bluuebunny/crossref_metadata_2025 huggingface dataset:
Increase in serum zinc levels after supplementation of zinc deficient nursing home elderly
Authors: Dao Maria Carlota , Barnett Junaidah Bajrai , Hamer Davidson , Kandel Ruth , Brandeis Gary , Fine Basil , Dallal Gerard , Jacques Paul , Schreiber Robert , Meydani Simin
Published: June 2021

Pneumonia is among the leading causes of morbidity and mortality in elderly. Zinc deficiency is associated with reduced immunity. Previously we showed that 30% of nursing home elderly (NHE) had low serum zinc levels, and zinc deficiency was significantly associated with higher incidence and duration of pneumonia. We conducted a randomized, double‐blind, placebo‐controlled study to determine if supplementing NHE with 30 mg/d of zinc for 3 months increases serum zinc levels. Participants (≥65 years) had serum zinc levels < 70 μg/dL, albumin > 3.0 g/dL, BMI > 18 kg/m2, and no preexisting conditions such as lung neoplastic disease. Participants were randomized into a zinc (30 mg/d zinc) or placebo (5 mg/d zinc) group; capsules also contained 1/2 DRIs of essential nutrients. Serum zinc, albumin, CRP, and copper levels were determined before and after supplementation. Using t‐test, the mean(±SD) serum zinc difference (baseline to month 3) is significantly higher by v17(±9) μg/dL in the zinc versus placebo group (p=0.004). The control group had a decrease of 7(±8) μg/dL, while the zinc group had an increase of 10(±10) μg/dL. No statistically significant changes in albumin, copper, or CRP levels were detected; correcting for these parameters did not impact zinc levels. Supplementation of zinc deficient NHE with 30 mg/day of zinc for 3 months increases serum zinc levels. Future studies are needed to determine if zinc supplementation can reduce incidence and duration of pneumonia in NHE. Supported by USDA (58‐1950‐7‐707)
Arginine utilization for citrulline synthesis in arginase II knockout mice
Authors: Marini Juan C , Didelija Inka Cajo , Castillo Leticia , Lee Brendan
Published: June 2021

The synthesis of citrulline (Cit) from arginine (Arg) in the small intestine depends on the activity of arginase II (ARG2). To test the hypothesis that Arg is the main dietary precursor for Cit synthesis, despite the lack of ARG2, tracer studies were conducted in WT and ARG2 ko conscious mice. WT mice had a higher rate of appearance (Ra) of Cit (130 vs. 118 μmol•kg−1 • h−1) than ARG2 ko mice, but no difference in Ra of ornithine (Orn; 278 μmol•kg−1 • h−1) was observed. There were significant differences in the utilization of dietary Cit precursors. The contribution of dietary Arg to the synthesis of Cit was reduced from 44 to 11 μmol•kg−1 • h−1 due to the lack of ARG2. No arginine was utilized at the site of Cit synthesis in ARG2 ko mice (WT = 26 μmol•kg−1 • h−1) and the contribution of Arg through Orn was reduced (18 vs. 14 μmol•kg−1 • h−1). Pro and Gln utilization increased in ARG2 ko mice from 5.7 to 8.1, and 0.7 to 1.9 μmol•kg−1• h−1, respectively. Most of the contribution of Pro and Gln was at the site of Cit synthesis, rather than through plasma Orn. Arg is still the main dietary precursor for Cit synthesis, despite the lack of ARG2 at the site of Cit synthesis. Arg is converted to Orn elsewhere in the body, presumably by action of ARG1, and then utilized for Cit synthesis. The contribution of dietary Gln and Pro increased in ARG2 ko mice, but these amino acids are minor sources for Cit synthesis. Support: USDA 2533771314 and NIH KO1 RR24173.
Prolonged leucine infusion differentially affects tissue protein synthesis in neonatal pigs
Authors: Suryawan Agus , Wilson Fiona A. , Gazzaneo María C. , Orellana Renán A. , Nguyen Hanh V. , Almonaci Rosemarie D. , Davis Teresa A.
Published: June 2021

Leucine (Leu) acutely stimulates protein synthesis by activating the mammalian target of rapamycin complex 1 (mTORC1) pathway. To determine whether Leu can stimulate protein synthesis in muscles of different fiber types and visceral tissues of the neonate for a prolonged period and to determine the role of other amino acids in the response, overnight fasted neonatal pigs were infused for 24 h with saline, Leu (400 μmol • kg‐1 • h‐1), or Leu with replacement amino acids to prevent the Leu induced fall in other amino acids. Protein synthesis rates and phosphorylation of 4E binding protein (4E‐BP1) and S6 kinase 1 (S6K1), indicators of mTORC1 activation, were measured. Leu increased 4E‐BP1 and S6K1 phosphorylation in the longissimus dorsi, gastrocnemius, and masseter muscles, liver, and pancreas, in both the absence and presence of amino acid replacement. However, protein synthesis was increased only when amino acids were infused to prevent hypoaminoacidemia. Leu had no affect on mTORC1 signalling or protein synthesis in the heart, jejunum, and kidney. Thus, prolonged infusion of leucine stimulates mTORC1 signaling in skeletal muscle and some visceral tissues but the Leu‐induced stimulation of protein synthesis in these tissues requires sustained amino acid availability. (Supported by Ajinomoto Amino Acid Research Program and USDA 58‐6250‐6‐001).
Alpha7nAChR modulates STAT3 activation to control inflammation and improve survival in sepsis
Authors: Ulloa Luis , Pena Geber , Cai Bolin , Liu Jun , vander Zanden Esmerij , Dejonge Woulter , Deitch Edwin
Published: June 2021

