bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessing angiogenic activity of cells in the presence of oxygen generating biomaterials
Authors: Ward Catherine L. , Haines Nathan , Patel Ravi , Eisele Evelyn , Harrison Benjamin S.
Published: June 2021

Upon implantation the center of a large (>1 cm3) cell seeded tissue scaffold typically becomes hypoxic and eventually necrotic. This is primarily due to the diffusion limitation of oxygen. We have developed a novel class of biomaterials capable of in situ sustained release of oxygen. Such materials would help improve cell viability within tissue scaffolds while the tissue becomes integrated with the host. However, angiogenesis is an oxygen dependent process. When hypoxia occurs, cells produce angiogenic factors, such as VGEF, to stimulate blood vessel growth to raise the oxygen tensions within the cellular environment. One concern in using materials capable of generating of oxygen is that it will hinder angiogenesis leading to the ultimate failure of the implant tissue. We examined the effect of oxygen generating materials on angiogenesis using an in vitro angiogenesis assays on HUVECs and have found that it does not suppress angiogenesis. Our results suggest that oxygen can be generated as a sufficient level that it can increase cell viability without suppressing angiogenesis. This was supported by grants from the NIH and DOD.
Evidence of apoptosis in skeletal muscle of young and old mice following ischemia‐reperfusion injury
Authors: Hammers David Wayne , Adamo Martin L , Walters Tom J , Farrar Roger P
Published: June 2021

We have previously shown that tourniquet (TK)‐induced ischemia‐reperfusion (I/R) injury results in larger decrements in mass, morphology, and function in aged skeletal muscle compared to young. To investigate whether apoptosis is also increased in muscles from aged rodents subjected to TK‐induced I/R, the gastrocnemius muscles of 6 mo (young) and 24 mo (old) C57BL mice were analyzed for biochemical markers of apoptosis, namely cleavage of caspase‐3 and poly(ADP‐ribose) polymerase (PARP), a downstream target of active caspase‐3, by Western blotting. I/R resulted in 22 and 7‐fold increases in caspase‐3 cleavage (compared to contralateral control limbs) in young at 5 and 7 days post‐TK, respectively, and 21 and 43‐fold in old at days 5 and 7, respectively (p = 0.05 for all groups). Similarly, there were significant I/R‐induced increases in PARP cleavage in young at day 5 (28‐fold), and in old at days 5 and 7 (26 and 28‐fold; p = 0.05). Total PARP increased in the young following TK but not in the old, resulting in a significant increase in the cleaved to total PARP ratio in old compared to the young I/R samples (p = 0.05) at day 5. These data demonstrate that apoptosis‐mediating pathways are highly activated in skeletal muscle following I/R injury, and provide evidence this may be exacerbated in aged muscle at the 5 day recovery time point. This work funded in part by the U.S. Army Medical Research and Materiel Command grant DAMD17‐03‐1‐0735 to RPF.
Biosynthesis of cyanobacterial phycobiliproteins in E. coli: examination of allophycocyanin subunit biosynthesis and creation of unique fluorescent phycobiliproteins containing phycoerythrobilin
Authors: Biswas Avijit , Schluchter Wendy M
Published: June 2021

Phycobiliproteins are soluble light‐harvesting proteins and are highly fluorescent due to the addition of a bilin attached at specific Cys residues, usually by enzymes called bilin lyases. An in vivo heterologous E. coli multiplasmid co‐expression system was created to test different phycobiliprotein bilin lyases and to produce large amounts of holo‐phycobiliproteins. Co‐expression of the bilin biosynthetic genes ho1 encoding heme oxygenase from Synechocystis sp. PCC 6803 and pcyA from Synechococcus sp. PCC 7002 (pPcyA) resulted in the production of large amounts of phycocyanobilin (PCB) from heme in E. coli cells as evidenced by the blue color of cells. The CpcS/CpcU bilin lyase was shown to recognize and attach PCB to HT‐ApcD/ApcB and to HT‐ApcF. However, ApcE (the allophycocyanin domain 1‐228) fused to glutathione S transferase had autocatalytic bilin lyase activity in the presence of PCB (pPcyA). A plasmid expressing ho1 and pebS to produce phycoerythrobilin (PEB) in E. coli was used to test the substrate specificity of the bilin lyase CpcS/CpcU from the cyanobacterium Synechococcus sp. PCC 7002 and of the bilin lyase CpcE/CpcF from Synechocystis sp. PCC 6803. We used this system to make unique phycobiliproteins containing PEB. This research was supported by National Science Foundation grant to W. M. S. (MCB‐0133441).
Effects of preconceptional through post‐partum vitamin A supplementation on intellectual, motor, and behavioural development of school‐aged offspring in rural Nepal.
Authors: Buckley Gillian J. , Murray‐Kolb Laura E. , Christian Parul , Khatry Subarna K. , Shrestha Sharada Ram , LeClerq Steven C. , West Keith P.
Published: June 2021

Vitamin A deficiency in pregnancy is a common nutritional problem in poor countries, especially in South Asia. Moderate‐to‐severe vitamin A deficiency in pregnancy causes gestational night‐blindness, a risk factor for increased mortality in pregnancy and post‐partum. In rural Nepal a randomized, double‐blind, placebo controlled trial carried out from 1994 through 1997 showed that preconceptional through post‐partum weekly supplementation with vitamin A or beta‐carotene reduced maternal mortality by >=40%, but had no effect on fetal loss, infant mortality, neonatal size or infant growth. Participants in this trial received a weekly dose of 7000 ug of retinol equivalents (RE) in an oil‐based capsule, approximating the RDA for pregnant or lactating women (~1000 ug RE). The weekly placebo capsule contained ~100 ug RE. The present study compared the development of peri‐adolescent children born to a subset of mothers randomized to receive vitamin A versus placebo during the trial. Trained assistants tested 381 ten to 13 year old children (mean age 12.3 years, 46% male) using the Universal Nonverbal Intelligence Test (UNIT) and the Movement Assessment Battery for Children (MABC) in local village test centers. Trained interviewers administered the Conners' Behavior Ratings to children's parents and teachers. Results to be presented will describe the treatment effect of vitamin A on the UNIT, MABC and Conners' Behavior Ratings, and also describe factors that predict test scores in this setting. Supported by the Bill and Melinda Gates Foundation, USAID, and the Sight and Life Research Institute.
Acute effects of increased hexosamine biosynthesis and protein O‐GlcNAcylation on cardiac gene expression
Authors: Chatham John C , Bray Molly S , Young Martin E
Published: June 2021

