bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Bases for Recognition of Short Peptides and Cysteine‐Directed Modifications of Insulin‐Degrading Enzyme
Authors: Ralat Luis Abel , Malito Enrico , Manolopoulou Marika , Tsay Julie L , Wadlington Natasha L , Tang Wei‐Jen
Published: June 2021

Insulin degrading enzyme (IDE) utilizes a catalytic chamber to cleave substrates like insulin and amyloid beta. Interactions with substrates occur at an exosite ~30 A away from the catalytic center that anchors the N‐term of substrates to facilitate binding and cleavages at the catalytic site. However, IDE also degrades peptide substrates that are too short to occupy both the catalytic and the exosite simultaneously. Here, we use kinins to address the kinetics and regulation of IDE with short peptides. IDE specifically degrades kinins at the Pro/Phe site. A 1.9 A structure of bradykinin‐bound IDE reveals the binding of bradykinin only to the exosite and not the catalytic site. This is in agreement with the high Km of bradykinin for IDE. We also found that human IDE is potently inhibited by physiologically relevant concentrations of S‐nitrosylation and oxidation agents. Cysteine‐directed modifications play a key role, since an IDE mutant devoid of all 13 Cys is insensitive to the inhibition by these reagents. Specifically, cysteine 819 of human IDE is located inside the catalytic chamber pointing toward an extended hydrophobic pocket and is critical for the inactivation. Thiol‐directed modification of this residue likely causes local structural perturbation to reduce substrate binding and catalysis.
C‐terminal labelling of recombinant proteins using an engineered split‐intein
Authors: Volkmann Gerrit , Liu Xiang‐Qin
Published: June 2021

Site‐specific labelling of proteins has great potential to aid in the characterization of protein function, three‐dimensional structure, folding behaviour, and interaction with other proteins or small‐molecules. However, new and more efficient labelling methods are needed to satisfy the increasing demand for creating site‐specifically modified proteins. Here, a novel technique is presented to specifically label recombinant proteins at the C‐terminus using a peptide‐protein trans‐splicing approach. A label is incorporated into a small synthetic peptide, and then transferred onto the C‐terminus of the protein of interest via a stable peptide bond formed by protein trans‐splicing. We applied this method to introduce fluorescent labels and biotin at the C‐terminus of recombinant proteins, with biotin being potentially useful in creating protein microchips. The gentle chemistry of the trans‐splicing method also allowed us to label receptor proteins on the surface of eukaryotic cells, indicating a broad applicability of our labelling technique. Research support was provided by CIHR and NSERC.
Structural basis for midbody targeting of spastin by the ESCRT‐III protein CHMP1B
Authors: Yang Dong , Rismanchi Neggy , Renvoise Benoit , Lippincott‐Schwartz jennifer , Blackstone Craig , Hurley James
Published: June 2021

The endosomal sorting complex required for transport (ESCRT) machinery, including ESCRT‐III, localizes to the midbody and participates in the membrane‐abscission step of cytokinesis. The ESCRT‐III protein chromatin‐modifying protein 1B (CHMP1B) is required for recruitment of the MIT domain‐containing protein spastin, a microtubule‐severing enzyme, to the midbody. The

2.5‐A structure of the C‐terminal tail of CHMP1B with the MIT domain of spastin reveals a specific, high‐affinity complex involving a noncanonical binding site between the first and third helices of the MIT domain. The structural interface is twice

as large as that of the MIT domain of the VPS4‐CHMP complex, consistent with the high affinity of the interaction. A series of unique hydrogen‐bonding interactions and close packing of small side chains discriminate against the other ten human ESCRT‐III subunits. Point mutants in the CHMP1B binding site of spastin block recruitment of spastin to the midbody.
HIV protease inhibitors activate the ER stress response and disrupt the lipid metabolism in 3T3‐L1 adipocytes
Authors: Pecora Beth S , Sun Lixin , Wu Xudong , Zha Weibin , Gurley Emily , Studer Elaine , Hylemon Phillip B , Pandak William M. , Zhang Luyong , Wang Guangji , Zhou Huiping
Published: June 2021

HIV protease inhibitor‐induced lipodystrophic syndrome is a major side effect of current anti‐HIV therapy. However, the underlying molecular mechanism remains unclear. We have recently shown that HIV PI‐induced ER stress and activation of the unfolded protein response (UPR) represents an important cellular mechanism by which HIV PIs disrupt lipid metabolism and induce inflammation in hepatocytes and macrophages. The aim of this study was to determine if HIV PIs also induce ER stress, activate the UPR, and affect the lipid metabolism and differentiation in adipocytes.

Methods

Mouse 3T3‐L1 adipocytes were used in this study. The effect of individual HIV PI on differentiation and lipid accumulation was examined by Oil red O staining. Activation of the UPR was determined by real time RT‐PCR and Western Blot analysis. The expression of adipokines/cytokines was determined by real‐time PCR and ELISA.

Results

Different HIV PIs had different effects on the UPR activation, cell differentiation, and lipid metabolism in adipocytes. Both lopinavir and ritonavir dose‐dependently activated the UPR, increased IL‐6 expression, reduced adiponectin expression, and induced cell apoptosis. But amprenavir had less effect.

Conclusions

HIV PI‐induced ER stress response and activation of the UPR may represent a critical molecular mechanism underlying HIV PI‐associated lipodystrophy and metabolic syndrome.
Towards a Crystal Structure of Complete Histidine Kinase E in Bacillus
Authors: Sergeeva Oksana , Bick Matthew J , Darst Seth A
Published: June 2021

Sporulation in Bacillus is controlled by five histidine kinases (HKs), KinA‐KinE, which are composed of an N‐terminal sensor domain and a C‐terminal kinase domain. Signals sensed in the sensor domain are transduced to the kinase domain, resulting in an autophosphorylation event on a conserved histidine. The crystal structure of the kinase domain from KinB in complex with the autophosphorylation inhibitor Sda, from the moderate thermophile Geobacillus stearothermophilis, has previously been solved in the Darst laboratory. The structure reveals a KinB dimer with Sda bound to a single monomer. Binding of Sda stabilizes the KinB dimer and prevents autophosphorylation.