The alpha7 nicotinic acetylcholine receptor plays a critical role in the immune system controlling systemic inflammation in sepsis. The signaling pathway of this mechanism has remained elusive possibly due to the lack of specific agonists and the irreconcilable results using different cell lines. Here, we report that the anti‐inflammatory potential of the alpha7nAChR is mediated by unphosphorylated STAT3, and that STAT3 can prevent systemic inflammation and improve survival in sepsis. Alpha7nAChR activation or JAK2 inhibition prevents STAT3 tyrosine phosphorylation in activated macrophages. Inhibition of STAT3 tyrosine phosphorylation mimics the alpha7nAChR‐signaling to control NF‐kB, cytokine production in macrophages, and systemic inflammation in sepsis by inhibiting cytokine production in the spleen. Consistently, inhibition of STAT3 expression prevents the alpha7nAChR‐signaling, but inhibition of STAT3 tyrosine phosphorylation enhances the anti‐inflammatory potential of alpha7nAChR. STAT3 prevents the production of late mediators of sepsis, including HMGB1 extracellular secretion from macrophages and rescues mice from polymicrobial sepsis. These results reveal the clinical implications and pharmacological advantages of unphosphorylated STAT3 in infectious diseases. Studies funded by USAMRMC#05308004, AHA06352230N, NIH‐GM084125.
Calcineurin homologous protein is required for a proton‐activated muscle contraction in Caenorhabditis elegans
Authors: Wagner Jamie , Taylor Ashley , Ulmschneider Kiri , Ulmschneider Bryne , Nehrke Keith , Peters Maureen A.
Published: June 2021

The Caenorhabditis elegans defecation cycle is a 3‐step motor program. Cycle timing is controlled by intestinal calcium flux. The first step, posterior body wall muscle contraction (pBoc), is elicited by protons1,2. Protons are transported from the intestine into the pseudocoelomic space via the sodium‐proton exchanger 7 (NHX‐7) and activate ion channels in body wall muscles, resulting in contraction1. A second exchanger, NHX‐2, allows proton movements between the lumen and intestinal cells2. We hypothesize calcineurin homologous protein (CHP), a calcium responsive co‐factor for sodium‐proton exchangers, activates these NHXs in response to calcium flux. Two CHP mutants that alter calcium binding motifs exhibit pBoc defects. Genetically encoded pH indicator proteins were used to monitor pH levels in vivo. CHP mutants display altered pseudocoelomic, cytoplasmic, and lumenal proton fluxes. Cumulatively, chp mutants' pH phenotypes encompass both nhx‐2's and nhx‐7's mutant traits, suggesting CHP activates these exchangers to coordinate the calcium wave and pBoc initiation. Support is from the NSF.
Saccharomyces cerevisiae responds to cold shock by inducing the transcription of genes required for zinc ion homeostasis
Authors: Buckmelter Kristen M , Infanzon Bianca A , Liu Elizabeth M , Sakhon Olivia S , Rodriguez Kenny R , Citti Wesley T
Published: June 2021

Previous studies on the global transcriptional response of budding yeast, Saccharomyces cerevisiae, to cold shock have revealed that the response can be divided into a set of early response genes (after 15 minutes to 2 hours of cold temperatures) and late response genes (after 12 to 60 hours of cold temperatures). The late response genes include the ESR genes induced by many environmental stresses, but less is known about the early response genes. We have characterized more fully the early transcriptional response at 15, 30, and 60 minutes of cold shock at 13°C and also the response to recovery after cold shock for 30 and 60 minutes at 30°C using DNA microarrays. Results were analyzed using the program GenMAPP to determine which biological processes were activated in response to cold shock and recovery. We found that genes involved in zinc ion homeostasis such as IZH1, IZH2, IZH4, and ZRT1 were induced by cold shock and then repressed during the recovery phase. These results were confirmed through quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) experiments. Furthermore, a yeast strain deleted for the transcription factor Zap1p which regulates zinc ion homeostasis genes was found to be impaired for growth at 15°C. This work was supported by a Loyola Marymount University Kadner‐Pitts Research Grant (K.D.D.) and NSF awards 0634613 (K.R.R. and K.D.D.) and 0921038 (K.M.B., B.A.I., and K.D.D.).
Probing the Active Site of Thermotoga Maritima ADPGlucose Pyrophosphorylase
Authors: Arteaga Adrianna , Matsui Mikiko , Orry Andrew , Meyer Christopher R
Published: June 2021

The T. maritima ADP‐glucose pyrophosphorylase (ADPG PPase) is comprised of both glgC and glgD subunits which form a heterotetramer. While glgD displays negligible activity alone, the low activity glgC showed ~20‐fold stimulation when combined with glgD and also responds to the activator FBP. Amino acid alignment and modeling studies indicated that glgC D126 may be a catalytic residue, so this position was probed by site‐directed mutagenesis. D126A was generated and the His tagged protein purified via a heat step and nickel affinity chromoatography. The D126A enzyme displayed a ~1400 fold decrease in activity compared to the native enzyme while the S0.5 for ATP only increased ~2‐fold. Native PAGE revealed that the D126A protein successfully formed a complex with WT glgD which was also activated by FBP. The glgC D126 is an important residue for catalysis indicating that the enzyme active site is similar to other bacterial ADPG PPases. Additional analyses of D mutations in combination with various C proteins are underway. Supported by NSF 0448676 and the CSUF HHMI Research Scholars Program.
SUMO (small‐ubiquitin‐like modifier) in Tetrahymena thermophila
Authors: Matejka Stephanie Lynn , Knobbe Amy , Johnson Jacob , Weeks Donald P. , Bailey Cheryl
Published: June 2021

