by NSF grants DMR‐1551095 to MDB, and NIH grants K25 GM110486 to MDB and R01 GM033787 to JRM.