In order to gain insights into how signals are communicated in HKs, we focused on the crystallization of a complete HK. Currently, a structure of an HK containing both sensor and kinase domains is unavailable. Availing ourselves of the fact that two of the five sporulation HKs in Bacillus are soluble, we choose one of these, KinE, for structural studies. A double expression construct containing KinE and Sda was made from three different thermophilic Geobacillus species. Of the three constructs tested for expression, one from G. thermodenitrificans gave considerable amounts of soluble protein following expression in E. coli Rosetta2 cells. We have purified the KinE/Sda complex via nickel affinity, ion exchange, and size‐exclusion chromatography. Biochemical assays have shown that autophosphorylation of KinE, like KinB, is inhibited by Sda. Initial crystallization trials in the presence of ADP have given several promising leads.

This work was supported by NIH GM081697 to S.A.Darst.
Does an Uncharacterized Gene/Protein Play a Role in Melanocortin Signaling?
Authors: Hughes Darden Cleo , Nyan Dougbeh , Wachira James , Redmond Brian
Published: June 2021

Melanocortin 3‐receptor (MC3R) is mainly expressed in the brain and plays an important role in a number biological processes, energy homeostasis, neuroendocrine regulation, sodium metabolism, and insulin signaling. Although many reports show that MCRs are involved in activating the cAMP pathway and they are important in a variety of cellular functions, post‐receptor mechanisms are not completely understood. As an initial approach, we performed microarray analyses to identify genes that are up‐regulated following MC3R activation/internalization by gamma‐melanocortin stimulating hormone (gamma‐MSH) in a mouse neuronal cell line over‐expressing MC3R. One gene, referred to as the uncharacterized gene (UNCG), had the highest expression (13 fold) in MC3R containing cells and was confirmed by real‐time PCR analysis. The UNCG mRNA is a single open‐reading frame of 415 bases that encodes a 137 amino acid protein with a predicted molecular mass of 16327 Daltons. Analysis of RNA from multiple mouse tissues reveals wide expression. A form of the gene has been identified in other organisms, rat and human. Preliminary localization studies indicate that the encoded protein is located in the cytoplasm in untreated cells. This data will enhance our knowledge of the molecular mechanisms involved in MC3R signaling. Supported: NIH/RCMI/G12RR17581‐05 (RCMI funded Histology and Molecular and Cell Biology core facilities).
Mechanisms underlying cAMP compartmentation
Authors: Feinstein Wei , Britain Andrea L , Rich Thomas C
Published: June 2021

There is mounting evidence suggesting that specificity within cAMP signaling pathways is caused by compartmentation. Mechanisms underlying compartmentation are poorly understood. The spatial spread of cAMP is affected by phosphodiesterase (PDE) localization, buffering, cell geometry, and restrictions on diffusion. We developed mathematical models to investigate potential contributions of these mechanisms in the compartmentation of cAMP. Models assumed realistic enzyme concentrations and homogenous rates of diffusion. Initial simulations used an effective diffusion coefficient (D) of 300 μm2/s ‐ D for cAMP in aqueous solution. Simulations of cAMP spread within a spherical cell indicated that PDE activity and buffering by PKA cannot solely account for the segregation of cAMP signals, regardless of their subcellular location. When D was lowered 10,000‐fold, significant cAMP gradients were observed. We then incorporated realistic geometries of endothelial cells into the mathematical model. In simulations of this model with D = 300 μm2/s and PDE activity concentrated at the nuclear membrane, 3‐fold cAMP gradients were observed between cellular processes and the nucleus. Lowering D 100‐fold increased the magnitude of cAMP gradients. These simulations are consistent with a conceptual model in which cell geometry and structural elements contribute to cAMP compartmentation.
The Roles of Serine/Threonine Protein Phosphatases in Regulation of PDE3B Signaling in 3T3‐L1 Adipocytes
Authors: Khan Faiyaz Ahmad , Zmuda‐Trzebiatowska Emilia , Degerman Eva , Manganiello Vincent
Published: June 2021

Using sucrose gradient centrifugation and confocal microscopy, PDE3B was localized in internal membranes and PM in adipocytes. PDE3B is phosphorylated/activated by insulin and CL316243 (b3‐receptor agonist) via PKB‐ and PKA‐ signaling, respectively. To study the role of phosphatases in the regulation of PDE3B, we investigated the phosphorylation/activation of PDE3B in response to calyculin A (PP2A inhibitor). Incubation of serum starved (16 h) adipocytes with calyculin A induced time and dose dependent phosphorylation/activation of PDE3B. Maximal effects were obtained after 30 min, using 1 uM okadaic acid (1.5 fold activation) or 100 nM calyculin A (2.4 fold). Superose 6 (S6) chromatography of solubilized membranes from serum‐treated adipocytes indicated that PDE3B primarily elutes in high molecular weight macromolecular complexes (HMW =3000 kD). After incubation of adipocytes in serum free media (6‐16 h), PDE3B moves from HMW to low molecular weight (LMW, ~900 kD) complexes. S6 chromatography of solubilized membranes from adipocytes (serum‐starved, 6‐16 h) incubated with calyculin demonstrated formation of distinct HMW complexes that contain 32P‐phosphorylated PDE3B. These studies suggest phosphorylation/activation of PDE3B and its recruitment into HMW can be regulated by calyculin‐induced inactivation of phosphatases, and that serum maintained HMW complexes that contained PDE3B.
Mechanistic implications of urate interaction with transcriptional regulator HucR from Deinococcus radiodurans
Authors: Perera Inoka , Lee Yong‐Hwan , Grove Anne
Published: June 2021