Cilia and flagella organelles serve important roles in a wide variety of organisms. Outside of Chlamydomonas reinhardtii flagella, there is limited understanding of ciliary protein composition and regulation. We investigate SUMO (Small ubiquitin‐like modifier) protein modification in the cilia of the single celled protozoan, Tetrahymena thermophila. SUMO is a member of the family of ubiquitin‐like polypeptides that become covalently attached to intracellular target proteins in eukaryotic organisms. Bioinformatics have shown a single SUMO gene in the genome of this organism. Real‐time RT‐PCR of T. thermophila samples has confirmed the expression of this SUMO gene. Additionally, western‐blot analysis of ciliary extracts has shown three SUMOylated proteins of approximately 35kD, 50kD and 135kD. The 135kD SUMOylated protein is preferentially found in ciliary extracts and not in cell body extracts. This implies there are specific SUMOylated target proteins in the cilia. Fractionations of ciliary extracts have been submitted for mass‐spectrometry analysis and reveal proteins not previously found in the T. thermophila ciliary proteome.

Supported by NSF and the Nebraska Research Council.
Small molecule inhibitors of PHLPP determined by chemical and virtual screening
Authors: Sierecki Emma , Sinko William , Mc Cammon J. Andrew , Newton Alexandra C
Published: June 2021

PHLPP (PH domain Leucine repeats Protein Phosphatase) dephosphorylates Akt and protein kinase C, poising this phosphatase on two major survival pathways in cells. The catalytic domain of PHLPP belongs to the PP2C family of serine/threonine phosphatases for which there are no specific inhibitors. Using a medium throughput chemical screening combined with a virtual approach, we tested compounds from the NCI compound library and identified small molecules that inhibit PHLPP in vitro at low micromolar range. Several clusters of structures could be identified and structure‐activity relationship studies were initiated. Competitive and uncompetitive inhibitors, specific for PHLPP compared to other serine/threonine phosphatases, were used on cells. Treatment with these molecules resulted in an increase in both basal and agonist‐evoked Akt phosphorylation and activity. These first inhibitors of PHLPP could find their therapeutic relevance in the treatment of diabetes or cardiovascular diseases, two pathologies in which Akt and/or PKC pathways are repressed.

This work was supported by the JDFR 3‐2008‐478, NIH 067946, NIH GM08326, NIH GM31749, NSF MCB‐0506593, and The Howard Hughes Medical Institute.
High Mobility Group Protein HMO2 promotes 3′ and 5′‐ homologous strand invasion in yeast
Authors: Ray Sreerupa , Grove Anne
Published: June 2021

Homologous recombination (HR) is the error‐free pathway involved in double strand break (DSB) repair. In Saccharomyces cerevisiae, the ATP‐dependent chromatin remodeling complex, INO80, has been reported to be involved in DSB repair. HMO2, a component of the INO80 complex, is a high mobility group (HMG) protein that binds to DNA in a non‐sequence specific manner. It has been shown recently that HMO2 interacts with γH2AX and helps recruiting INO80 to the region of DSB. HMO2 binds and protect DNA ends, both blunt and cohesive, and sequence of the single stranded overhang affects binding significantly. Here we show using D‐loop assay that HMO2 has the ability to mediate DNA strand invasion and annealing to produce a D‐loop‐ a crucial step in recombination repair. Further we also found that HMO2 mediates 3′ homologous strand invasion more efficiently than the 5′ strand, indicating that the polarity of the ssDNA is important for the process. This suggests that HMO2 might have pivotal implications on DSB repair in yeast beyond just INO80 recruitment. This work is supported by NSF (MCB‐0744240).
Structural insights into DNA polymerase X from African swine fever virus in the presence of oxoG lesions
Authors: Benitez Benedetta Alessandra Sampoli , Arora Karunesh , Bogdanovic Jasmina , Schlick Tamar
Published: June 2021

DNA polymerase X (pol X) from the African swine fever virus is a 174‐amino‐acid repair polymerase that participates in a viral Base Excision Repair pathway and is characterized by low fidelity. Pol X is homologous to human DNA polymerase β, but lacks the fingers subdomain. An essential feature of pol X is that insertion rate is of the G:G mispair is comparable to that of the four Watson Crick base pairs. Molecular dynamic studies of polX/DNA complexes suggest an induced fit mechanism for this enzyme. Interestingly, an active closed conformation is achieved also in simulations that contain the G:G mispair, in agreement with the kinetic data. Here we present molecular dynamic simulations of pol X/DNA complexes in which the templating base pair opposite to the incoming nucleotide contains the damaged base 7,8‐Dihydro‐8‐oxoguanine (oxoG). OxoG is known to be one of the major known products of oxidation of DNA by reactive oxygen species (ROS). Since the African swine fever virus's components are in an active state of respiration, it is not unreasonable to think that the virus may encounter this common ROS‐induced DNA lesion. According to kinetics data, pol X readily accommodates A opposite to oxoG, with efficiency even higher than the non‐damaged G:C base pair. The goal of this molecular dynamic study is to help interpret, at an atomistic level, the kinetics data. Support provided by NSF grant MCB‐0316771 and NIH grant R01 ES01269.
Lutein and Zeaxanthin in Red Blood Cells as a Measure of Their Status in Humans
Authors: Evans Julie A. , Johnson Elizabeth J.
Published: June 2021

Background

Intake of lutein and zeaxanthin (L+Z) may reduce age‐related macular degeneration (AMD) risk. Plasma L+Z, which reflects short‐term intake, is often used to assess status. Red blood cell (RBC) level may reflect long‐term intake. Use of RBC to assess status has not been explored. Macular pigment (L+Z in retinal tissue) is considered reflective of long‐term L+Z status. Evaluation of relationships of L+Z in plasma and RBC with macular pigment density (MPD) may be useful.