Increased levels of O‐linked N‐acetylglucosamine (O‐GlcNAc) on nucleocytoplasmic proteins have been implicated in attenuating ischemic injury as well as mediating the adverse effects of diabetes on the heart. However, little is known regarding the effects of altered protein O‐GlcNAcylation on cardiomyocyte gene expression. Therefore, the purpose of the present study was to examined the acute (6 hours) effects of increasing protein O‐GlcNAcylation on global gene expression in neonatal rat ventricular cardiomyocyte (NRVMs), by activating the hexosamine biosynthesis pathway (5mM glucosamine), or inhibiting O‐GlcNAcase (100μM PUGNAc). Glucosamine significantly altered the expression of 1650 genes of which 775 were induced and 845 repressed. Gene ontology analysis revealed effects on stress response pathways following glucosamine treatment. KEGG pathway analyses indicated that MAPK signaling and cell cycle pathways were significantly modified by glucosamine (p<0.05) there was also a trend towards modulation of PPAR and mTOR pathways. PUGNAc significantly altered 653 genes of which 253 were induced and 400 repressed; pathway analysis indicated that these genes were associated with p53 and Wnt signaling pathways. These data suggest that alterations in O‐GlcNAc turnover contribute to regulation of cardiac gene expression. Grants: USDA/ARS 6250‐51000‐044 (MEY) & NIH HL067464/HL079364 (JCC).
Using Physiological Structure to Integrate across Biological Scales
Authors: Ayati Bruce
Published: June 2021

We present a modeling and simulation approach for integrating smaller scale biological processes into studies of larger scale systems. We use so‐called structured variables, which represent physiological traits such as age, size or senescence, to integrate the important properties of the smaller scale into mathematical models of the larger scale. The methodology allows for hypothesis‐based modeling and simulation by permitting the inclusion of significantly more information with a modest increase in computation.

We discuss our approach in the context of understanding the role phenotypic diversity plays in the development of bacterial biofilms, and the role of cell heterogeneity in tumor invasion.

The author is partially supported by the NSF under award DMS‐0914514.
Mass spectrometric analysis of biotinylated peptides and histones
Authors: Kuroishi Toshinobu , Cerny Ronald L. , Zempleni Janos
Published: June 2021

Previously, we reported a novel post translational modification of histones: binding of biotin to distinct lysine residues. In these previous studies, biotinylation sites were identified by incubating synthetic histone‐based peptides with histone biotinyl ligases, followed by probing with streptavidin, HPLC, and mass spectrometry (MS); subsequently, biotinylated (bio‐)histones were detected by biotinylation site‐specific antibodies. Objective of this study is to detect bio‐histones by MS in human cell extracts. First, we calibrated our system by using synthetic bio‐peptides. MS analysis by Quadrupole Time‐of‐Flight LC/MS with electrospray revealed that biotinylation of lysine prevented tryptic digestion of the C‐terminal peptide bond; also, the amide bond between biotin and the epsilon amino group in lysine was resistant to tryptic digestion. MS/MS analysis revealed two signature peaks (m/z = 310 and 227) that were detected in bio‐peptides but not in non‐biotinylated peptides. Second, we optimized enrichment protocols for bio‐peptides. Enrichment with immobilized monomeric avidin and 1% formic acid was associated with a >99% loss of bio‐peptides. Currently, we are testing alternative enrichment strategies using other derivatives of avidin and anti‐biotin. (Supported by UNL ARD, NIH DK 063945, 077816, 082476 and ES 015206, USDA 2006‐35200‐17138, and NSF EPS 0701892.)
Pharmacokinetics of flavonoids and phenolic acids from cranberry juice cocktail in humans
Authors: McKay Diane L. , Chen Chung‐Yen Oliver , Blumberg Jeffrey B
Published: June 2021

Putative health benefits attributed to cranberry consumption include a reduced risk of urinary tract infections, stomach ulcers, and cardiovascular disease. However, data on the bioavailability and metabolism of cranberry bioactives, including flavonoids and phenolic acids, are required to better understand their impact on pertinent risk factors for chronic disease, and inform the design of relevant clinical trials. We conducted a single‐dose, pharmacokinetic study to characterize the bioavailability of cranberry flavonoids and phenolics acids from cranberry juice cocktail (CJC) in 10 healthy adults with BMI 18.5–29.9, and age 50–70 y. After a 2‐d run‐in period following a low polyphenol diet, fasted subjects consumed 237 mL low‐calorie, sugar‐free CJC (54% juice). Blood samples were collected at specified time points before and up to 24 h after beverage consumption. We found marked variation in the maximal plasma concentration (Cmax) and time to reach Cmax (Tmax) of these cranberry bioactives, e.g., sinapic acid with 18.6 ng/mL at 30 min, homovanillic acid with 54.1 ng/mL at 6 h, protocatechuic acid with 1.3 μg/mL at 10 h, myricetin with 18.3 ng/mL at 2 h, quercetin with 0.4 μg/mL at 8 h, and kaempferol with 18.4 ng/mL at 2 h, respectively. These data suggest that different phenolic constituents in CJC are absorbed and metabolized at different locations in the gastrointestinal tract. (Supported by USDA and Ocean Spray, Inc.)
Tactile tips, toes & tails
Authors: Organ Jason M.
Published: June 2021