The hypothetical uricase regulator (HucR) is a ligand‐responsive MarR family transcriptional regulator from D. radiodurans that regulates uricase expression. Regulation of uric acid levels may contribute to the bacterium's extreme resistance to reactive oxygen species. Molecular docking was used to predict the urate binding site in HucR. A site was predicted above the DNA recognition helices, and mutation of residues predicted to contact uric acid, R80, D73, and W20, shows that all three residues are required for DNA binding antagonism in presence of urate. Although DNA binding is not antagonized by urate, the R80 mutant shows Trp fluorescence quenching at 338 nm on urate binding, indicating ligand binding to the predicted site. In contrast, mutation of D73 abolishes fluorescence quenching with increasing urate concentrations. Other purines with the exception of xanthine have no effect on DNA binding affinity of HucR and display no concentration‐dependent fluorescence quenching. Based on these observations we propose that urate‐dependent perturbation of DNA binding by HucR may be due to the binding of N3‐deprotonated urate changing the orientation of D73 which in turn affects the disposition of the recognition helix due to altered interaction with R106. Due to high DNA and ligand binding affinity, HucR serves as a model to study mechanistic implications of ligand binding to MarR type transcriptional regulators.
Assay platform for screening and identifying allosteric and catalytic site inhibitors of viral polymerases.
Authors: Gregory Kalvin , Sun Ye , Golovlev Val
Published: June 2021

An assay platform consisting of two luminescent‐based assays has been developed for screening inhibitors of viral polymerases and for studying mechanisms of action of the prospective pharmaceutical compounds. The assays from this platform are designed to measure the activity of DNA‐directed DNA polymerases, RNA‐directed DNA polymerases, and RNA‐directed RNA polymerases, and can be used for identifying the mechanism of action of a prospective viral polymerase inhibitor in a single volume experiment and operating in real‐time detection mode. The assays have been demonstrated for detection of allosteric vs. catalytic site inhibition (e.g., NNRTI vs. NRTI) of HIV‐1 reverse transcriptase and for the study of the inhibition of RNA‐directed RNA polymerases. The assay platform addresses the drawbacks of the existing methods in that it reduces the cost of reagents and reduces the assay time. The technology is adaptable to high‐throughput format for application in large and small scale drug discovery projects.
Recent Insights into the Regulation of mRNA Splicing
Authors: Guthrie Christine
Published: June 2021

The spliceosome is a highly dynamic RNA‐protein machine requiring the ordered binding and release of 5 small nuclear ribonucleoprotein particles (snRNPs) to the intron‐containing pre‐mRNA. Following the recognition of the 5' splice site by base‐pairing to U1 snRNA and the intron branchpoint sequence by base‐pairing to U2 snRNA, the U4/U6.U5 triple snRNP joins the assembling spliceosome. In this complex, U4 snRNA is extensively base‐paired to U6 snRNA. Yet despite the presence of all 5 essential snRNAs, this form of the spliceosome is catalytically inactive. The 2 chemical steps require the unwinding of U4 from U6 and the coordinated release of U1 snRNP. This strategy presumably ensures that catalytic activation is restricted until the appropriate intronic signals have been accurately identified. We are using a combination of biochemical and genetic approaches to understand the mechanistic underpinnings of this fidelity mechanism. Key players are Brr2, an RNA‐dependent ATPase, Snu114, an EF2‐like GTPase, and Prp8, the largest and most evolutionarily conserved spliceosomal protein. Brr2 is required for the ATP‐dependent unwinding of U4/U6 and we have recently recapitulated this reaction in a minimal in vitro system using synthetic RNAs. Notably, this reaction requires the presence of a C‐terminal fragment of Prp8 and this stimulation is inhibited by mutations that give rise to retinitis pigmentosa in humans. Since Prp8 and Brr2 are both constitutive components of the spliceosome, we now seek to understand how the productive interaction of the C‐terminus of Prp8 with Brr2 is itself regulated. An interesting hypothesis is that this requires the GTP‐dependent conformational rearrangement of Snu114 with respect to Prp8.
Evaluation of manganese balance status in Korean adults
Authors: Choi Mi‐Kyeong , Kim Mi‐Hyun
Published: June 2021

Few data are available to establish recommendations for manganese in Korea. This study assessed the status of manganese balance among 51 free‐living Korean adults. We measured dietary manganese intake using 3‐day dietary records, and analyzed the manganese contents in 24 hr urine, feces and serum of the subjects. Average age, height, weight and body mass index were 24.7 years, 173.2 cm, 73.6 kg and 24.6 kg/m2 in males and 22.8 years, 160.8 cm, 55.5 kg and 21.6 kg/m2 in females. The daily manganese intakes of male and female subjects were 3.2 mg and 3.7 mg, respectively. Average serum manganese level was 10.49 ug/dl in male and 6.11 ug/dl in females, and there was no significant difference. Urinary manganese excretion in male (344.5 ug/day) was significantly higher than that in female (184.7 ug/day). However, fecal manganese excretion was not significantly different between male (2078.0 ug/day) and female (1950.6 ug/day). Also, retention, retention rate and apparent digestibility of manganese were 765.6 ug/day, 18.4% and 30.9% for male, and 596.6 ug/day, 19.4% and 26.0% for female, it turned out that there was no significant difference. By continuous evaluating balance status of manganese, it is anticipated that reference intakes of manganese will be established. <'This work was supported by the Korea Research Foundation Grant funded by the Korean Government (KRF‐2006‐531‐C00069).'>
Measurement of the body composition of small piglets by quantitative magnetic resonance (QMR) and dual‐energy X‐ray absorptiometry (DXA)
Authors: Mitchell Alva , Taicher Gersh , Kovner Israel
Published: June 2021