Objective

To determine the relationship between L+Z in plasma and RBC with MPD.

Methods

Blood and MPD were obtained from 15 healthy adults. Extraction and measure of L+Z in plasma and RBC were performed with standard lipid extraction methods and reverse‐phase, gradient HPLC. MPD was measured with a psychophysical method at a 1‐deg target and a 7‐deg parafoveal reference. Pearson correlation coefficients were used to measure relationships with MPD.

Results

Plasma and RBC levels of L+Z were significantly related to MPD (p = 0.050 and 0.018, respectively). The correlation was greater for RBC than plasma (r = 0.622 and 0.532, respectively).

Conclusions

A greater correlation of RBC L+Z with MPD may be due to RBC and MPD reflecting long‐term intake and plasma reflecting short‐term intake. Measures of L+Z in RBC in studies examining the relationship between L+Z status and AMD may be useful. Support: USDA grant# 58‐1950‐7‐707
Differential antioxidant and anti‐inflammatory activities of dietary polyphenols and vitamins
Authors: Hapner Christopher , Page Sabina , Deuster Patricia , Chen Yifan
Published: June 2021

The objective of this study was to determine if the anti‐oxidative and anti‐inflammatory activities of plant polyphenols and vitamins are associated with their antioxidant efficacy. We compared the inhibition of curcumin (CU), muscadine (MU), quercetin (Q), ascorbic acid (AA), α‐tocopherol (TP) and α‐tocotrienol (TT) on free radicals, oxidative hemolysis and neutrophil oxidative bursts. Fresh blood cells were obtained from healthy volunteers and used immediately for chemiluminescence and spectrophotometric analyses. MU was the most potent scavenger of superoxide, hydrogen peroxide and peroxyl radicals, whereas TP and TT were the least effective. Exposure of red blood cells (RBCs) to free radicals for 2 hours resulted in 41.2±9.0% hemolysis. Pre‐incubation of RBCs with Q (10μM) for 30 min significantly reduced the oxidative hemolysis to 4.8±1.3% (p<0.001), while no significant effects on hemolysis was found with other compounds. Pre‐incubation with quercetin caused greater inhibition of neutrophil burst (34.7±6.9% of control) induced by fMLP than with CU or MU (45.5±10.9 and 47.7% ±10.2 of control), whereas AA, TP and TT were ineffective. These results suggest that anti‐inflammatory rather than antioxidative action by polyphenols is more likely mediated by antioxidant capacity. Neither action can be explained through antioxidant potency solely. (This work was supported by USUHS Grant R091EH)
Prolonged sulforaphane treatment activates extracellular‐regulated kinase 1/2 signaling in nontumorigenic colon cells but not colon cancer cells
Authors: Zeng Huawei , Trujillo Olivia , Moyer Mary P. , Botnen James H.
Published: June 2021

Sulforaphane (SFN) is a naturally occurring member of the isothiocyanate family of chemopreventive agents and the induction of cell cycle arrest and apoptosis is a key mechanism by which SFN exerts its colon cancer prevention. However, little is known about the differential effects of SFN on colon cancer and normal cells. In this study, we demonstrated that prolonged SFN (5, 10 or 15 _mol/L) exposure (72 h) inhibited cell proliferation up to 95% in colon cancer cells (HCT116) and 52% in normal colon mucosa‐derived (NCM460) cells, respectively. Our data also showed that prolonged SFN exposure led to the significant induction of apoptosis and G2/M cell cycle arrest in HCT116, but to a much lesser extent in NCM460 cells. Furthermore, the examination of mitogen‐activated protein kinase (MAPK) signaling status revealed that prolonged SFN treatment strongly up‐regulated the extracellular‐regulated kinase ½ (ERK1/2) in NCM460 but not HCT116 cells. Taken together, the activation of ERK1/2 in NCM460 but not HCT116 cells may play a critical role in SFN's stronger potential of inhibiting colon cancer cell proliferation when compared with normal colon cells. Supported by USDA/ARS
Adiponectin and body weight regulate colon cancer tumorigenesis in obese mice
Authors: Boddicker Rebecca Luchtel , Whitley Elizabeth , Nilsen‐Hamilton Marit , Birt Diane F. , Spurlock Michael E.
Published: June 2021

Obesity is associated with downregulation of the anti‐inflammatory adipokine, adiponectin (Adn), and concomitant upregulation of proinflammatory cytokine production in adipose tissue. Recently, the chronic inflammatory state characteristic of obesity was causally linked to development of colon cancer. It has been suggested that Adn plays a regulatory role in this association, although results remain controversial. Consequently, we used an Adn knockout (KO) mouse model and wild type C57BL/6J mice (Wt), coupled with a diet high in saturated fat, to investigate the role of Adn in colon tumorigenesis in obesity. Mice were first administered an injection of azoxymethane (10 mg/kg body weight) and provided 1% dextran sodium sulfate in drinking water for 4 wks to induce tumorigenesis, and then provided a high fat lard (HFL) or control (Cont) diet ad libitum for 7 wks. The HFL mice gained more weight than mice fed the Cont diet, regardless of genotype. However, the KO mice had lower blood glucose concentrations, perhaps indicative of early stage hyperinsulinemia. Interestingly, total lesions per mm colon length were higher in Wt mice than KO, although lesions correlated only with body weight. Taken together, these results suggest a role for adiponectin status and body weight in colorectal tumorigenesis. Supported by grant 2008‐34115‐19372 to the Nutrition and Wellness Research Center from the USDA.

