bluuebunny/crossref_metadata_2025 huggingface dataset:
Human DNA2/BLM and EXO1 participate in parallel longrange resection pathways for repair of DSB due to replication stress
Authors: Karanja Kenneth , Cox Stephanie , Campbell Judy
Published: June 2021

Double stranded breaks (DSB) are lesions repaired by the homologous recombination (HR) pathway during S/G2 phase. MRN/CtIP nuclease activity initiates a short 5′ end resection followed by a longer resection to generate a recombinogenic 3′ ssDNA sufficient to activate a cell cycle checkpoint and ensure HR. It is postulated that the long‐range resection occurs via two pathways. One requires BLM helicase and the other utilizes EXO1. Here we show that DNA2 nuclease may function with BLM to effect the long‐range 5′ resection. Using camptothecin to induce DSBs, we have shown that DNA2 and EXO1 function in two parallel pathways. In the absence of both DNA2 and EXO1, DNA resection is markedly impaired in comparison to instances where DNA2 and EXO1 are depleted individually. S‐phase checkpoint is probably impaired due to diminished resection, RPA and Chk1 phosphorylation. Cells also exhibit reduced RPA2, RAD51 and ssDNA foci after S‐phase specific DNA damage. Moreover, DNA2 and EXO1 knockdown cells have increased DNA‐PK phosphorylation, suggesting that interference with HR may activate NHEJ. In clonogenic assays, DNA2 and EXO1 knockdown cells are sensitive to DNA damaging agents camptothecin and cisplatin. Overall, we demonstrate that DNA2/BLM is important for HR in human cells undergoing replication stress.
The function of Deinococcus Radiodurans SSB protein in RecA‐mediated DNA strand exchange
Authors: Koo Sunghyu , Ngo Khanh , Cox Michael M
Published: June 2021

Deinococcus Radiodurans (Dr or D. Radiodurans) is a bacterium with an extraordinary resistance to radiation. Strand exchange promoted by the RecA protein is an important process in homologous recombination, which is essential for maintenance of accurate DNA replication. Along with RecA, the single strand DNA binding protein (SSB) is critical to DNA replication, recombination, and repair in all bacteria including Deinococcus Radiodurans. in Deinococcus Radiodurans, SSB is absolutely required for RecA‐mediated DNA strand exchange. We present results indicating that the primary function of DrSSB is to sequester the displaced strand during DNA strand exchange. Furthermore, DrRecA pre‐incubated with ssDNA appears not to work efficiently in the strand exchange reaction, supporting the inverse DNA strand exchange pathway. Further experiment on inverse DNA strand exchange must be done to establish the role of DrSSB and the correct strand exchange model in D. Radiodurans.
Double‐stranded breaks in S. cerevisiae: recruitment of TFIIH and stability of DNA ends
Authors: Wolstencroft Paige Wheaton , Negritto M. Cristina
Published: June 2021

Transcription factor IIH (TFIIH), with known functions in the nucleotide excision repair pathway (NER) and transcription initiation, has been implicated as having a role in the repair of Double Strand Breaks (DSBs) in S. cerevisiae. The Bailis research group identified the Rad3 helicase, a subunit of TFIIH, as an important factor in suppressing short sequence recombination (SSR). The RAD3 mutant, rad3‐G595R, has been shown to increase the frequency of SSR by increasing the stability of DSB ends. In this study we set to determine if TFIIH is physically present at DSBs such that it could be directly affecting DNA end processing. Chromatin immunoprecipitation (ChIP) assays were used to study the recruitment of TFIIH to the site of DSBs in both wildtype and rad3‐G595R mutant strains. Our results indicate that in wild type strains the level of TFIIH at the site of the break increases after a DSB has been induced and then gradually declines once the break stops being induced. However, in a rad3‐G595R mutant there is no significant increase in TFIIH localization following DSB induction. By verifying the presence of TFIIH at DSBs, a role for this complex in dealing with the repair of DSBs has been confirmed. The faulty recruitment observed in the mutant strain helps postulate new ideas on the exact role TFIIH may be playing in DSB repair.

(Funding: Pomona College Summer Undergraduate Research Program)
The role of autophagy in erlotinib‐resistant lung cancer and the synergistic induction of cell death with combination cisplatin and erlotinib treatment by Atg3 modulation
Authors: Lee Jasmine G , Wu Reen
Published: June 2021

Objective

Erlotinib resistance (ER) in non‐small cell lung cancer (NSCLC) is a major problem clinically. Therefore, in this study we tested whether combination of low dose erlotinib and cisplatin is effective against ER and specifically examined the role of autophagy in ER.

Methods

PC9 cells were used to establish erlotinib resistant cells (PC9ER). Cell survival was quantified with Alamar blue assay. LC3II and regulatory proteins of autophagy were measured by western blot. SiRNA was used to modulate protein translation.

Results

Combination of low dose erlotinib‐cisplatin induced synergistic cell death in PC9ER cells and this response was related to a decrease in autophagy. Concordantly, rapamycin increased ER and 3‐MA sensitized cell death response in PC9ER cells. Western blot analysis of autophagy regulatory proteins showed significantly higher baseline levels of LC3II and Atg3 in ER cells as compared to the sensitive PC9 and a significant decrease in Atg3 level was seen with combination treatment. Atg3 SiRNA transfection resulted in the reversal of the resistance in PC9ER cells to both erlotinib‐alone and combination treatment.