During studies of the growth of neonatal piglets it is important to be able to accurately assess changes in body composition. The purpose of this study was to compare the in vivo measurements of body composition of small piglets using QMR and DXA and to validate those results by chemical analysis. A total of 25 pigs (1.7 ‐ 4.1 kg) were measured by QMR (EchoMRI) and by DXA (Lunar Prodigy). Both instruments were tested as supplied by the manufacturer, with QMR measuring total body fat, water, and lean and DXA measuring total body fat and lean. The chemical measure of total body fat was highly correlated with both the QMR (r = 0.96) and DXA (r = 0.98) fat measurements and the correlation (r) between QMR and DXA was 0.93. However, DXA overestimated the amount of fat by 31% (P<0.05), whereas QMR overestimated the amount of fat by only 3.4% (P>0.05). The chemical measure of total body water was highly correlated with both the QMR (r = 0.98) and DXA (r = 0.99) measurements of total body lean mass and the correlation between QMR and DXA was 0.99. The DXA measure of total body lean was 18% more (P<0.05) than that measured by QMR. The QMR measure of total body water was 8% less (P>0.05) than that measured by chemical analysis. These studies indicate that QMR represents a potentially accurate method for measuring the body composition of piglets offers the advantages of no exposure to X‐ray radiation and measurement without the pig being anesthetized.
Video recording of school meals – Implications for whole grain eating behavior
Authors: Rosen Renee A , Peters Patrica , Lacey Alexandra , Marquart Len
Published: June 2021

More than 29 million children participate in the National School Lunch Program (NSLP). Recent plate waste studies indicate that whole grain products are accepted as well as their refined counterparts. To further investigate children's eating behaviors of whole grain products, cafeteria lunches were video recorded in a local school district. Approximately 80 children were taped ~1 to 6 times eating a variety of grain foods including whole grain products served in a typical elementary school lunch. Focus groups (n~30) were conducted to exam students' responses to introductions of whole grain products. DVD analysis used a modified Dyadic Interaction Nomenclature for Eating (DINE) to investigate eating patterns and behaviors of whole grain products. Results indicate that at higher grade levels children decrease the number of food items selected and time spent eating. Fruit desserts compared to desserts made with grains, such as cookies and cakes, are taken half as often by all grades when served. Entrée waste is significantly higher when grain desserts are served compared to fruit desserts. This baseline data provides a foundation for further exploring the use and acceptability of foods in school meals at the elementary level. This information can be helpful in facilitating product development and introduction of school foods, such as whole grains with children in an interactive and realistic cafeteria environment. This research was funded by ConAgra.
Analysis of NHANES Data to Estimate Long‐Term Fish Intake Rates
Authors: Bi Xiaoyu , Tran Nga L. , Barraj Leila M.
Published: June 2021

Fish and shellfish are excellent sources of nutrients, but exposure to environmental contaminants can also occur from fish consumption. Estimates of usual fish and shellfish intake are needed to assess chronic exposure to environmental contaminants. While consumption rates based on short testing periods provide useful information on consumption over short periods, the intra‐individual variation of consumption from day to day presents unreliable information on chronic exposure. Using a probabilistic approach, data from the National Health and Nutrition Examination Survey (NHANES) 1999‐2000, 2001‐2002, 2003‐2004, and 2005‐2006, conducted by the Center for Disease Control (CDC) National Center for Health Statistics (NCHS), on the frequency of intake of fish and shellfish over a 30‐day period were combined with the dietary recall (24 hr) of fish and shellfish data from NHANES 2003‐2004 to provide estimates of chronic average daily intake of fish and shellfish. Over 300 NHANES food codes were mapped to 31 fish and shellfish categories; for each category, a weighted distribution of amounts consumed per eating occasion was generated from NHANES 2003‐2004 intake records. Long‐term (30‐day average) daily fish/shellfish intake rates were derived by combining randomly sampled NHANES individual records of 30‐day fish/shellfish frequency of consumption with amounts consumed per eating occasion randomly sampled from the intake distribution. The usual intake rates for all fish, all shellfish, and all fish and shellfish combined, as well as for individual fish/shellfish species for both children (1 to <2 years, 2 to <3 years, 3 to <6 years, and 6 to <11 years) and adolescent women (11 to <16 years, 16 to <18 years, and 18 to <21 years), as practicable (i.e., depending on sample sizes) are presented in this study.
Association of micronutrient status and early childhood stunting with cognitive performance among school children in Northeast Thailand
Authors: Pongcharoen Tippawan , DiGirolamo Ann M , Ramakrishnan Usha , Winichagoon Pattanee , Flores Rafael , Martorell Reynaldo
Published: June 2021

Nutritional deficiencies and stunting are reported to impair cognitive performance in children. This study explored the relationship between micronutrient deficiencies (iron, zinc, iodine) and stunting with cognitive performance in 9‐y old children. Data were collected in 560 children who as infants had participated in a randomized controlled iron and zinc supplementation trial in Khon Kaen, Thailand. Cognitive performance was measured using the Wechsler Intelligence Scale for Children, III (Thai version). Biochemical (hemoglobin, transferrin receptor, plasma zinc, urinary iodine (UI)) and anthropometric data were obtained using rigorous methods. Data were analyzed by generalized linear model controlling for other confounders. Stunting at 1 y was included among the predictors. At 9 y, neither iron nor zinc deficiency were associated with Full Scale, Verbal, or Performance IQ. For each 100 μg/L UI increased, the verbal IQ was 2 points higher (95% CI: 0.2, 3.0). Stunting at 1 and 9 y were associated with reductions of 3‐4 points on all IQs. The association between stunting at 9 y and IQ was explained by stunting at 1 y. In conclusion, UI at 9 y was the only micronutrient indicator associated with cognitive performance. Stunting and attending an urban school had the largest and most consistent associations of any nutritional or social indicators.
No effect of 6‐month zinc supplementation on anthropometric measures in 6‐11 year‐old urban school children in Guatemala
Authors: Bui Vinh Quang , DiGirolamo Ann M. , Stein Aryeh D. , Ramakrishnan Usha , Ramirez‐Zea Manuel , Flores‐Ayala Rafael C. , Martorell Reynaldo
Published: June 2021