The prehensile tail, capable of suspending the entire body weight of an animal, is thought to have evolved twice (in parallel) in New World monkeys (Platyrrhini): once in the monophyletic Atelinae (Alouatta, Ateles, Brachyteles, Lagothrix), and once in Cebus. This line of reasoning derives primarily from osteologic evidence suggesting similar mechanical structure of the prehensile tail in these two groups – both atelines and Cebus have caudal vertebrae capable of withstanding significantly higher bending stresses than nonprehensile‐tailed platyrrhine caudal vertebrae. This study focuses on the soft‐tissue similarities between atelines and Cebus, when compared against nonprehensile‐tailed platyrrhine taxa. Specifically, atelines and Cebus share similar pinnation angles, fiber lengths, and physiologic cross‐sectional areas of the prime lateral flexors of the tail (mm. intertransversarii caudae). The similarities in structure of these muscles indicate that the mm. intertransversarii caudae of atelines and Cebus are capable of producing higher contractile forces than those of nonprehensile‐tailed platyrrhines without any trade‐off in contraction velocity. These parallels are remarkable given that the skin of the tail is thought to differ dramatically between atelines and Cebus vis‐à‐vis Meissner's corpuscle density – anatomy associated with high tactile precision and prehension. Supported by NSF BCS‐0550676.
Chronic Enteral Leucine Supplementation of a Low Protein Diet Increases Skeletal Muscle Protein Synthesis in Neonatal Pigs by Stimulating mTOR‐Dependent Translation Initiation
Authors: Torrazza Roberto Murgas , Suryawan Agus , Gazzaneo Maria C. , Orellana Renan A. , Nguyen Hanh V. , Davis Teresa A.
Published: June 2021

Leucine appears to be the key amino acid that positively regulates mTOR signalling. We hypothesized that prolonged feeding (24 hours) of a Leu supplemented low protein (LP) diet in neonatal pigs will increase protein synthesis in skeletal muscle to a rate similar to that of a high protein diet (HP). Fasted 5‐d‐old piglets (n=17) were gavage fed every 4 hours either: 1) LP diet, 2) low protein diet supplemented with Leu (LP+L) to equal HP diet, or 3) HP diet. Diets were isocaloric and lactose was equal. Fractional protein synthesis rates and translational control mechanisms were examined after 24 hours. Protein synthesis increased in the skeletal muscle of the LP+L group compared to LP group alone and did not differ from HP group. LP+L and HP increased the phosphorylation of S6K1 and 4E‐BP1, decreased inactive 4E‐BP1 eIF4E complex abundance, and increased active eIF4E eIF4G complex formation in skeletal muscle. Our results suggest that chronic feeding of a low protein diet supplemented with leucine stimulates and maintains the protein synthesis response in skeletal muscle to a rate similar to that achieved by feeding a high protein diet and this stimulation involves increased eIF4F complex assembly. (Supported by Ajinomoto Amino Acid Research Program and USDA 58‐6250‐6‐001).

Grant Funding Source: Ajinomoto Amino Acid Research Program and USDA 58‐6250‐6‐001
High‐frequency oscillatory ventilation (HFOV) associated with inhaled nitric oxide (iNO) attenuates oxidative lung injury in a saline‐lavaged rabbit model of acute respiratory distress syndrome (ARDS)
Authors: Ronchi Carlos Fernando , Fioretto Jose Roberto , Ferreira Ana Lucia dos Anjos , Campos Fabio Joly , Kurokawa Cilmery Suemi , Carpi Mario Ferreira , Moraes Marcos Aurelio , Bonatto Rossano Cesar , Defaveri Julio , Yeum Kyung‐Jin
Published: June 2021

ARDS has been associated with high mortality rate despite a better understanding of its pathophysiology and recent advances in treatment.

Objective

To compare protective conventional mechanical ventilation (CMV) and HFOV associated or not with iNO for lung oxidative stress.

Methods

Lung injury was induced by tracheal infusion of warm saline. Fifty rabbits were randomly assigned to: a) Control (CG): without ARDS + CMV; b) ARDS + CMV (MVG); c) ARDS + CMV with iNO (MVGNO); d) ARDS + HFOV (HFG) and e) ARDS + HFOV with iNO (HFGNO). In addition, ten rabbits were used for oxidative stress analysis without ARDS and MV treatment, Healthy group (HG). Lung oxidative stress was assessed by total antioxidant performance (TAP) assay.

Results

All ARDS groups showed worse PaO2/FiO2 (CG: 438.4±87.1 > MVG: 79.9±12.7 = MVGNO: 80.7±24 = HFG: 66.2±34.2 = HFGNO: 56±21; p<0.05), as compared to those of CG. After 4 hours of MV, HFOV groups showed better oxygenation (PaO2/FiO2: CG: 465.9±30.5 = HFG: 399.1±98.2 = HFGNO: 326.9±119 > MVG: 232.7±104 = MVGNO: 218.3±118; p<0.05) and reduced oxidative stress in lung (TAP, % protection: HG: 67.3±4.21 = HFG: 69.0±4.99 = HFGNO: 68.9±4.14 > MVG: 47.6±2.58 = MVGNO: 56.3±2.40 = CG: 59.4±4.52, p<0.05) compared to CMV groups.