Grant Funding Source: USDA
The Dietary Supplement Ingredient Database: Comparison of adult and children's multivitamin/mineral (MVM) products
Authors: Andrews Karen , Roseland Janet , Middleton Angela , Feinberg Matthew , Zhao Cuiwei , Holden Joanne , Dwyer Johanna , Picciano Mary Frances , Saldanha Leila , Sempos Christopher , Bailey Regan , Douglass Larry
Published: June 2021

In the Dietary Supplement Ingredient Database (DSID), ingredients in dietary supplement (DS) products are chemically analyzed and the results statistically evaluated for nutritional applications. The first data release, DSID‐1, reported on 18 ingredients in adult multivitamin/mineral (MVM) products. Recently, children's MVMs were studied. Representative products were identified using National Health and Nutrition Examination Survey (NHANES) 2003–06 and current market data. Differences between adult and children's MVM products include product forms, nutrient ranges and total nutritional content. Adult MVMs reported in NHANES had median values of 12 vitamins and 7 minerals; children's MVMs had median values of 10 vitamins and 2 minerals. Multiple lots of >60 children's MVMs were purchased in 2008 using a nationwide sampling plan and analyzed with certified reference materials. Adult products showed a wider range of nutrient values than the children's. For 14 nutrients, the average range for adult MVMs was 2.7–4.5 times the average range for children's products. For 4 B vitamins (B12, B6, riboflavin, thiamin), the average range for adult MVMs was 40 times that of the children's MVMs. Preliminary analytical results for 18 nutrients in children's products, calculated as % difference from label, show some distinct differences when compared to adult MVM products. Funding: ARS/USDA & ODS/NIH Y1CN501006
Probing the Role of the Extreme N‐terminus in Rhodobacter sphaeroides ADPGlucose Pyrophosphorylase
Authors: Tran Margaret , Matsui Mikiko , Meyer Christopher R
Published: June 2021

The allosteric enzyme ADPGlucose Pyrophosphorylase (ADPG PPase) catalyzes the rate‐limiting step in glucan biosynthesis. The enzyme from Rhodobacter sphaeroides is perhaps the most highly regulated ADPG PPase and is activated by F6P, FBP, and pyruvate. Given the diverse sequences present in the ADPG PPase family at the N‐terminus and the lack of structural information about this region, it was of interest to probe the role of the extreme N‐terminus by truncation mutagenesis. Three truncation constructs have been generated (initiating at the third, fifth, and ninth codons) and the altered proteins have been successfully expressed and purified to homogeneity using anion exchange, hydrophobic interaction, and Cibacron Blue chromatography. Truncation 1 displayed a Vmax ~20% of wild‐type and S0.5 and A0.5 values for substrates and activators ~2–3 fold higher than wild type with ~2‐fold higher activation by activators. Truncation 3 exhibited only ~1% of wild type activity and also an extremely diminished response to activators. The N‐terminus is clearly important for optimal activity and regulation. Complete characterization of the altered proteins is in progress. Supported in part by NSF grant 0448676.
Association of Bioscavengers to Human Red Blood Cells
Authors: Calderon Frances Suiyin , Gauchan Sangeeta , Garcia Gregory Edward
Published: June 2021

We are developing technologies to increase the in vivo retention of bioscavengers by attaching them to red blood cells (RBCs). We have examined cell‐penetrating peptides (CPPs) of 5‐40 amino acids that potentially could either anchor the bioscavengers to the cell surface or internalize them by associated transport. In addition, we tested the binding ability of RBC surface antigens (anti‐type A and B blood group) antibody‐bioscavenger conjugates. RBCs obtained from human blood were incubated with CPPs such as the protein transduction domain of Tat‐HIV (Tat‐PTD), calcitonin, and inverse antennapedia (Inverse‐Antp) biotin congeners in complex with Neutravidin‐fluospheres. No significant differences were observed between control RBCs and CPPs‐incubated RBCs. RBC binding of blood group antibodies was initially monitored with secondary antibodies conjugated to fluorescent marker CY2. Bioscavenger BChE was bound to RBCs by a bridge formed of primary antibody, biotinylated secondary antibody, Neutravidin, and finally biotinylated bioscavenger butyrylcholinesterase (BChE). The preparations were washed and the bound BChE was detected by fluorescence microscopy following incubation with CY2 labeled anti‐butyrylcholinesterase antibodies and BChE enzymatic activity. Our results show that there was significant binding of BChE to the RBCs demonstrating that antibodies can be used as an effective means to bind BChE to RBCs surfaces.

This work was supported by DTRA#1.D0011_08_WR_C.
Hypothalamic Angiotensin Type 1 Receptors and Sodium Chloride Intake in Genetically Hypertensive Rats Raised on High Salt Diets
Authors: Bradshaw Timothy , Osborn Jeffrey L
Published: June 2021

Spontaneously Hypertensive Rats (SHR) may have impaired titration of ingested sodium chloride. The role of Angiotensin II (AII) and hypothalamic angiotensin (AT1) receptor expression in regulating salt and water intake was evaluated in SHR raised from weaning on high salt. Female SHR were crossed with normotensive, Brown Norway (BN) males and hypertensive offspring then were sib/sib mated yielding hypertensive F2 rats that were raised to adulthood on either 4.0% (HS; n=7) or 0.8% (NS; n=8) NaCl chow. NS and HS rats were placed in metabolic pens and offered ad lib access to 2% saline, distilled H2O and sodium free chow and daily water and sodium intakes were measured. After control (2 days), AII synthesis was blocked with Captopril (1 g/L) in the saline and H2O (5 days). Blockade of AII increased NaCl ingestion in both NS (2.63 + 1.57 to 5.20 + 1.66 mmol/day) and HS (3.90 + 1.88 to 7.65 + 3.29 mmol/day) rats compared to control. Captopril decreased H2O ingested by HS rats (20.47 + 3.95 ml/day to 8.29 + 2.78 ml/day) but not by NS rats (16.39 + 4.86 ml/day vs.15.09 + 4.19 ml/day). Hypothalamic AT1 receptor density (western blot) was 60% lower in HS rats compared to NS rats (p<0.05). Thus, reduced AT1 receptor expression in SHR raised on high salt intake may limit HS rats from appropriately adjusting water intake during elevated salt intake. (Funded by NSF #0437768)
The substitution of NAC fragment for the NQ domain of Sup35p generated a novel yeast prion
Authors: Peng Weilin , Qi Xiaojie , Conticello Vince
Published: June 2021