Zinc deficiency is reported to lead to poor growth. We conducted a double‐blind randomized controlled trial of 10 mg chewable zinc oxide vs. placebo 5 days/wk for 6 months among 6‐ 11 year‐old Guatemalan children from 5 urban public schools. Weight; standing and seated height; head, arm, abdominal, and calf circumferences; triceps and subscapular skin folds; and knee‐heel and leg lengths were measured at baseline and at close‐out. Among 750 children randomized, 8% were underweight, 19% were stunted, 7% were obese (BMI‐z score > 2), and 22% had serum zinc <65 mcg/dL. The two groups, 378 receiving zinc and 372 receiving placebo, were balanced at baseline on zinc status and anthropometry. At the 6‐month follow‐up (n=715) mean serum zinc was significantly higher (p=0.04) in the zinc (111.5± 31.0 mcg/dL) compared to placebo group (106.6 ± 32.3 mcg/dL). There were no differences between the groups in changes in weight (1.77±1.19 kg vs. 1.77±1.22 kg, respectively for zinc vs. placebo group; p=0.94); height (2.79±0.79 cm vs. 2.87±0.86 cm; p=0.15); or any other anthropometric measures examined. Zinc supplementation for six‐months did not impact growth among 6‐11 year‐old Guatemalan urban children at moderate risk of zinc deficiency.
Maternal HIV status is associated with food insecurity at 12 months post‐partum in Ghana
Authors: Perez‐Escamilla Rafael , Lartey Anna , Mazur Robert , Sellen Daniel , Hromi‐Fiedler Amber , Marquis Grace
Published: June 2021

Food insecurity is prevalent in HIV affected communities in Sub‐Saharan Africa. We examined household food insecurity status at 12 months post‐partum among women from Ghana's Eastern region who were diagnosed prenatally as HIV‐positive (HIV‐P; n=102), HIV‐negative (HIV‐N; n=150) or of unknown HIV status (HIV‐U; n=106).Women responded to a modified version of the US Household Food Security survey module. The reference time period was changed to reflect only the month preceding the survey. This analysis used a summative score based on 8 adult‐related questions (Cronbach's alpha=0.92). Households were classified as food secure (score=0), experiencing mild food insecurity (FI) (score =1‐3), moderate FI (score=4‐6), or severe FI (score =7‐8). The proportion of households experiencing severe adult FI was 8.8% among HIV‐P and HIV‐U women and 1.3% among HIV‐N women (p<0.05), The corresponding values for moderate adult FI were 14.5%, 15.7%, and 10.7%, respectively (p<0.05). Findings suggest that special attention is needed regarding the additional food security needs of HIV‐affected households. Funded by NIH grant HD43620.
The determination of a fecal D‐lactate threshold in neonatal ruminants with diarrhea
Authors: Zello Gordon A , Abeysekara Saman , Ewaschuk Julia B , Alcorn Jane , Naylor Jonathan M , Lohmann Katharina L
Published: June 2021

We have documented D‐lactic acidosis secondary to diarrhea in neonatal calves and lambs. The acidosis only appears after sufficiently high levels of D‐lactate are produced by microbial action in the gut. Measurements of serum and fecal lactate led to a determination of a fecal threshold for D‐lactate systemic absorption from the gut in neonatal ruminants. Jugular blood and feces were collected from 27 diarrheic and 4 healthy lambs, and 19 diarrheic and 4 healthy calves. Blood was analysed for pH, bicarbonate concentration (HCO3−), base excess (BE), hemoglobin (Hb), Na+, K+, Ca2+, Cl−, anion gap, and glucose. Serum, feces, and urine were analyzed for lactate enantiomers. A range of mild to severe diarrhea, as well as metabolic acidosis was observed with a corresponding wide range of D‐lactate concentrations in both serum (< 0.05‐24.0 mmol/L) and feces (< 0.05‐31.0 mmol/L). The fecal D‐lactate threshold was determined statistically using biphasic (break‐point) regression analysis (serum vs fecal D‐lactate). This fecal threshold concentration for absorption of D‐lactate was 10.2 and 8.8 mmol/L in lambs (n=31) and calves (n=23), respectively. These similar values in different species support a lumen D‐lactate threshold (~10 mmol/L) for monocarboxylic (i.e. lactate) transporters and uptake into the mucosal cells and then blood circulation. Therefore, controlling the amount of D‐lactate produced by gut bacteria, and hence lowering levels below threshold, may be key in preventing the metabolic acidosis common in enteritis.
Iron status and LDL ‐ oxidation in a group of healthy adults from Argentina. Preliminary results
Authors: Langini Silvia , Lardo Mabel , Ghersevich Sergio , Aragone María , Carbia Claudio , Pandolfo Marcela , Fleischman Silvana , Lazarowski Alberto , Río María E
Published: June 2021

Excess body iron (Fe) has been proposed to oxidize low ‐density lipoproteins (LDL). Blood samples from 109 non‐institutionalized healthy adults attending Hospital Italiano, Buenos Aires during 2006 were obtained. Samples negative for C‐reactive protein (PCR‐latex, Wiener lab) and risk population were studied: men (M) 23‐95y (n=30) and postmenopausal women (PostW) 51‐83y (n=31). Serum ferritin (SF) (quimiolumminescence analyzer‐ DPC‐Siemmens); unsaturated iron binding capacity (UIBC) (FerroZine‐) (Hitachi 917; automated systems‐ Roche diagnostics) and plasma oxidized LDL (ox‐LDL) (competitive ELISA developed at UNR, School of Biochemical and Pharmaceutical Sciences, Argentina, normal range 2.2‐3.8 U) were measured. Mean values ± SD, (range) and % of individuals with normal values were:

No correlation was found between ox‐LDL and SF (r= 0.02) nor UIBC (r=0.006) levels in M and PostW. However, increased risk of chronic diseases may be possible, as wheat flour fortification with Fe is mandatory in Argentina since 2002 in spite of high meat intake (54.7 Kg /capita /y, FAO 2003). Partially supported by UBACyT B414, CONICET PIP 5068 & ROCHE, Argentina
Zinc supplementation does not alter plasma trace elements in Kenyan adults
Authors: Scrimgeour Angus , Lukaski Henry C , Polhemus Mark E , Otieno Lucas , Young Andrew J , Bovill Maria E
Published: June 2021

Multiple micronutrient deficiencies constitute a significant public health problem in Kenya. Low iron (Fe) and zinc (Zn) status are frequent. As micronutrient homeostasis may be compromised when single‐micronutrients are used in intervention studies, we examined the effects of dietary Zn supplementation on other plasma trace elements (Ca, Cu, Fe, K, Mg, Mn and Se) in free‐living adults participating in an anti‐diarrheal study.

Methods

A double‐blind, RCT involving 500 adults, aged 18‐55 years, were supplemented with either 20 mg elemental Zn (as Zn‐sulphate) or placebo, per day for 90 days. Plasma trace elements were analyzed by ICP‐OES. Indices of inflammation (serum ferritin, C‐Reactive Protein) and vitamin A (VA) were also analyzed, using standard kits.

Results

At baseline, 99% of subjects had elevated plasma Mn (>0.15 µg/dL), 33% were Zn‐deficient (<65 µg/dL), 32% were VA‐deficient (<30 µg/dl), and 14% were Fe‐deficient (<60 µg/dL). After 90 days of supplementation, concentrations of plasma Zn increased 18.5% (p<0.0001) in subjects receiving Zn‐supplements, while it was not different in the placebo group (p=0.30). Zn‐supplementation did not modify Ca, Cu, Fe, K, Mg, Mn, Se, ferritin or CRP values.

Conclusion

Zn supplementation at moderate doses does not lead to deleterious effects on plasma trace elements, or indices of inflammation, in adults in western Kenya.

The study was funded by the U.S. Army MRMC
Nuclear and mitochondrial functions of p53 in zinc deficient human neuronal precursor cells
Authors: Corniola Rikki S , Levenson Cathy W
Published: June 2021

Our previous work has shown a role for zinc and the tumor suppressor protein p53 in the regulation of neuronal precursor cell proliferation and survival in vivo and in vitro. In human neuronal precursor cells (NT‐2) zinc deficiency results in increased p53 nuclear abundance, where it acts as a DNA‐binding transcription factor to regulate 14 downstream target genes, including cell cycle arrest genes (eg 1.8‐fold up‐regulation of reprimo mRNA) and a number of pro‐apoptotic genes including transforming growth factor‐β (+1.4‐fold) and retinoblastoma‐1 (+4.5‐fold) leading to decreases in BrdU labeling and increases in cell death. A dominant‐negative p53 gene construct confirmed the p53‐dependent nature of this regulation during zinc deficiency. In addition to the nuclear translocation of p53, we have also shown that p53 translocates to the mitochondria where it plays a role in the generation of mitochondrial reactive oxygen species (ROS) as measured by dihydrorhodamine fluorescence. Pre‐treatment with pifithrin‐α, a p53 inhibitor, prevents the generation of ROS. Additionally, there is 50% decrease in total superoxide dismutase activity (p<0.05), and a 2‐fold increase in glutathione peroxidase mRNA. These data identify a role for zinc in the nuclear and mitochondrial mechanisms that control neuronal stem cell proliferation and survival. Supported by Susan E. Lucas Fellowship in the Neurosciences.

Grant Funding Source

Phoenix Foundation
Soy diet inhibits expression of inflammation‐induced vascular cell adhesion molecules in endothelial cells
Authors: Goodwin Van B , Burris Ramona L , Nagarajan Shanmugam
Published: June 2021

Recently we reported that dietary soy attenuated atherogenesis in apolipoprotein E knockout (apoE‐/‐) mice. However, the molecular mechanisms contributing to the atheroprotective effect of soy diet is not clear. Since interactions between endothelial cells and monocytes play a pivotal role in the initiation of atherosclerosis, we hypothesize that components in soy diet prevent monocyte adhesion to endothelial cells by inhibiting vascular cell adhesion molecule expression on endothelial cells. Genistein, a major soy isoflavone, inhibited expression of TNF‐α‐induced vascular adhesion molecules CD62E (E‐selectin) and CD106 (VCAM‐1) in HUVEC cells in a concentration‐dependent (1‐100 µM) manner, with no effect on CD54 (ICAM‐1) expression. Interestingly, equol, a metabolite of the soy isoflavone daidzein produced by gut microflora, also showed significant inhibition of inducible CD62E and CD106 expression. The effect of soy feeding on expression of CD106 was further addressed in apoE‐/‐ mice. Quantitative RT‐PCR analyses of proximal aorta showed reduced expression of CD106 in soy‐fed compared to casein‐fed mice. These findings suggest that the atheroprotective effect of soy diet is mediated, in part, by inhibition of vascular cell adhesion molecule expression that could lead to reduced monocyte adhesion and migration, an early event in atherogenesis. USDA‐CRIS‐6251‐5100002‐06S.
Different FGF‐2 isoform patterns influence drug sensitivity in human melanoma and murine fibroblast cell lines
Authors: Dini Germana , Schiavone Nicola , Magnelli Lucia
Published: June 2021