Conclusion

HFOV improves oxygenation and attenuates oxidative lung injury in saline‐lavaged rabbit model of ARDS. [Supported by FAPESP: 2008/08199‐2, USDA 1950‐51000‐065‐08S]
Expression of wheat eukaryotic initiation factor (eIF) 3 in E. coli
Authors: Wilkerson Chris , Cho Peter , Mu Kathy , Shie Jessica , Tzeng Shinetin , Fontenot Miles , Koythong Tamisa , Lay Erica , Browning Karen S. , Choy Grace
Published: June 2021

The eukaryotic initiation factor 3 (eIF3) complex is comprised of 13 non‐identical subunits. eIF3 interacts with several other initiation factors and controls the assembly of the 40S ribosomal subunit on mRNA. It would be useful to express the subunits for eIF3 in E. coli and purify a functional complex for further analysis. A Freshman Research Initiative (FRI) group prepared de novo all the subunits for wheat eIF3 in a format that optimized codon usage for E. coli. The goal was to clone all 13 eIF3 subunits into three plasmid cassettes for simultaneous expression and self‐assembly into the functional eIF3 complex. The FRI has students from the spring of the freshman year to the fall of the sophomore years participate in goal oriented research. Over a period of 2 years, FRI students prepared coding regions by overlap extension PCR of oligonucleotides, cloned the PCR products into vectors and showed expression of the individual subunits in E. coli. The 13 subunits were cloned into one of three plasmids, pET22b (subunits i,g,h,f), pRSFDuet‐1 (subunits c,d,l,k,e), and pACYCDuet‐1 (a,b, m). Each of the subunits in the plasmids were shown to express. Additional orientations will be tested for optimized expression of the complex.

Grant Funding Source: NSF
Kinetic and structural studies of Haemophilus influenzae carbonic anhydrase with altered proton transfer capacity
Authors: Lam Gretl , Ciaccia Jessica , Rowlett Roger S.
Published: June 2021

The rate‐determining step for CO2 hydration by β‐carbonic anhydrase (β‐CA) is believed to be the loss of a hydrogen ion from the zinc‐bound water to regenerate the zinc‐bound hydroxide ion. Intramolecular acceptors, including a Tyr residue in the active site cleft of Haemophilus influenzae β‐CA (HICA) and a His residue near the exterior of the active site cleft of plant β‐CAs, are hypothesized to facilitate proton transfer from the zinc‐bound water. We report that in HICA the replacement of Tyr83 with Phe decreased kcat ≈30% of wild type, and displays substrate inhibition similar to the analogous Y212F variant of Arabidopsis thaliana β‐CA. Conversely, the replacement of Val87 with His increased the kcat to ≈250% of wild‐type, similar to the kcat of plant β‐CAs. X‐ray crystallographic structures of the V87H variant reveal a unique orientation of Tyr181 to hydrogen bond to the “unique” water molecule in HICA, and alternate “in” and “out” conformations of His87 that could facilitate intermolecular proton transfer to bulk solution; V87H crystals soaked in bicarbonate ion demonstrate that the enzyme still retains the ability to bind bicarbonate in the allosteric site. These results support the hypothesis of proton transfer as the rate‐determining step of β‐CA, and that Tyr and His residues can play important roles in this proton transfer step. This work is supported by NSF grants CHE‐0819686 and MCB‐0741396.
Reversible negative design destabilization of hemolysin A
Authors: DeVilbiss Andrew , Weaver Todd
Published: June 2021

Hemolysin A (HpmA) from Proteus mirabilis is a member of the two‐partner secretion (TPS) pathway found in Gram‐negative bacteria. The TPS pathway provides an energy independent mechanism for secretion of large virulence factors, like adhesions and hemolysins. A truncated form of HpmA, termed HpmA265, containing the first 265 residues, facilitates biphasic, cooperative, heat stable, and template‐assisted hemolytic activity. The crystal structure of HpmA265 was recently solved, revealing a right‐handed beta‐helix fold, and a dry dimeric interface formed between C‐terminal adjacent HpmA265 monomers. To investigate the role of the C‐terminal dry dimeric interface during template‐assisted hemolysis, a negative design concept was employed. A positively charged lysine residue was used to replace Leu263 located within the dry, dimeric interface. The resultant mutant has been termed L263K. Template‐assisted hemolytic assays confirmed a decreased ability to activate full length HpmA, while circular dichroism (CD) confirmed structural destabilization within L263K. Also, SDS‐PAGE, mass spectrometry, N‐terminal sequencing, and surface plasmon resonance confirmed a degradation event at the C‐terminus of L263K. Re‐analysis via template‐assisted hemolytic assays and CD confirmed the restoration of template‐assisted hemolysis and beta‐signal within degraded L263K. These data demonstrate the C‐terminal dry dimeric interface is the important site of interaction between HpmA265 and full length HpmA. The research was supported by the UW‐La Crosse Dean's Distinguished Undergraduate Research Fellowship to AD, along with an NSF grant (MCB0744754) to TW.
Structural studies of an RNA aptamer‐with high affinity for the B. anthracis ribosomal protein S8.
Authors: Davlieva Milya G , Doneske James , Michnicka Malgorzata , Nikonowicz Edward P. , Shamoo Yousif
Published: June 2021

The bacterial S8 protein is a ribosomal protein which binds to the 16S rRNA in the central domain and plays a critical role in 30S ribosomal subunit assembly. An RNA aptamer with high affinity for B. anthracis S8 protein was developed using the SELEX methodology and crystals of the complex were obtained. To understand how S8/RNA recognition occurs for this apatamer, we are determining the structure of the complex by X‐ray crystallography.

Diffraction data for the S8/RNA crystal were collected to 2.6Å resolution at the Advanced Light Source synchrotron (Berkeley, CA). The crystal belonged to the space group P212121 with unit cell parameters a= 55.41 b=59.27 c=92.25Å, α=90, β= 90, γ=90. The data were processed using the program d*TREK with Rmerge=9.2 % and completeness = 99.9 %. A molecular replacement solution for S8/RNA complex was found with the program Phaser for MR from the CCP4 suite using S8 protein (unpub.data) as a search model. The initial solution suggested one monomer per asymmetric unit consistent with a Matthews coefficient of 3.2 (65% solvent). The molecular replacement solution was further confirmed by the initial (2Fo‐Fc) maps generated using Coot that clearly indicated electron density for the RNA aptamer. A model is being constructed for the protein‐RNA complex.