Prions in fungi are not pathogenic as mammalian cells but act as cell physiology regulator and provide models to study mechanisms involved in prion propagation. The Sup35p, which is the protein account for the yeast prion [PSI+], could be dissected and reconstructed to chimeric proteins with the replacement of N/Q domain to aggregation‐prone sequences. NAC, a peptide fragment from a‐synuclein, is suggested to be responsible for a‐synuclein aggregation and toxicity. The N/Q domain of Sup35p was switched to NAC. Such substitution generated a novel prion with a non‐Q/N‐rich sequence as the aggregation domain. The chimeric yeast prion might be capable of serving as a model to study the property of the aggregation region in vivo. This research was funded by the DOE.
Omega‐3 Fatty Acids Mitigate Inflammation in Felines with Chronic Kidney Disease
Authors: Harris Mary A , Lunn Katharine , Quimby Jessica , Kim Susan , Mulligan Christopher
Published: June 2021

The objective of this study was to examine the relationship of fish oil omega‐3 (n‐3) fatty acids (FA) on inflammatory markers and clinical indices of malnutrition:inflammation syndrome in a feline model of chronic kidney disease. 63 client owned cats (mean age 14.04 ± 3.86 y), with stage 2 and 3 chronic kidney disease (CKD) were studied. Dietary intake of n‐3 docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were obtained by diet record. Erythrocyte (RBC) membrane n‐3 DHA and EPA were measured by gas chromatography. Markers of inflammation, serum amyloid A (SAA) and alpha‐1 acid glycoprotein (AGP) were measured by feline specific immunoassay and correlated with clinical variables: serum albumin, body condition score (BCS), hemoglobin (Hgb), hematocrit (Hct) and urine protein:creatinine (P:C). Intakes of DHA and EPA were 42.77 ± 74.34 and 36.02± 69.74 mg/day (mean ± SD) respectively. DHA intake was correlated with BCS (r=.299, p=.05) controlled for age, gender and indicators of CKD (BUN, calcium, Hct, potassium and bicarbonate). Mean AGP was 446.98 ± 220.48 ug/ml (range 140 – 1460) and SAA was 11.75 ±25.78 ug/ml (range 0.1 – 151). AGP was inversely correlated with serum albumin (r=−.321, p=.01). The P:C ratio was positively and highly correlated with SAA (r=.338, p=.008) and approaching significance with AGP (r=.237, p=.06). Hgb and Hct levels were negatively and highly correlated with AGP (p=.000) and SAA (p=.04). The results indicate that adequate DHA status may decrease inflammation in CKD and that higher DHA intakes may protect against malnutrition:inflammation syndrome as indicated by BCS and serum albumin in felines with stage 2 and 3 CKD. This research was supported by the USDA, AES Project #COL00631.
Investigation of mechanical properties of breast cancer cells using micropipette aspiration technique
Authors: Mohammadalipour Ameneh , Choi Young Eun , Benencia Fabian , Burdick Monica M. , Tees David F. J.
Published: June 2021

Characterization of the mechanical properties of cancer cells is a promising diagnostic method for recognizing cancerous cells in the very early stages of the disease. In this study, the micropipette aspiration technique was used to investigate deformability differences between two types of breast cancer cell line: BT‐20 and Hs578T. Hs578T breast cancer cells have been reported having stem cell‐like properties, whereas BT‐20 cells are more recognized as non‐stem cell‐like cancer cells. Using a controlled aspiration pressure, a cancer cell was aspirated into a small glass micropipette while the length of the aspirated section of the cell inside the micropipette was measured. For a solid‐like cell, the Young's modulus can be calculated from the slope of a pressure versus aspiration length graph. The Hs578T cell line was found to have a Young's modulus larger than that of the BT‐20 cell line. This supports our hypothesis that the technique can be used for the characterization of cancer cells by their mechanical properties, which could lead to a new method of detecting circulating tumor cells. This work was supported by grant CBET‐1106118 from the National Science Foundation.
Dietary supplement education for the military: An education module for healthcare providers
Authors: Attipoe Selasi , Costello Rebecca , Kohlmeier Martin , Deuster Patricia
Published: June 2021

There is a high prevalence in the use of dietary supplements (DS) to promote health, boost energy, and enhance performance in the military. With the ease of acquiring DS, even in austere environments, and the lack of credible information on appropriate use, individuals may be at significant risk for adverse events. Thus, it is imperative to prepare healthcare providers on how to address DS use during patient encounters. To help address this problem, an interactive web‐based education module on DS for military healthcare providers was developed using the well‐established platform from the Nutrition in Medicine project at the University of North Carolina. The module, located at www.nutritioninmedicine.net/neppphp/champ.php, provides excellent training on how to: evaluate and communicate evidence‐based DS literature, obtain DS history, and appropriately report adverse events. It also contains downloadable summaries, interactive exercises, and a final exam. An evaluation feature is included and continuing education credits may be earned. The module helps bridge the training gap and provides adequate information to prepare military healthcare providers to address DS‐related issues in their daily practice. Funding: The module was supported by the Consortium for Health and Military Performance and Human Performance Resource Center, Uniformed Services University; and the NIH Office of Dietary Supplements.
Perinatal high fat diet dysregulates GABAergic signaling to vagal efferent motoneurons regulating gastric motility
Authors: Reagan Zachary K , Browning Kirsteen N
Published: June 2021