The effect on drug sensitivity of FGF‐2 expression was evaluated in a murine fibroblast (Balb/A31) and in a human melanoma (MC) cell line. Human metastatic melanoma cells, negative for FGF‐2 expression, were transfected in order to express low (18 kDa), high molecular weight (HMW) and all (ALL) isoforms. Clonogenic assay showed that 18 kDa expression was related to chemoresistance to Dacarbazine (DITC), both in 18 kDa and ALL MC cells with respect to the mock transfected cell line. In contrast, the expression of High Molecular Weigth FGF‐2 isoforms alone produced a chemosensitive phenotype, even if compared to that of FGF‐2‐negative clones. Dacarbazine‐resistant clones were obtained following drug selection confirming that resistance was induced by 18 kDa low molecular weight isoform while high molecular weight isoforms produced a sensitive phenotype. Accordingly high molecular weight isoforms were lost and low molecular isoform was gained from DITC resistant ALL and 18 kDa cell line, respectively. Also we failed to obtain resistant clones from HMW cells.

Balb/A31 exogenously expressing different human FGF‐2 isoforms were treated with increasing concentration of doxorubicin, cisplatin and 5‐fluorouracyl. Clonogenic survival was evaluated to determine drug resistance. Results showed a highly variable response dependent on both FGF‐2 isoform expression pattern and treatment.

Grant: CRF 2003.0808
Inhibition of DNMT1 with 5‐aza‐2′‐deoxycytidine induces expression of tumor antigens (GAGE, MAGE, PAGE, and CT45) in MCF7 cells
Authors: Lobo Rebecca C. , Lee Kyung‐Seon , Green Ralph , Miller Joshua W.
Published: June 2021

Cancer cells often express tumor antigens that are recognized by host cytolytic T lymphocytes. Such antigens include GAGE (G antigen), MAGE (melanoma specific antigen), PAGE (prostate specific antigen), and CT45 (cancer/testis antigen). Repression of tumor antigens is a mechanism by which cancer cells may evade recognition by the immune system. Methylation of CpG sites in promoter regions of specific genes leads to recruitment of methylated DNA binding proteins and co‐repressors that cause chromatin compaction and gene repression. This is a key mechanism contributing to abnormal gene expression patterns observed in cancer cells. We are investigating the effect on gene expression of 5‐aza‐2′‐deoxycytidine (ADC), an inhibitor of DNA methyltransferase 1 (DNMT1), in the human breast cancer cell line, MCF7. Cells were treated in vitro for 72 hours with 1 µM ADC in DMSO. RNA was then isolated for microarray analysis (Affymetrix). Compared with cells treated with DMSO alone, ADC‐treated cells exhibited increased expression (≥2‐fold) of 38 genes. Ten of these genes were members of the GAGE, MAGE, PAGE, or CT45 families, with all 10 exhibiting >5‐fold increased expression. These results suggest that tumor antigens are repressed epigenetically in MCF7 cells. De‐repression of these antigens using drugs that target epigenetic factors may provide an effective strategy for cancer treatment. American Cancer Society
AN IN VITRO STUDY OF EPIGENETIC CHANGES DUE TO ETHANOL TREATMENT
Authors: Bardag‐Gorce Fawzia , Nguyen Sheila , Oliva Joan , French Barbara A , French Samuel W
Published: June 2021

High levels of ethanol in liver cells cause significant epigenetic changes affecting histone post translational modifications and histone modifying enzymes responsible for histone acetylation and methylation. The present study is an in vitro study using HepG2 human cells transduced to over express CYP2E1 (E47) or not (C34), which were first treated with arachidonic acid, then Fe‐NAT prior to ethanol exposure (100 mM) for 24h. These HepG2 cells showed an increase in histone acetylation (H3K9 and H3K18) as well as an increase in histone methylation (H3K27me3), which was similar to the effect of ethanol on the liver in the in vivo study. In addition, the treated cells showed a significant increase in the levels of histone modifying enzymes, particularly the histone deacetylase Sirt1. With respect to methylation, the re‐methylation pathway, responsible for generating the methyl donor S‐adenosylmethionine, was also changed in this in vitro study. Methionine adenosyltransferase II, alpha (Mat2a) was significantly decreased in the ethanol treated cells compared to the controls. The increase in H3K27me3 indicated that other methylation mechanisms, such as an increase in the activity of an histone methyltransferase Ezh2, are involved in the increase of histone methylation. In addition, there was no difference in the response to ethanol between the E47 cells and the C34 control cells, which suggests that CYP2E1 induction by ethanol does not contribute to the changes in histone post translational modifications. Supported by NIH/NIAAA Grants 8116 and P50‐011999 Alcohol Center Grant, Liver/Pancreas.
A variant in 5‐UTR of ERCC6 protects against age‐related macular degeneration in European decent
Authors: Tuo Jingsheng , Cho Youngeun , Chew Emily , Chan Chi‐Chao
Published: June 2021

Age‐related macular degeneration (AMD) is the leading cause of visual loss in aged people and has genetic components in its pathogenesis. ERCC6 functions in DNA repair and the disruption of ERCC6 is causal to Cockayne syndrome, a disorder featuring accelerated aging and progressive multi‐organ degeneration including retina. Previously we reported that a single nucleotide polymorphism (SNP) in ERCC6 regulatory region (rs3793784) was moderately associated with AMD. This study is to investigate the association of a set of SNPs in ERCC6 with AMD. We selected 12 common SNPs across ERCC6 and evaluated them in 162 advanced AMD cases and 189 controls. We identified that a C/A SNP in near 5′‐UTR of ERCC6 is highly associated with protection against AMD (odds ratio=0.33, p<0.01 after permutation correction). The SNP was close to the transcriptional starting site, suggesting a function of gene regulation. The SNP was in linkage disequilibrium with rs3793784 but has 2‐folds higher impact odds ratio. Interestingly, the control subjects with AA alleles (homozygous protective) had a higher average age than the controls with CC or CA. The genotypes were distributed evenly in dry and wet types of AMD. The SNP could result in the loss of Sp1 binding site and in the generation of a C/EBPalpha binding site. The underlying mechanism of this protective association may involve a SNP alteration of the regulatory function by an ERCC6 activator element.
The Levels of Calcitonin Gene‐Related Peptide and Substance P in the Plasma of Rats with Traumatic Brain Injury and the Role of Neurogenic Inflammation in the Pathogenesis of TBI
Authors: Duan hubin , Hao chunyan , Li shuzhen
Published: June 2021