This work was supported by NSF (0641792) to Y.S. and The Robert A. Welch Foundation (C‐1584 to Y.S. and C‐1277 to E.P.N.) and NIH(U54 AI057156).
Vitamin D Status in Overweight and Obese Individuals During A Controlled Calorie Restricted Intervention
Authors: Piccolo Brian D , Gertz Erik R , Hall Laura , Cheema Minnu , Woodhouse Leslie , Souza Elaine , Keim Nancy L. , Adams Sean H , Stephensen Charles B , Van Loan Marta D
Published: June 2021

Vitamin D deficiency is common in obese subjects and status may be improved with dietary interventions. We examined serum 25(OH)D in overweight and obese individuals enrolled in a controlled calorie restricted intervention. Serum samples from overweight and obese (BMI = 28–37) subjects (F=53, M=18), participating in a study examining dairy's role in weight and fat loss, were taken pre‐ and post‐intervention. Participants were randomly assigned to either a high dairy (HD: 3–4 servings; average 333IU D) or low dairy (LD: ≤ 1 serving/d, average 151 IU D) for 12 wks. Energy balance was achieved in wks 1–3 followed by 12wk of 500 kcal/d reduction. All foods were supplied by the metabolic food lab. Outcome variables were body weight (WT), body fat (BF) determined by DXA and serum 25(OH)D measured by RIA. When controlling for total body fat mass, ANOVA showed a significant treatment effect with a greater increase in the %change of serum 25(OH)D in the high dairy group (27.27 ± 37.47 nmol; p = 0.05) compared to the low dairy group(13.16 ± 32.7 nmol; p = .09). The inclusion of 3–4 servings of vitamin D fortified dairy rich foods in a moderate energy restricted diet may contribute to an improved vitamin D status in overweight and obese individuals.

Funding: National Dairy Council; USDA, ARS, WHNRC; DairyCouncil of California; CTSC, CCRC University of California (1M01RR19975‐01), and National Center for Medical Research (UL1 RR024146).
Riboflavin protect against lipopolysaccharide ‐induced shock via HSP25 in mice
Authors: Chen Chung‐Yen Oliver , Chen Chiao‐Ming , Shih Chun‐Kuang , Chou Hung‐Tsung , Liu Jen‐Fang , Li Sing‐Chung
Published: June 2021

Riboflavin (B2) has been found to increase survival rate of mice with endotoxin‐induced shock via an anti‐inflammatory mechanism. Heat shock proteins (HSP) are produced in response to stress, injury, and shock. However, the protective effect of B2 on shock through this mechanism remains to be explored. Thus, we examined the effects of B2 on survival rate, and expressions of HSPs and heat shock factor 1 (HSF1) in mice with lipopolysaccharide (LPS) induced shock. Mice were treated with saline, LPS, LPS + low dose of B2 (LB2, 2 mg/kg BW) or LPS + high dose of B2 (HB2, 20 mg/Kg). Riboflavin was administered intraperitoneally at 2 and 0 hr before LPS administration (20 mg/kg). Survival rate monitored at 12 h intervals for 7 d was 10, 20, 60, and 100% for LPS, LB2, HB2, and saline mice, respectively. HSP25 expressions in heart and lung were significantly enhanced in a time dependent manner in HB2 mice as compared to saline mice (P ≤0.05), but not altered in LB2 mice. HSF1 expression was not altered in all mice. Plasma B2 concentrations were increased in time and dose dependent manners. In HB2 mice, B2 level reached 300 nM at 6 h post LPS and returned to the 0 h level at 72 h. Our results suggest that B2 could decrease LPS‐induced mortality through increased expression of HSP25 protein. Supported by the National Science Council of Taiwan and Taipei Medical University‐Wan Fang Hospital, and USDA.
Effect of Zinc Supplementation on Diarrhea and Malaria Morbidity in Adults in Rural Kenya
Authors: Scrimgeour Angus G , Lukaski Henry C , Polhemus Mark E , Otieno Lucas , McGraw Susan M , Young Andrew J , Bovill Maria E
Published: June 2021

Zinc (Zn) supplementation has been associated with reductions in the incidence and severity of diarrheal diseases and malaria in young children, but not in adults. We evaluated the potential role of Zn for treatment of diarrhea and malaria in adults living in western Kenya, a malaria and HIV‐prevalent setting.

Methods

A double‐blind, RCT was conducted in 500 adults (18–55 yr of age), supplemented daily with 20 mg elemental Zn (as Zn‐sulphate) or placebo for 3 months. Trace elements and indices of inflammation (ferritin and CRP) were determined before and after the study.

Results

At baseline, 33% of subjects were Zn‐deficient (<65μg/dL). Plasma Zn increased by 18.5% in subjects receiving Zn (P<0.0001), and did not change in the placebo group (P=0.30). Zn‐supplementation did not modify Ca, Cu, Fe, Mg, Mn, Se, ferritin or CRP values, and did not decrease overall diarrhea or malaria indices. However, compared to placebo, Zn supplementation decreased diarrhea prevalence (−48%, P=0.20), incidence (−50%, P=0.17), and malaria prevalence (−26%, P=0.06) in co‐infected adults (malaria and HIV‐positive).

Conclusions

In Kenya, Zn decreased diarrhea and malaria morbidity in co‐infected adults, but not in malaria‐only, HIV‐only, or “healthy” subjects. The observation is biologically plausible, has potential for public health impact, and should be explored in future studies.