Conclusions

Our data suggests that ER lung cancer cells are responsive to combined low dose erlotinib‐cisplatin treatment by targeting the baseline over production of Atg3 and that Atg3 is a key regulator/promoter of ER in NSCLC.

This research was supported by NIH‐T32 grant.
Defying the proteasome, autophagy and convention: S. cerevisiae dodges the isoaspartyl aging bullet
Authors: Patananan Alexander , Clarke Steven
Published: June 2021

The spontaneous degradation of aspartyl and asparaginyl protein amino acids to isoaspartyl residues is one of the most common types of protein damage in aging organisms. Although the protein isoaspartyl methyltransferase (PIMT) repairs this detrimental modification and is essential to many organisms, no gene homolog or equivalent enzymatic activity is present in Saccharomyces cerevisiae. We therefore used biochemical approaches to quantify isoaspartyl damage and elucidate how isoaspartyls are repaired and/or degraded in a system absent of PIMT. Surprisingly, cytosolic isoaspartyl damage quantified by base labile volatility assays was strikingly low (~70 pmol/mg of total protein) compared to cells with PIMT and decreased as the cells aged. Proteasome and autophagy inhibition by MG‐132 and 3‐methyladenine, respectively, did not significantly alter isoaspartyl protein damage levels. Yeast lacking the catalytic and regulatory proteasome proteins Rpn4p, Ubr2p, Pre3p and Pup1p, or the autophagy Atg8p protein, also did not exhibit elevated damage. However, depleting cells of ATP did increase isoaspartyl damage 5‐fold. Perturbing the intracellular pH homeostasis, an ATP‐dependent process, by incubating exponentially growing yeast in water buffered to pH 3 or 9 resulted in significant isoaspartyl accumulation (>200 pmol/mg of total protein). Further analysis under these conditions using pH‐sensitive plasma membrane and vacuolar H+‐ATPase knockout cells revealed additional increases in protein isoaspartyls, suggesting that pH is a vital factor controlling damage, particularly in the vacuole. Finally, enzymological approaches with synthetic peptides have potentially identified an isoaspartyl‐targeted protease. Future investigations will identify and characterize this isoaspartyl‐directed proteolytic activity, in addition to elucidating the role of cytosolic and vacuolar pH on isoaspartyl protein damage. Ruth L. Kirschstein National Research Service Award GM007185.
Recombinant Expression of Human Enteropeptidase Light Chain in Pichia pastoris
Authors: Smith Eliot T , McGhee Mallory , Johnson David A
Published: June 2021

The catalytic light chain of the serine protease human enteropeptidase (enterokinase, hEKLC) was expressed as an active recombinant protein in the yeast Pichia pastoris. EKLC shows high degrees of specificity and selectivity for the amino acid sequences DDDDK~X and DDDDR~X, making it useful for separating fusion domains of recombinant proteins. The amino acid sequence of hEKLC was altered to remove N‐linked glycosylation sites by changing Asn to Gln; similarly, an Arg in a potential kexin protease cleavage site was changed to Gln to preclude proteolysis. The cDNA encoding hEKLC was codon‐optimized for expression in P. pastoris and commercially synthesized. Fusion to α‐mating factor targeted the protein for secretion. Active recombinant hEKLC (rhEKLC) was purified from fermentation medium by liquid chromatography using the enzyme's affinity for soybean trypsin inhibitor (STI). Western blot results confirm the identity of rhEKLC using an antibody to bovine enteropeptidase. Enzymatic activity was characterized via cleavage of the synthetic substrates z‐Lys‐SBzl, GDDDDK‐p‐nitroanilide, and GDDDDR‐p‐nitroanilide. Since the enzyme is secreted in active form, its expression is useful for testing P. pastoris expression media and it has proven useful for activating other proteases expressed as fusion proteins linked via DDDDK sequences. NIH grant R15HL091770.
Lipocalin 2, an innate immune protein, plays a key role in immune‐complex (IC) mediated inflammation
Authors: Aitken Jesse D , Srinivasan Gayathri , Sashidhramurthy Rangaiah , Parkos Charles A , Selvaraj Periasamy , Vijay-Kumar Matam
Published: June 2021

Lipocalin‐2 (Lcn2), an innate immune protein predominantly secreted by neutrophils, is upregulated by several logs during inflammatory conditions, including autoimmune diseases. However, the defined role of Lcn2 in IC‐mediated inflammation is largely unknown. We investigated the role of Lcn2 in an acute model of IC‐mediated inflammation (Reverse‐Passive Arthus Reaction) and a chronic model of Serum Induced Arthritis (SIA) using Lcn2 knock out (Lcn2KO) and their wild type (WT) littermates. In the acute model of IC‐mediated inflammation, Lcn2KO mice showed approximately 50% reduced inflammation when compared to WT mice. Histopathologic analysis of skin biopsies showed substantially reduced immune cell infiltration at the site of inflammation in Lcn2KO mice. Similarly, neutralization of Lcn2 in WT mice significantly rescued from IC‐mediated inflammation and, further, supplementing rec‐Lcn2 to Lcn2KO mice resulted in inflammation similar to that seen in WT mice. In contrast, in the chronic SIA model, Lcn2KO mice suffered significantly as measured by arthritic disease severity and paw thickness. These results suggest that Lcn2‐dependent immune cell recruitment plays a role in the initiation, perpetuation and, finally, resolution of inflammation. Collectively, our studies demonstrate that targeting Lcn2 may be a promising approach for treating autoimmune diseases.
Targeting Metabolism in Liposarcomas
Authors: Braas Daniel , Wu Hong , Christofk Heather
Published: June 2021