Parasympathetic innervation to the stomach is supplied by dorsal motor nucleus of the vagus (DMV) neurons. Studies have demonstrated that ongoing GABAergic inputs to DMV neurons play a major role in regulating vagal efferent output. The aim of this study was to investigate whether perinatal HFD affects the GABAergic regulation of vagal efferent output to the stomach. Male Sprague Dawley rats were fed a HFD (60%kcal from fat) from E13 until experimentation at 12 weeks of age. Miniaturized strain gauges sutured to the ventral surface of the gastric corpus were used to record gastric motility. The GABAA receptor antagonist, bicuculline methiodide (BMI; 60nl) was microinjected into the dorsal vagal complex and the resulting increase in gastric motility was measured at 3 time points after injection (1.5, 4 and 6.5min) and averaged. Responses were expressed relative to the maximal increase in motility in response to i.v. injection of the non‐selective muscarinic agonist, bethanecol (353pmol; 1ml), which itself was unaffected by diet (9.08±1.20g in control vs 9.27±1.03g in HFD; P>;0.05).

In control rats, BMI induced a dose‐dependent increase in gastric motility (3.1±0.6%, 4.8±1.1% and 5.7±1.7% at 25, 50 and 100pmol, n=8,8,4, respectively, P<0.05 for each). In contrast, following HFD, the BMI‐induced increase in gastric motility was significantly attenuated (0.004±0.0001%, 1.2±0.3% and 1.25±0.3% at 25, 50 and 100pmol, n= 4,5,4, respectively, P<0.05 vs control). These data suggest that exposure to a HFD during the perinatal period alters GABAergic activity within brainstem neural circuits controlling gastric functions, which may contribute to the dysregulation of homeostatic reflexes, including appetite and metabolic regulation.

Supported by NSF IOS 1148978
Chronology of Early Heat Stress Mediated Changes in Oxidative Skeletal Muscle
Authors: Volodina Olga , Ganesan Shanthi , Pearce Sarah , Gabler Nicholas , Baumgard Lance , Rhoads Robert , Selsby Joshua
Published: June 2021

We have previously reported that 24 hours of heat stress (HS) induced free radical injury in oxidative skeletal muscle. In addition, we found widespread aberrant intracellular signaling following 12h of HS. The aim of this investigation was to extend our knowledge regarding the early chronology of HS‐mediated intracellular signalling in oxidative skeletal muscle. We hypothesized that 2, 4, and 6 hours of HS would result in increased autophagy and decreased mitochondrial content in oxidative skeletal muscle compared to muscle maintained under thermoneutral (TN) conditions. To address this hypothesis, pigs were assigned to four groups (n=8/group) and housed under TN conditions (21°C) or exposed to HS (37°C) for 2, 4, or 6 hours. Next, animals were euthanized and the semitendinosus red (STR) was collected and analyzed. Compared to TN, relative protein abundance of autophagy initiators phosphoinositide 3‐kinase Class III and Beclin‐1 were increased by 57% and 56%, respectively (p<0.05), following 6 hours of HS. At the same time point, relative abundance of the pre‐autophagosomal structure stabilizer, autophagy‐related protein 16‐1 (ATG16), was increased 59% (p<0.05). Microtubule‐associated protein A/B light chain 3 (LC3)‐I was similar between all four groups but the autophagosome formation marker LC3‐II was increased by 94% (p<0.05) following 6 hours of HS compared to TN. This resulted in a 2‐fold increase in the LC3II/I ratio (p<0.05), which is indicative of increased autophagic flux, though sequstome‐1 (p62) protein abundance was similar between groups. In addition, the mitophagy marker, BCL2/adenovirus E1B protein‐interacting protein 3‐like (BNIP3), was increased by 41% (p<0.05) following 6 hours of HS compared to TN. Relative protein abundance of TCA enzymes, electron transport chain components, and mitochondrial markers was similar between groups. These data demonstrate that short‐term HS induced autophagy/mitophagy. Together with our previous work we also suggest thatbetween 6 and 12 hours of HS the intracellular environment of oxidative skeletal muscle switches from one that promotes autophagy to one that inhibits it.

Support or Funding Information

This work supported by USDA grants 2014‐67015‐21627 and 2011‐67003‐30007.
Take Your Medicine; It's Good For You. Teaching Quantitative Principles To Biologists
Authors: Springer Michael
Published: June 2021

Recent advances in automation and technology have changed the nature of life science research. Experiments now often generate large amounts of quantitative data – far too much for a person to sort through or to analyze by hand. Both academic institutions and funding agencies have recognized the need for programming and quantitative skills, but most life sciences educational programs have not yet integrated these skills into their curriculum. Compounding this problem, the life sciences, and in particular biology has historically attracted students who were often the least quantitatively inclined science majors. I will discuss our efforts to integrate quantitative methods into our curriculum, with an emphasis on using active learning approaches to engage and motivate students.