Study funded by the U.S. Army MRMC
Effect of Soy Diets and Equol on Female Depressive Behaviors and Body Weight in Long‐Evans Rats
Authors: Blake Crystal , Setchell Kenneth DR , Lund Trent D , Lephart Edwin D
Published: June 2021

In five different studies we examined the effect of soy‐containing diets and equol injections on depression, serotonin levels, weight gain and white adipose tissue (WAT) deposition of female Long‐Evans rats in various stages of life. In these experiments the rats were intact, ovariectomized or experienced natural ovarian failure (NOF). The results of all the experiments will be presented, but experiment 5 is covered here due to space limitations. From conception the female rats were exposed to either: a) a soy‐rich (Phyto‐600) or b) low‐soy diet (Phyto‐low). After approximately 290 days all the rats experienced NOF. At 330 days‐old the animals were examined in the Porsolt forced swim test (PFST). One month later the animals were again tested in the PFST [after the animals fed the Phyto‐low diet were injected with equol (@ 5 mg/kg/day) for one week]. At the first PFST, serotonin levels and mobility were decreased in the Phyto‐low fed animals compared to the Phyto‐600 animals. After the equol injections the differences in the measured parameters by treatment disappeared. Equol increased mobility in aged‐female rats that had undergone NOF and increased serotonin levels comparable to Phyto‐600 fed animals. In all the studies, animals fed a soy‐rich diet decreased body weight and adipose tissue deposition vs. a low‐soy diet. (Grant Support: USDA 2004‐01811)
Intake of added sugars is not associated with weight measures in children 6 to 18 years: NHANES 2003–2006
Authors: O'Neil Carol E , Nicklas Theresa A , Zanovec Michael , Liu Yan
Published: June 2021

The association between intakes of added sugars and weight measures in children continues to be under scrutiny because the evidence is inconclusive. This study examined the association between intake of added sugars and five weight measures using a nationally representative sample of children. NHANES, 2003–2006, 24‐hour recall data from children aged 6–18 years (n = 3,136) were used in the analyses. USDA's definition of added sugars and MyPyramid Equivalents Database were used to estimate daily intake. Multiple linear regression and squared partial correlation coefficients were used to estimate the strength of association between weight, body mass index, waist circumference, triceps skinfold, and subscapular skinfold as dependent variables with added sugars as the independent variable. Covariates included age, gender, race‐ethnicity, poverty income ratio, total energy intake (kcals), and physical activity. Mean intake of added sugars was 21 tsp accounting for 16% of total kcals. The percent variance explained in the weight measures ranged from 18%–59%; with none of the variance explained by intakes of added sugars. No significant associations were observed between intakes of added sugars and weight measures. Results are limited to cross‐sectional comparisons and the assessment measures used. Additional longitudinal studies are needed to confirm the findings.

Funding: USDA
Antioxidant defense in renal proximal tubular cells from normal and diabetic rats
Authors: Lash Lawrence Harold , Putt David A. , Terlecky Stanley R. , Zhong Qing
Published: June 2021

Primary cultures of proximal tubular (PT) cells from streptozotocin (STZ) diabetic rats had higher basal and toxicant‐stimulated reactive oxygen species and mitochondrial membrane potential and were more susceptible to oxidant injury as compared to those from age‐matched control rats. Both N‐acetyl‐L‐cysteine (NAC) and a cell‐permeable catalase derivative (Cat‐SKL) protected. Despite higher basal oxidant stress, mitochondria of diabetic PT cells had higher GSH content. Protein levels of mitochondrial GSH transporters were slightly higher in diabetes, that of superoxide dismutase 2 (SOD2) was modestly elevated, and that of total thioredoxin 2 (Trx2) was decreased at 30 days and increased at 90 days. Levels of 3‐nitrotyrosine‐modified proteins in mitochondria were somewhat higher at both times in diabetic rats. 4‐Hydroxy‐2‐nonenal (HNE)‐modified proteins were mostly increased at 30 days but decreased at 90 days. Thus, redox processes and mitochondrial energetics are markedly altered due to diabetes and change as nephropathy progresses. (Supported by DOD Grant PR64340)
Undergraduate research and active learning opportunities in biochemistry at UNL
Authors: Matejka Stephanie Lynn , Jimenez Ignacio , Bailey Cheryl
Published: June 2021

At large universities and some smaller institutions, not all undergraduate biochemistry majors are involved in a research project. This is unfortunate, since the scientific method and research process provides an excellent learning opportunity. Three models are currently being implemented to provide alternate opportunities to undergraduate life science majors at the University of Nebraska, Department of Biochemistry. These include the Genomics Education Partnership and the Joint Genomic Institute Annotation Collaborative Tool, both undergraduate research classes. In addition, short bioinformatics modules have been implemented in existing laboratory and large traditional lecture classes, to facilitate a mini research experience for a large number of students. We report methods, implementation strategies, student assessment results, and confidence in project findings.

Supported by HHMI and NSF
Subdivision of the echinoderm nervous system as defined by antibody probes
Authors: Diaz‐Balzac Carlos A. , Lázaro‐Peña María I. , García‐Arrarás José E.
Published: June 2021

Echinoderms are an enigmatic group of deuterostomes that present some unusual properties in many cells and organs, including those of the nervous system. Nonetheless, studies of their nervous system have lagged behind when compared to those in other invertebrates groups due to the lack of nervous system markers. We have now used 8 commercially available antibodies and 3 antibodies produced by our group, which specifically label nervous system components in the echinoderms, to study the neuroanatomy of the sea cucumber Holothuria glaberrima. Our studies have produced three important findings. First, each antibody labels specific regions of the radial nerve, suggesting that the radial nerve cord is subdivided into distinct regions with specialized tracts or networks. Second, the level of complexity of the enteric nervous system is higher than described in the past, since our antibodies label many more cells and nerves than previously documented. Third, labeling of the different muscular tissues showed the presence of a large number of nerve fibers that appear to be innervating the muscle fibers, thus identifying the possible motor pathway that innervates the echinoderm muscles system. By combining our observations, we have begun to assemble a map of the neural circuitry of the holothurians. Our findings suggest that the echinoderm nervous system is more similar to that of other deuterostomes than previously thought.