Positron emission tomography (PET) FDG uses cellular metabolism to localize malignant tissue. However, some cancers have a low glycolytic phenotype. Here, we analyzed the metabolic requirements of liposarcomas that frequently show low FDG uptake by PET. We established xenograft and cell culture models of three liposarcomas and investigated changes in the culture medium over time by LC‐MS/MS. Among those metabolites consumed are substrates for the nucleoside salvage pathway. Deoxycytidine kinase (dCK) catalyzes the rate‐limiting step in the nucleoside salvage pathway. We found high dCK activity and uptake of FAC, a cytidine derivative, in all three liposarcoma cell lines in vitro. We also tested the sensitivity of the liposarcoma cell lines to the cytidine‐derived nucleoside pro‐drug gemcitabine. To test if nucleoside salvage pathway is also active in vivo, we established xenograft tumors in NSG mice and imaged them with [18F]‐labeled FAC. Furthermore, gemcitabine treatment of the liposarcoma xenografts led to complete tumor regression. Measurment clinical liposarcoma samples show high dCK activity in 10–15% of liposarcoma patients. Together our results suggest that a subset of liposarcomas show high nucleoside salvage pathway activity which could potentially be employed to select for liposarcoma patients who respond to gemcitabine treatment.
AnnexinA2 regulates beta 1 integrin internalization and intestinal epithelial cell migration
Authors: Rankin Carl Robert , Hilgarth Roland S , Leoni Giovanna , Beste Kyle Den , Kwon Mike , Parkos Charles A , Nusrat Asma
Published: June 2021

The gastrointestinal epithelium functions as an important barrier that separates luminal contents from underlying tissue compartment and is vital in maintaining mucosal homeostasis. Inflammatory disorders induce epithelial injury and mucosal wounds that compromise the critical epithelial barrier. In response to injury, intestinal epithelial cells (IECs) rapidly migrate to reseal wounds. We have previously observed that a membrane associated and actin binding protein, Annexin 2 (AnxA2) is up‐regulated in migrating IECs and promotes wound closure. To address the mechanisms by which AnxA2 promotes cell migration, we generated IEC lines with stable down regulation of AnxA2 using shRNA. Loss of AnxA2 increased IEC cell‐matrix adhesion that was associated with increased β1 integrin protein expression with no change to mRNA levels. Since cell migration requires dynamic turnover of integrin based adhesions, we determined if AnxA2 influences β1 integrin internalization from the cell surface thereby regulating IEC migration. Indeed, pulse‐chase biotinylation assays identify a 6 fold increase in β1 integrin at the cell surface and significantly decreased integrin internalization and degradation in IECs lacking AnxA2. These findings support an important role of AnxA2 in controlling dynamics of β1 integrin, cell –matrix adhesion turnover, cell migration and, therefore, wound closure.
Paradoxical reduction in cardiac O‐GlcNAcylation following short‐term high fat feeding
Authors: Hughey Curtis C , Zadeh Zarah Ezzat , Johnsen Virginia L , Belke Darrell D , Hittel Dustin S , Shearer Jane
Published: June 2021

Chronic high fat feeding (HFF) leads to hyperglycemia which elevates cardiac O‐linked B‐N‐acetylglucosamine glycosylation (O‐GlcNAcylation) of mitochondrial proteins and leads to reduced oxidative phosphorylation. This impairs the hearts ability to oxidize surplus fatty acids perpetuating substrate toxicity associated with lipid excess. We evaluated short‐term HFF on C57BL/6 cardiac O‐GlcNAcylation and mitochondrial function. Following 20 wk HFF, mice were obese (49.12 +/− 0.73 vs. 31.82 +/− 0.95 mg; n=10), but had similar fasting blood glucose (13.98 +/− 1.45 vs. 16.56 +/− 0.91 mM; n=10). High‐resolution respirometry exhibited increased state 3 oxygen consumption supported by complex I substrates in HFF permeabilized cardiac fibers (288.82 +/− 24.81 vs. 188.80 +/− 16.48 pmol/s/mgww; n=8). This was associated with ~1.5‐fold greater mitochondrial density in the HFF group as determined by electron microscopy (n=5). Additionally, global O‐GlcNAcylation decreased ~7‐fold in the HFF mice (n=6) and immunoprecipitation experiments show complex I subunit NDUFA9 O‐GlcNAcylation is reduced ~2.5‐fold (n=6). In conclusion, prior to the onset of hyperglycemia, cardiac mitochondrial oxidative phosphorylation increases to accommodate excess substrate availability due, in part, to enhanced mitochondrial density and decreased O‐GlcNAcylated NDUFA9. This work was supported by CIHR, AHFMR, CDA and HSF.
The role of G0/G1 switch gene 2 in mitotic clonal expansion during adipogenesis
Authors: Choi Hyeonjin , Lee Hye-Min , Kim Hyo Jung , Kim Jae-woo
Published: June 2021