Support or Funding Information

This work was supported by the Bertarelli Program in Translational Neuroscience and Neuroengineering and by NSF grant 1349248
A Chemical Glycoproteomics Platform To study the Human Prostate Secretome
Authors: Spiciarich David R. , Purcell Sean , Maund Sophia L. , Peehl Donna M. , Bertozzi Carolyn R.
Published: June 2021

Accurate diagnosis and treatment of prostate cancer demands a highly specific biomarker of both the disease, progression and prognosis. As glycoproteins have proven to be altered in most cancers, we have focused on studying glycoproteins in human prostate tissue samples. We have developed an efficient method for labeling and enriching glycoproteins, followed by MS‐based analysis of normal and carcinoma tissue. We sought to selectively enrich secreted glycoproteins because of the potential of secreted glycoprotein biomarkers in non‐invasive prostate cancer detection. This is accomplished by culturing human prostate tissue derived from radical prostatectomies, at varying states of prostatic disease, and collecting the cultured media which samples secretome proteins. During culturing, tissue is chemically tagged with an azide functionalized monosaccharide, N‐azidoacetylmannosamine‐tetraacylated (ManNAz). Labeled proteins are subsequently reacted with a biotin‐functionalized probe for the capture of azido‐sialylated glycoproteins. After analysis of confirmed azide‐dependent labeling, glycoproteins were digested and analyzed on a linear ion trap mass spectrometer with collision‐induced dissociation (CID). Greater than 80% of the proteins identified by LC‐MS/MS were secreted proteins, indicating that this strategy achieves enrichment of low abundance proteins. Additionally, a comparative proteomics analysis between diseased and healthy tissue identified proteins unique to each tissue type, which are undergoing validation as potential biomarkers. Virtually all clinically approved cancer biomarkers are cell‐surface or secreted glycoproteins and this work extends the application of existing metabolic labeling techniques, while developing new methodologies for cell surface labeling to discover biomarkers associated with prostate cancer.

Support or Funding Information

This work was funded by grants from the NIH to C.R.B. (GM066047 and GM058867) and to S.L.M. (2T32DK007217‐37). D.R.S. is supported by NSF predoctoral fellowships.
Systematic Review of Child Obesity Prevention and Treatment Trials Addressing Parenting
Authors: Hubbs‐Tait Laura , Kimble Ashley , Hingle Melanie , Novotny Rachel , Fiese Barbara
Published: June 2021

A systematic review was conducted to determine whether child obesity/overweight treatment and prevention trials that address parenting result in significant changes or differences in child BMIz, BMI percentile, weight status, or related outcomes. On May 14, 2015 Pubmed, PsycINFO, Web of Science, and Scopus were searched with the following search strategy “parenting OR parent OR mother OR father AND intervention OR RCT OR trial AND parenting style OR feeding style OR responsive feeding OR restrictive feeding AND child obesity OR child overweight OR child weight status OR child waist circumference OR child BMI OR child BMI percentile OR child BMI% or child BMI z OR child BMI centile OR child BMIZ or child BMI‐z,” yielding 244 unique articles. Abstract and title screening (α = .90) excluded all protocols as well as completed studies restricted to adolescents. Full text review resulted in 16 studies that included some aspect of parenting in the trial and also provided weight outcomes for children. Study sample was obese and/or overweight children in 12 studies; the remaining four included children from the full range of weight statuses. Ten of 16 studies reported a significant effect of intervention on child BMIz, BMI percentile, or other weight outcome. All 10 studies with significant effects were focused on intervention and treatment of obese/overweight children rather than on obesity prevention. Only two of the 10 were focused solely on children ages 5 and under. Results suggest obesity intervention trials that address parenting styles and similar constructs have been successful, particularly for older children. Additional efforts to increase the success of interventions for the toddler and preschool periods are needed.

Support or Funding Information

USDA/NIFA Hatch/Multistate and Oklahoma Agricultural Experiment Station OKLO2849; Oklahoma Agricultural Experiment Station OKLO2868
Alterations of Gut Microbiota Prior to Changes of Paneth Cell‐antimicrobial Peptides in High Fat‐induced Obese Mice
Authors: Guo Xiulan , Li Jinchao , Tang Renyong , Liu Wei , Liu Zhenhua
Published: June 2021

It is under intensive argument about the reciprocal relationship between gut microbiota and the altered intestinal metabolic and inflammatory status in the obese state. Gut microbiota composition and Paneth cells is closely associated, especially in obesity. This study aimed to investigate the mutual interaction between the Paneth cell‐produced antimicrobial peptides expression and shift of the intestinal microbiota in obesity. Eight male C57BL/6 mice were fed a low fat diet (LF, 10%) as the control and 24 animals were fed a high fat diet (HF, 60%) and sacrificed sequentially with 8, 12, 16 weeks of feeding. The expression of antimicrobial peptides and the composition of gut microbiota were determined at each stage. The mice with 8‐wk HF feeding had higher fat percentage and significant changes of the gut microbiota comparing to the LF control group (p < 0.05). The abundances of Firmicutes and Bacteroidetes from 8‐ to 16‐wk HF feeding were steady, with a higher abundance of Firmicutes and a lower of Bacteroidetes. However, 8‐wk HF diet feeding did not changed the Paneth antimicrobial peptides: lysozyme (LYZ), α‐defensin 5, and RegIIIγ (p > 0.05), but reduced LYZ and RegIIIγ mRNA expression was observed in animals with 12‐ and 16‐wk HF feeding (p < 0.05), and the results for LYZ protein level, measured by immunohistochemistry, also indicated a decrease after 16‐wk HF feeding. These results indicated that the high fat diet initially alters the profile of gut microbiota in obese mice, and changes in the expression of antimicrobial peptides occurs secondary to alterations in the composition of the intestinal microbiota.

Support or Funding Information

This project is supported by National Natural Science Foundation of China (Grant No.31401488), USDA grants (2014‐67017‐21762 and MAS00454), and Deans Incentive Grant.