Grant Funding Source: This work was supported by NSF (IBN‐0110692) and NIH‐MBRS (S06GM08102). CADB was funded by the UPR‐RP MARC Program (5T34GM007821) and MILP was funded by the UPR‐RP RISE Program (2R25GM061151). We also acknowledge partial support from NIH‐RCMI (RRO‐3641‐01) and the University of Puerto Rico.
Open complex contacts: an analysis of the interaction between T5 N25 discriminator region and E. coli RNA polymerase
Authors: White Cassandra L , Hsu Lilian M
Published: June 2021

The discriminator (DIS) region referring to the 6–8 bp between the +1 start site and the −10 box of the promoter has been shown to make direct contact with RNA polymerase (RNAP) to stabilize the open complex. Previous research showed that the second nucleotide on the non‐template strand downstream of the −10 box, when it is a G, in the rrnB P1 C−7G and λ PR promoter contacts the σ1.2 region (Haugen et al. 2006 Cell 125, 1069; 2008 PNAS 105, 3292), but in the T5 N25 promoter contacts the β’ subunit. These promoters differ in the three nucleotides immediately downstream of the −10 box: GGG in rrnB P1 C−7G, GGT in λ PR, and AGA in N25. To determine if the G‐content in these three bases affect the contact of the G−5 residue with RNAP, we constructed three promoters – the wild type N25 (where DIS is A−6GATTC−1), A−6G N25, and A−6G/A−4G N25 – and subjected them to UV‐induced crosslinking to RNAP via their −5 S6G residue. Preliminary results showed that all three promoters transcribe very similarly, indicating that the stability of the open complex is minimally affected by the DIS mutations. In crosslinking, while both the wild type N25 and the A−6G N25 were found to contact the β’ subunit, the A−6G/A−4G N25 promoter failed to show any crosslinking. Our results suggest that the path of the DIS DNA may differ from promoter to promoter, thus lending different degree of stabilization to the open complex. Sponsored by an NSF grant (RUI‐0841452) to L. M. Hsu
Investigating the structural flexibility of intrinsically disordered proteins
Authors: Grese Laura Nicole , Stanley Christopher , Rowe Erica , O'Neill Hugh , Berthelier Valerie
Published: June 2021

Intrinsically disordered proteins (IDPs) either possess an unstructured domain or are fully disordered until recognizing a target binding partner, upon which a coupled folding‐binding event occurs. Exactly how this mechanism is linked to protein signaling and recognition is still poorly understood, primarily because the structural flexibility of IDPs limits the number of suitable characterization techniques. We are using circular dichroism (CD) spectroscopy and small‐angle neutron scattering (SANS) to investigate IDP regions of the transcription co‐activator: CREB binding protein (CBP), and its interactions with the binding partner: activator for thyroid hormone and retinoid receptors (ACTR). CBP is essential to several biological processes such as embryonic development, growth control, and homeostasis, and its dysfunction is implicated in Huntington's disease and some cancers. CD spectroscopy allows us to study changes in protein secondary structure. With SANS we are able to directly probe structural changes on the nanometer length‐scale and in solution, which is essential for capturing the flexibility of IDP structures. SANS experiments have been performed using the BioSANS instrument at HFIR, ORNL to characterize the CBP‐ACTR complex. Ultimately, this research should provide new possibilities for the study of disordered protein regions and yield unique insights into the mechanism of IDP function.

This research is supported by a Laboratory Directed Research and Development (LDRD) grant from Oak Ridge National Laboratory, managed by UT‐Battelle for the Department of Energy.
Identifying the Cox24 Protein as a Factor Involved in Mitochondrial Protein Synthesis
Authors: Shin Jane , Tibbetts Anne S. , Appling Dean R.
Published: June 2021

The current understanding of mitochondrial protein synthesis is far from complete; it is likely that many accessory factors required for this process have not yet been identified. The FMT1 gene encodes the S. cerevisiae methionyl‐tRNA formyltransferase, which formylates the mitochondrial initiator Met‐tRNA. A genetic screen was designed to identify accessory factors involved in mitochondrial translation, particularly in the absence of formylated Met‐tRNA. This screen yielded yeast mutants that in combination with the fmt1 deletion, cause dysfunctional mitochondrial respiration. One mutant strain, s.p. 106–1, was transformed with a yeast genomic DNA library to identify open reading frames that complement the synthetic petite phenotype. Library plasmids that complemented the mutation were sequenced and resulting open reading frames were subcloned and expressed in the mutant strain. Expression of the COX24 gene rescued respiratory growth, and sequencing of s.p. 106–1 yeast genomic DNA revealed a T‐to‐C mutation in COX24 that leads to a leucine to proline substitution at amino acid 93. Studies indicate that Cox24p is required for proper splicing of mitochondrial COX1 pre‐mRNA. Our results indicate that Cox24p may also play a role in the initiation of mitochondrial translation, particularly when formylated Met‐tRNA is not available. Work supported by HHMI and NSF grants to the Freshman Research Initiative.
Mechanical Ventilation and Sepsis Impair Protein Metabolism in the Diaphragm of Neonatal Pigs
Authors: Srivastava Neeraj , Gazzaneo Maria C , Torrazza Roberto Murgas , Suryawan Agus , Nguyen Hanh V , El‐Kadi Samer W , Fiorotto Marta L , Davis Teresa A , Orellana Renan A
Published: June 2021