One of the earliest events occurring in adipogenesis is mitotic clonal expansion which is required for differentiation of 3T3‐L1 cells. In this study, we carried out microarray analysis and analyzed genes which might play an important role during this process. Among them, we sorted out candidates, G0/G1 switch gene 2 (G0S2) which is highly expressed in brown and white adipose tissue and is greatly up‐regulated during mouse 3T3‐L1 adipogenesis. When knockdown of endogenous G0S2 inhibits differentiation and decreases the expression of adipocyte specific genes. Moreover, its knockdown induced apoptosis. G0S 2 is a novel putative target gene of PPAR¥ã. PPAR¥ã, which is most highly expressed in adipose tissue, is known as the master regulator of adipogenesis. The absence of PPAR¥ã in 3T3‐L1 fibroblasts by RNA‐mediated interference knockdown impaired adipocyte differentiation same as knockdown of G0S2. And its knockdown caused apoptosis in 3T3‐L1 preadipocytes. These results show that G0S2 is an important role for differentiation of 3T3‐L1 preadipocytes into adipocytes.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011‐0030711 and 2011‐0015665) and by Korean Research WCU grant (R31‐2008‐000‐10086‐0) from the Korean Ministry of Education, Science, and Technology.
Membrane localization of a novel regulator of actin depolymerizing factor (ADF)
Authors: Cuddy Katrina K , Grey Paris H , Zhang Xiaoguo , Oppenheimer David G
Published: June 2021

Actin filament turnover is required for many actin‐dependent cellular processes including cell motility and membrane trafficking. Members of the actin depolymerizing factor/cofilin (ADF) family of actin binding proteins are essential for severing/depolymerizing actin filaments. Because ADF plays a central role in severing actin filaments, understanding the regulation of ADF activity is essential for understanding actin dynamics. We recently identified a new regulator of ADF in plants named ITB for REGULATOR OF PLANT ADF. Our in vitro analysis of ITB3 function showed that it interacts with ADF and inhibits actin binding to ADF. Interestingly, about half of the ITB members possess a putative signal sequence (SS). Given that signal sequences direct proteins for secretion, we have to reconcile the fact that ADF, which interacts with ITB3, is not secreted. We thus hypothesize that the putative signal sequences are novel N‐terminal transmembrane anchors. To test this hypothesis, we constructed GFP fusions to the ITB3L‐10 protein, which contains a putative SS, and used particle bombardment to transiently transform Arabidopsis epidermal cells. Results show localization to motile structures that are likely Golgi bodies. We are also conducting in vitro transcription/translation of ITB3L‐10 in the presence of eukaryotic microsomes to test for co‐translational membrane insertion.
Differential uptake of functionalized polystyrene nanoparticles by human macrophages and monocytic cells
Authors: Lunov Oleg , Syrovets Tatiana , Loos Cornelia , Nienhaus G. Ulrich , Mailänder Volker , Landfester Katharina , Simmet Thomas
Published: June 2021

Tumor cell lines are frequently used as models to analyze the cellular uptake of nanoparticles. Here we show that carboxy‐ (PS‐COOH) and amino‐functionalized (PS‐NH2) polystyrene nanoparticles of ~100 nm in diameter are internalized by human macrophages, THP‐1 monocytic leukemia cells, and by PMA‐differentiated THP‐1 cells via different mechanisms. Macrophages and THP‐1 rapidly internalized PS‐COOH and to a lesser extent PS‐NH2 particles when incubated in buffer. The uptake of both nanoparticles was drastically reduced in media containing serum. Using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between the CD64 receptor and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization in THP‐1 cells occurred via dynamin II‐dependent endocytosis. PMA‐differentiated THP‐1 cells took up the particles via macropinocytosis. In line with the in vitro data, more PS‐COOH particles were accumulated in liver tissue, whereas PS‐NH2 particles were preferentially targeted to tumor tissue. These data show that the extent of the particle internalization, the uptake kinetics and its mechanism differ significantly among primary cells and model tumor cells, whether differentiated or not, and are further critically dependent on the particle opsonisation by serum proteins. Supported by the DFG, SPP1313.
The Effect of the Lipid Environment on Drug Binding and Transport by the P‐Glycoprotein Multidrug Transporter
Authors: Sharom Frances Jane , Clay Adam T.
Published: June 2021

The P‐glycoprotein multidrug transporter (Pgp, ABCB1) is an ABC protein that effluxes hundreds of structurally unrelated compounds from cells, and is implicated in multidrug resistance in many human cancers. The membrane environment is critically important for Pgp function since lipophilic substrates partition into, and are pumped out of, the membrane. Using purified Pgp reconstituted into proteoliposomes of defined lipids, the effect of the bilayer melting transition on the lipid partition coefficient, binding affinity, and initial rate of transport was measured for Hoechst 33342, LDS‐751 and MK‐571. All drugs partitioned best into liquid crystalline lipid, and bound to Pgp with millimolar affinities within the membrane, suggesting that they interact with the protein weakly. Pgp drug binding affinity was modulated by both the lipid phase state and acyl chain length, and was higher for all drugs in the rigid gel phase. The kcat values for Pgp‐mediated drug transport were also sensitive to lipid melting, however, they were higher in the liquid crystalline phase. Transition state analysis showed the existence of entropy‐enthalpy compensation and an isokinetic relationship between ΔH‡ and ΔS‡. The transport rate is proposed to be controlled by the drug binding affinity and a large conformational change, rather than the rate of ATP hydrolysis.