Background and Purpose

Substance P (SP) is thought to play a crucial role in the traumatic brain injury (TBI). Calcitonin gene‐related peptide (CGRP) is an inflammatory neuropeptide found in neurons. The purpose of research is to explore the correlation between SP and CGRP in the rats plasma after TBI and to study their significance in the neuropathic mechanism of TBI.

Methods

l68 male Wistar rats were randomized into the injury group and control group.Mild,moderate and severe injury models (0.5‐1.0 atm,1.5‐2.0 atm and 2.5‐3.0atm ) was produced with the fluid percussion brain injury (FPI) technique in anesthetized adult rats equipped with a closed cranial window.All groups were again stratified into 7 sub‐groups according to the time after TBI(0.5,2,6,12,24,48 and 72 h).The plasma levels of SP and CGRP were measured with ELISA at specific time after the injury.

Results

There was a positive correlation between the plasma levels of SP and CGRP (r=0.879.P<0.01)after TBI.The plasma levels of SP and CGRP varied significantly across all groups with different severity of TBI(P<0.01) and time after injury (P<0.01).The peak and trough of plasma levels of SP were recorded in 0.5 h‐moderate (1.46±0.23ng)and 24h‐severe (0.43±0.17ng) sub‐groups,and those of CGRP were recorded in 2 h‐moderate (5.9±0.9ng)and 24 h‐severe sub‐groups(0.7±0.4ng) respectively.

Conclusion

These data show that both plasma levels of SP and CGRP changed regularly with the severity of injury and the time after TBI. A positive correlation also be observed between the plasma levels of SP and CGRP.Therefore, detection of plasma SP and CGRP should be helpful to estimate the severity of injury and pathological changes of the TBI.
Characterizing the sympathetic neuropeptide Y system in 4T1 murine mammary carcinoma model
Authors: Medeiros Philip John , Novielli Nicole , Jackson Dwayne Neville
Published: June 2021

Neuropeptide Y (NPY) released from the sympathetic nervous system (SNS) is a potent mitogenic and angiogenic factor. Recent studies have illustrated the prevalence of NPY receptors in many cancer cell lines. Remarkably, little is known of the role that NPY and its Y1‐ and Y2‐receptors play in the progression of breast cancer tumor development. Moreover, no in vivo models have addressed the impact of NPY in breast cancer. In this study we used the murine 4T1 cell line as it mimics growth and metastatic patterns seen in human breast cancer. Cells (103) were injected into the inguinal mammary fat pad of female BALB/c mice (n=10), after 29 days tumors were harvested and prepared for immunofluorescence and molecular analysis (Western blot and qRT‐PCR). 4T1 tumors were characterized by: 1) extensive vascular development that was innervated by sympathetic neurons, and 2) robust expression of NPY, Y1 and Y2 receptors. Additionally, Y1 receptor mRNA was augmented in 4T1 tumors compared to cultured cells (p<0.05), suggesting potential for a Y1‐mediated functional role of NPY in this cancer model. Based on these data we show for the first time that breast cancer tumors raised from the 4T1 cell line have a complete and intact sympathetic NPY system. Thus, we conclude that this is an ideal model to investigate the functional impact of the SNS on breast cancer growth. CIHR
Chronic serotonin infusion leads to prolonged fall in blood pressure in the spontaneously hypertensive rat
Authors: Davis Robert Patrick , Szasz Theodora , Linder Elizabeth , Burnett Robert , Watts Stephanie Wengert
Published: June 2021

Chronic serotonin, or 5‐hydroxytryptamine (5‐HT) infusion results in a prolonged fall in blood pressure (BP) in the deoxycorticosterone acetate (DOCA)‐salt hypertensive model. The role of 5‐HT in BP regulation and hypertension remains largely unknown. To further our understanding of the hypotensive role of 5‐HT, we investigated chronic 5‐HT infusion in the spontaneously hypertensive rat (SHR). We hypothesized that 5‐HT infusion in SHR would result in a fall in BP when compared with DOCA‐salt model. 5‐HT and Vehicle (Veh) were administered subcutaneously via osmotic pump (25μg/kg/min) for 7 days to 14 week old male SHR. Free circulating plasma 5‐HT levels increased in both SHR and DOCA model with 5‐HT infusion. Platelet poor plasma levels (PPP) were higher in the 5‐HT‐infused SHR (23.1± 16.2 ng/ml), compared with Veh‐infused SHR (0.33 ± 0.1 ng/ml). PPP levels were also higher in 5‐HT‐infused DOCA (114.5 ± 21.1 ng/ml), compared with Veh‐infused DOCA (24.9 ± 5.0 ng/ml). Mean arterial BP was monitored via radiotelemetry. Resting SHR BP was 145± 1.61 mmHg, while DOCA BP was 166 ± 7.58 mmHg. After 24 hours of 5‐HT infusion, SHR BP fell to 111 ± 2.45 mmHg, while DOCA pressure fell to 139 ± 3.15 mmHg. This study demonstrates a similar hypotensive role for 5‐HT in the SHR compared to DOCA and a potentially beneficial role for 5‐HT in cardiovascular system. This is the first study to investigate chronic 5‐HT‐infusion in the SHR model.