Mechanical ventilation (MV) impairs diaphragmatic function and diminishes the ability to wean from ventilatory support in adult humans. In normal neonatal pigs, animals that are highly anabolic, endotoxin (LPS) infusion induces sepsis, reduces peripheral skeletal muscle protein synthesis rates, but increases protein synthesis in the diaphragm. To determine whether MV and sepsis lead to protein catabolism in the diaphragm, neonatal pigs (n=5–6/group) were subjected to MV and infused with LPS (0 and 10 μg•kg−1•hr−1), as well as dextrose and a balanced amino acid mixture. After 9 hours, fractional protein synthesis rates and translation and degradation signals were determined in diaphragm muscle. Compared to controls, MV alone decreased protein synthesis in the diaphragm by 16%, and this effect was not further aggravated by the addition of LPS. MV alone and in the presence of LPS decreased eIF4G·eIF4E association and eIF4G phosphorylation in similar proportion when compared to controls. MV alone increased MURF‐1 abundance by 240% and by 340% in the presence of LPS. These findings suggest that MV induces diaphragmatic wasting due to both decreased protein synthesis and increased proteolysis, and the presence of sepsis enhances the MV‐associated induction of proteosomal degradation. NIH AR51563, NIH AR44474, and USDA 6250‐51000‐040

Grant Funding Source: NIH AR51563, NIH AR44474, and USDA 6250‐51000‐040
Regulation of protein turnover in porcine intestinal cells by L‐glutamine (Gln)
Authors: Xi Pengbin , Jiang Zongyong , Dai Zhaolai , Li Xilong , Yao Kang , Wang Junjun , Wu Guoyao
Published: June 2021

Intestinal porcine epithelial cells (IPEC‐1) from newborn pigs were seeded in 6‐well culture plates with DMEM‐F12 containing 5% FBS, 1% antibiotics and 5μg/ml insulin. For determining protein degradation, after 16‐h culture, cells were cultured for 3 days in DMEM‐F12 containing 0.5 mM Gln. Beginning on day 4, cells were cultured for 24 h in DMEM containing 0.5 mM Gln and 0.1 mM L‐phenylalanine (Phe) plus L‐[3H]Phe. After 24‐h culture, the medium was removed and cells were washed 3 times with PBS. The cells were then cultured for 3 h in 2 ml DMEM containing 1 mM Phe and 0.03, 0.5 and 2 mM Gln. At the end of 3‐h culture, medium and cells were analyzed for 3H‐Phe. For determining protein synthesis, after 16‐h culture, the cells were cultured for 3 days in DMEM‐F12 containing 0.5 mM Gln. On day 4, cells were cultured for 3 h in 2 ml DMEM containing 1 mM Phe plus 0.1 μCi L‐[ring‐2,4‐3H]Phe, 0.03, 0.5 and 2 mM Gln. At the end of 3‐h culture, cells were collected to determine protein‐bound 3H‐Phe. Compared with 0.03 mM Gln, addition of 0.5 and 2 mM Gln to culture medium dose‐dependently decreased (P < 0.05) protein degradation in IPEC‐cells by 19% and 23%, respectively, while increasing protein synthesis by 28% and 36%, respectively, in IPEC‐1 cells. Likewise, 0.5 and 2 mM Gln enhanced the growth of IPEC‐1 cells. These results indicate that Gln stimulates net protein synthesis in intestinal cells. Supported by grants from USDA and NSFC.
Modeling dietary fiber intakes in U.S. adults in National Health and Nutrition Examination Survey (NHANES) 2003–2006
Authors: Fulgoni Victor L , Liska DeAnn J , Almedia Nelson G , O'Neil Carol E , Nicklas Theresa A
Published: June 2021

Over 90% of adults do not obtain the Adequate Intake (AI) for dietary fiber (DF). Using only reliable recalls in NHANES 2003–2006, we modeled the following changes to assess impact on usual DF intakes in US adults 19+ yrs: 1) increase all fiber containing foods by 10, 25, 50, or 100%; 2) increase fiber content of low fiber grain products to a) a good source level (2.5 g/serving) or b) an excellent source level (5.0 g/serving); and 3) increase currently available whole grain foods to meet recommendations. With each scenario, usual DF intake was determined using the National Cancer Institute method. Baseline DF intake was 15.7 g/d with 96% of adults not obtaining the AI for DF and 92% not obtaining the Daily Value of 25 g/d. Increasing DF containing foods 10, 25, 50, or 100% increased DF intake to 16.9, 18.9, 22.1, and 28.5 g/d, respectively with a concomitant increase in calories of 104, 260, 521, 1042 kcal/d, respectively. Adding fiber to low DF grain foods to 2.5 or 5.0 g/serving resulted in DF intakes of 24.7 and 39.1 g/day, respectively without an increase in calories. Increasing consumption of currently available whole grain foods could be expected to increase fiber intake to 25.3 g/day but with an additional 1266 kcal/d. Consuming the recommended level of DF is challenging for most people; adding fiber to low fiber grain products and increasing DF concentration in foods selected will be essential to meet recommendations without increasing calories. (Supported by USDA and Kellogg Company)
Chronic Alpha‐1A Adrenergic Receptor Stimulation Reduces Anxiety in Mice
Authors: Wood Sarah , Luger Elizabeth , Davis Bethany , Haselton James , Perez Dianne , Doze Van
Published: June 2021

Tricyclic antidepressant drugs are used for the treatment of chronic depression, obsessive‐compulsive disorder, and anxiety‐related disorders. Chronic use of these drugs increases the expression of alpha‐1 adrenergic receptors (α1‐ARs). Yet, it is unclear whether increased α1‐AR expression contributes to the anxiolytic effects of these drugs or if this effect is unrelated to their therapeutic benefit. In this study, mice expressing constitutively active mutant (CAM) α1A‐ARs or CAM α1B‐ARs were used to examine the effects of chronic α1A‐ and α1B‐AR signaling on rodent behavioral models of anxiety. When compared to normal wild type mice, CAM α1A‐AR mice, but not CAM α1B‐AR mice, exhibited anxiolytic‐like behavior (i.e., spent more time in the open sections) in both the elevated zero maze and elevated plus maze. These results not only demonstrate that α1A‐ and α1B‐ARs differentially reduce anxiety in the mouse, but that α1A‐ARs may be a useful therapeutic target for the treatment of anxiety‐related disorders. Supported by NSF 0347259, NSF 0639227, NSF0851869 and NIH P20RR0167141