This research is funded by the Canadian Cancer Society (grant #700248).
Stress‐activated cellular senescence as a mechanism of melanocytes degeneration in vitiligo
Authors: Bellei Barbara , Pitisci Angela , Ottaviani Monica , Ludovici Matteo , Cota Carlo , Fabiola Luzi , Dell'Anna Maria Lucia , Picardo Mauro
Published: June 2021

Several studies show evidence of an alterated redox status and an increased susceptibility of vitiligo cells to pro‐oxidant agents, supporting the idea that an imbalance of the cellular redox status might be the pathogenic mechanism in vitiligo. In order to study intracellular pathways involved in stress‐dependent vitiligo cell damage, we focused on cAMP/PKA/CREB and mitogen activated protein kinases (MAPK)‐dependent signal transduction. We found correlation between intrinsic oxidative stress, activation MAPK and p53, hyperphosphorylation of CREB and increased cholesterol levels. Notably, these long‐term effects of subcytotoxic oxidative stress are also biomarkers of premature cellular senescence. Consistent with this, vitiligo cells showed a significant increase in p16 that did not correlate with the chronological age of the donors. Chronic stimulation of MAPK and downstream inhibition of cyclin‐dependent kinases in the presence of elevated levels of cyclinD1 suggest that vitiligo melanocytes are prone to entering a hypermitogenic cell cycle arrest. Accordingly, vitiligo cells displayed attenuated responses to mitogens. Moreover, vitiligo melanocytes produced biologically active proteins among the senescence‐associated secretory phenotype, such as MMP3, Cox‐2, IGFBPs. Together, these data argue for a pathologic predisposition of vitiligo cells to stress‐induced premature senescence.
Probing and improving students’ understanding of protein alpha helix structure using targeted assessment and classroom interventions
Authors: Loertscher Jennifer , Minderhout Vicky , Lewis Jennifer E , Villafane Sachel M
Published: June 2021

Molecular visualization is central to the study of biochemistry. Fundamental skills in this area are often taught in introductory biology and chemistry courses. As biochemistry instructors, we hope that students are able to apply visualization skills learned in earlier coursework to advanced coursework including biochemistry, yet many of us know from experience that this is not always the case. A pre/post‐test we developed revealed a robust incorrect idea about the location of the amino acid side chains in the protein alpha helix structure (1,2). Since this incorrect idea was present even after a semester of instruction in biochemistry at a range of institutions, an intervention was necessary. We worked collaboratively with a community of twenty expert biochemistry instructors to design two active learning classroom activities that examine alpha helix structure more systematically. Several of these faculty used one or both of the activities in their classrooms. Through this process we were able to use assessment data to make targeted changes in teaching. Student responses to the activities revealed further issues related to molecular visualization and students’ ability to properly interpret space‐filling molecular diagrams.
The total amino acid profile of human milk is stable over the first three months of lactation
Authors: McMahon Robert Joseph , Gray Michael , Adelman Alicia , Herbers Patricia , Woo Jessica , Morrow Ardythe
Published: June 2021

The amount and composition of human milk protein is thought to be critical in the development of breastfed infants. While the amino acid profile of human milk has been previously reported, there have been methodological advances which may result in more accurate amino acid determinations, and we are interested in the variability of the protein level and amino acid profile between mothers and over the course of lactation in rigorously collected samples. Here we report total nitrogen, as assessed by the Dumas method, as well as the protein associated amino acid profile over the course of the first three months of lactation (5 time points) in a cohort of 10 mothers in the US. Total nitrogen decreased by about half over the first three months of lactation (p<0.001) and exhibited inter‐individual variation early on, while the amino acid profile, expressed as each amino acid in proportion to the total amino acids, remained remarkably consistent both between mothers as well as over time. Supported by Mead Johnson Nutrition and NIH HD 13021.
Effects of milk collection and processing methods on origin and integrity of RNA in milk
Authors: Lemay Danielle G , Hinde Katie , Hovey Russell C , Smilowitz Jennifer T , Hartono Stella R , Ventimiglia Frank , Schmidt Kimberli A , Lee Joyce W.S. , Korf Ian , Barry Peter A , German J Bruce
Published: June 2021

Given the potential diagnostic utility of RNA in milk, it is important to establish best practices for its collection and processing. Milk was collected from 5 human participants and 6 rhesus macaques at 2 time points and each macaque underwent a mammary biopsy. Degradation of human milk fat RNA was positively correlated with distance from collection site and time stored in TRIzol at −80 C. RNA integrity also varied by sample type: milk fat RNA was significantly more degraded than RNA from cells in milk or mammary tissue. Intriguingly, there were no differences in RNA degradation in macaque milk collected after 10 mins or 4 hours of accumulation, suggesting that milk fat RNA degradation may not occur in the breast. Origin of RNA was determined by applying our automated method to images of milk stained with acridine orange. Human whole milk contained more cytoplasmic crescents and fewer nucleated cells than macaque milk. A repeated milking protocol increased crescent‐derived RNA in some, but not all, macaque milk samples. Using RT‐PCR, expression of a transcript specific to milk‐producing cells, alpha‐lactalbumin, was significantly higher in milk fat compared to mammary tissue. Milk fat RNA may more accurately portray the RNA profile of milk‐producing cells in vivo compared to RNA from mammary tissue; however, this sample type requires adherence to protocols that minimize RNA degradation. Funded by NIH P51RR000169.
Identification of environmental supports for healthy eating in older adults
Authors: Sylvie Amanda , Cohen Nancy L
Published: June 2021

The nutritional status of older adults (OA) correlates to their quality of life, ability to live independently, and risk for developing costly chronic illnesses. An OA's nutritional wellbeing can be impacted by multiple socio‐environmental factors including access to healthy and affordable foods, congregate meal sites, and nutritious selections at restaurants. Research related to improving the nutritional status of OA through environmental changes is limited. The objective of this study was to identify priority areas within a community's environment to enable healthier food choices in OA.

Twelve experts in aging and nutrition participated in an online brainstorming session (as two panels of six experts). Transcripts were analyzed to determine primary factors that enable healthy eating in OA, and behavioral settings that are changeable and would impact OA nutrition. Major enablers identified were accessibility, affordability, social support, and living accommodations. The panels identified critical behavioral settings as food stores, restaurants, religious settings, congregate nutrition sites, senior housing, and health care settings. The enablers and behavioral settings will be prioritized based on their contributions to the goal of improving OA nutrition using the Analytic Hierarchy Process. Priority ratings and implications for community public health planning will be discussed.

Grant Funding Source: USDA/NIFA NE 1039
Integrating nutrition and physical activity education into a food pantry
Authors: El-Kabany Salma , Jara Eddy A , Abdrabalnabi Ahmed Al
Published: June 2021

In 2010, about one out of 20 households utilized food pantry assistance one or more times. With over 40,000 pantries in the US, this presents an opportunity to address the high prevalence of household food insecurity while simultaneously promoting healthful eating and exercise habits in low‐income communities. The objective of this project is to implement nutrition and physical activity education in the Helping Hands Pantry, a center that provides groceries to over 7,000 people each week. As individual clients waited to receive food, healthy food samples were distributed by nutrition students to draw clients’ attention to the nutrition education display in the waiting room. University nutrition students provided adults with nutrition information, meal planning, and exercising tips and children were given nutrition‐related coloring sheets. Through conducting oral assessments, authors found that many children lacked proper knowledge in nutrition and many adults seemed to know about nutrition, but discounted the importance of exercise. Authors found that targeting educational interventions at food pantries to individual clients is a promising strategy. To promote sustainability and effectiveness, pantry volunteers can be trained to implement the nutrition education interventions described above on a frequent basis.
A study on the nutritional status and effect of iron and calcium intervention in Korean preschool children
Authors: Shin Kyung Ok , Jung Tae Hwan , Hwang Hyo Jeong , Choi Kyung Soon , Park Hyun Suh , Chung Keun Hee
Published: June 2021

In a previous study, the Fe, Ca, Zn, niacin contents of preschool children were found to be deficient. Therefore, in this study, Fe and Ca were supplied through 4 different diet types: A, B, C, and D (A : Fe 0.63mg, Ca 27.5mg, B : Fe 15mg, Ca 232mg, C : Fe 30mg, Ca 232mg, and D : Fe 15mg, Ca 462mg). In total, 135 childrenˈs blood samples were analyzed in order to determine anemia status. Ferritin levels increased significantly in the group fed B, C, and D (Fe and Ca supplemented) but not in group A (p<0.05). Children with ferritin levels less than 20 ng/ml before intervention showed a significant increase after intervention, and ferritin levels significantly increased in the group supplied Fe 30mg and Ca 232mg (C group). Ferritin levels decreased in children with ferritin levels higher than 30 ng/ml before intervention. Therefore, intervention was not necessary for children whose ferritin levels were high enough before intervention. Children with Hb levels less than 12g/dl showed a significant decrease, but children who were supplemented with 30mg of Fe did not show a significantly higher level of Hb than those supplemented with 15mg of Fe. The value of Fe saturation(%) was significantly increased by 15.2% in children with transferrin Fe saturation(%)<20% after intervention of Fe 15mg & Ca 232mg, but the saturation of Fe was rather decreased by the intervention of Fe 30mg & Ca 232 mg or Fe 15 mg & Ca 464mg(C and D groups). The mean value of TIBC was in the normal range after intervention, and there was no significant difference among the groups. Therefore, Fe intervention increased ferritin, Hb, transferrin, and Fe saturation, and TIBC levels in anemic children.

Grant Funding Source: Sahmyook University
Korean office workersˈ recognition and use of health functional foods
Authors: Chang Kyung Ja , Choi Sang Hee
Published: June 2021

In order to provide proper guideline for Korean office workers’ use of health functional foods, this cross‐sectional survey was conducted using a questionnaire. The subjects of this study were 624 office workers (male 291, female 333) in the Gyeonggi‐Incheon district, Korea. Data were analyzed with SPSS 12.0 program. Office workers aged over 40 years(p<0.05), married(p<0.001), with higher educational background(p<0.05), with higher interest in health(p<0.05) or who were obesity(p<0.01) showed significantly more interest in health functional foods. Office workers with higher educational background(p<0.01), with higher interest in health(p<0.05), who were obesity(p<0.001) or who were residents of apartment or single house(p<0.05) recognized significantly effect of health functional foods. Also 65.6% of the subjects answered that nutrition education on health functional foods is necessary and office workers with higher educational background(p<0.01) significantly more answered the necessity of that education. As for use of health functional foods, 42.4% of the subject had the experience using them and 40.7% of the subject used them now. Office workers aged over 40 years(p<0.001), married(p<0.001), with higher income(p<0.001), or who were residents of apartment or single house(p<0.01) used significantly more health functional foods. As for the reason for using health functional foods, fatigue‐recovery(34.1%), fitness(30.1%) and prevention from disease(22.1%) were most answered and there were significant differences in gender(0.05), age(0.001), marital status(0.001) or job type(0.05). As for satisfaction in health functional foods, ‘convinience’ was the highest and ‘price’ was the lowest. Therefore, proper guideline based on social‐demographic variables is necessary for Korean office workers’ use of health functional foods.
Daily Intakes of Iron, Zinc and Manganese in Korean Children Aged 6 to 11 Years
Authors: Choi Mi-Kyeong , Bae Yun-Jung , Kang Myung-Hwa , Kim Mi-Hyun
Published: June 2021

The essential trace elements such as Fe, Zn, and Mn play an important role in human growth and development. The purpose of this study was to estimate the daily intakes of Fe, Zn, and Mn in Korean elementary school students. A total 1,320 elementary school students aged 6 to 11 years participated in this study. A 24‐hr recall method for 3 days was conducted and daily intakes of Fe, Zn, and Mn were calculated. The mean daily intakes of energy, Fe, Zn, and Mn were 1852.4 kcal, 13.4 mg, 9.1 mg, and 4.5 mg for boys aged 6 to 8 years and 1764.3 kcal, 13.0 mg, 8.6 mg, and 4.2 mg for girls aged 6 to 8 years and 1830.1 kcal, 13.0 mg, 9.2 mg, and 4.4 mg boys aged 9 to 11 years and 1783.1 kcal, 12.5 mg, 9.0 mg, and 4.2 mg girls aged 9 to 11 years, respectively. The major food groups consumed Fe and Mn were cereals, vegetables, and pulses and Zn were cereals, meats, and vegetables. The median intakes of Fe, Zn, and Mn in total subjects were 12.7 mg, 8.8 mg, and 4.2 mg and were more than their recommended intakes (RIs of Fe and Zn) and adequate intake (AI of Mn). Daily intakes of Fe, Zn, and Mn of elementary school students participated in this study were sufficient compared with the dietary reference intakes. However, in order to assess accurately trace elements in elementary school students, systematic balance study examining their intakes and excretions is needed in future.
Korean Elementary School Students’ recognition, preference and nutrition knowledge on seafood
Authors: Chang Kyung Ja , Joo Ran
Published: June 2021

In order to provide information on the efficient utilization of nutritious seafood in school food service, this cross‐sectional study was conducted using a questionnaire. The subjects of this study were 493 fifth‐ and sixth‐grade elementary school students in Incheon metropolitan city, Korea. Data were analyzed with SPSS 12.0 program. As for preference for seafood in school food service, 79.9% of the students had a moderate or higher preference. The boys had significantly greater preference for seafood compared to the girls(p<0.001). As for the reason of preference for seafood in school food service, most of the boys and girls answered good taste. As to the reason of disliking seafood in school food service, there was no significant difference between genders. But many of the boys answered that the types of seafood in school food service weren't what they liked and many of the girls answered that they didn't like the taste and smell of seafood in school food service. As for reason why they should eat seafood, most of the boys and girls answered excellent nutrition. As for nutrition knowledge on seafood, the boys(8.13) showed a significantly higher knowledge score compared to the girls(7.54)(p<0.05). Their favorite seafood was dried laver regardless of gender, followed by shrimp, blue crab, squid and tuna, and their unfavorite seafood was Alaska pollack, followed by oyster, green laver and mussel. In case of seafood cooked in different ways, most of the boys and girls preferred fried seafood, boiled rice with seafood, noodles with seafood, porridge with seafood, panbroiled seafood and Korean Jeon, and they didn't prefer soup and stew with seafood, baked and boiled seafood, seasoned seafood and salted seafood. Therefore, it is required to study how to cook seafood in a way preferred by Korean students to let them eat more seafood in school food service.
Food insecurity is associated with poor social capital, perceived health, and perceived diet among adult food bank users in and around the lower mainland of British Columbia, Canada
Authors: Hill Ryan Wayne , Holben David H
Published: June 2021

Adult clients from 4 food banks in British Columbia (Surrey, Richmond, TriCities, Nanaimo) were surveyed for differences between food security situation among adults (FSSAA), social capital (SC), perceived health (HLTH), and perceived diet (DIET). Of 1,064 invited, 528 (49.6% response rate) completed the study. For FSSAA, only 5.5% were food secure; 26.3% and 68.2% were food insecure (moderate) and food insecure (severe), respectively. 42.2% had high SC, while 57.8% had low. 34.9% considered their HLTH to be poor/fair, while 65.1% considered it to be good/very good/excellent. 50.3% considered their DIET to be poor/fair, while 49.7% considered it to be good/very good/excellent. FSSAA [Kruskal‐Wallis (K‐W), p=.046], SC (K‐W, p=.003), and DIET (K‐W, p=.030) significantly differed by food bank, while HLTH did not (K‐W.341). Considering all participants, FSSAA was significantly related (Kendall's Tau b) to SC (−0.141, p<.001), HLTH (−0.196, p<.001), and DIET (−0.290, p<.001). This study confirms that food bank users are food insecure and have poor SC, HLTH, and DIET. It also underscores the negative relationship of food insecurity to those constructs.
Impact of Eat Well…For your Self, For the World text and curriculum on college students’ attitudes and eating habits
Authors: Morris Michelle Neyman , Rios Letisia , Bacon Linda , Clifford Dawn , Gray Katharine
Published: June 2021

The objective of this study was to examine the impact of a nutrition text and curriculum on the attitudes and eating habits of community college students in northern California. The nutrition curriculum introduced emphasized the relationships between food choices and personal health, environmental sustainability, and social justice issues. A convenience sample enrolled in an introductory nutrition course between fall 2007 and spring 2009 served as the intervention group (n=448) while students enrolled in a physiology course served as controls (n=134). Intervention participants read Eat Well…For your Self, For the World and completed associated curriculum over the course of a four month semester. All participants completed an on‐line survey assessing demographics, eating attitudes and habits at the beginning (pre) and end (post) of the semester they were enrolled. Participants were primarily single, Asian/Pacific Islander and Caucasian, aged 15 to 58 years. Intervention participants significantly improved their mean Intuitive Eating Scale, Pleasure Subscale, and Health Consciousness Subscale scores (p<0.001), significantly decreased their BMI (p<0.001), and reported positive changes in their shopping and cooking habits and motivators for food choices post intervention compared to controls. Future research should include follow‐up assessment to test for sustainability of these results.
Comparison of the anti‐inflammatory activities among water extracts of grapefruit, green tea, and cochineal and their microencapsulation particles in RAW 264.7 macrophages
Authors: Jun Yoon-Kyung , Jeon Yeong-Hui , Jiang Yi-Zhang , Kim Myung-Hwan , Nam Seong-Pil , Chang Moon-Jeong
Published: June 2021

Microencapsulation is a promising processing methods that can increase the surface area, so we expected to increase the physiological effects. Our study aims to evaluate the anti‐inflammatory effects of microcapsules containing water extracts of green tea(WGT), grapefruits(WGF) or cochineal(CCN) in LPS activated RAW 264.7 macrophages. WGT and CCN at the concentration of 0.1–0.3mg/ml, WGF at the concentration of 0.01–0.03mg/ml, that inhibited NO production and decreased the expression of inducible nitric oxide syntase(iNOS) and nuclear factor κB(NF‐κB) without showing cytotoxicity. WGF was considerable more potent than the WGT or CCN. The productions of prostaglandin E_(PGE_) were reduced by the treatments of WGT, WGF or CCN. WGF inhibited LPS‐induced nuclear factor κB to a much greater extent than did WGT or CCN. The processing of the microencapsulation for WGF potentiated the inhibition of anti‐inflammatory effects by water extracts samples, whereas the effects of microencapsulation for WGT or CCN were minimal. The findings presented here suggest that the anti‐inflammatory effect of a grapefruit extract was remarkable and the most potent compared to green tea or cochineal and the microencapsulation enhanced these effects. The results of these investigations indicate possible protective effects against cardiovascular disease and chronic inflammatory diseases.

This study was supported by 15 agenda of Korea Rural Development Administration in 2011.
The effects of resistance exercise and post‐exercise meal timing on the iron status in iron deficient rats
Authors: Fujii Takako , Matsuo Tatsuhiro , Okamura Koji
Published: June 2021

Resistance exercise increases heme synthesis in the bone marrow and the hemoglobin (Hb) in iron deficient rats. Post‐exercise early nutrient provision facilitates skeletal muscle protein synthesis compared to late provision. However, the effects of post‐exercise nutrition timing on Hb synthesis are unclear. This study investigated the effect of post‐exercise meal timing on the activity of the key enzyme involved in hemoglobin synthesis, δ‐aminolevulinic acid dehydratase (ALAD), in the bone marrow and examined the Hb concentration in iron deficient rats. Male 4‐week‐old Sprague‐Dawley rats were fed an iron deficient diet containing 12 mg iron/kg and performed climbing exercise (5 min × 6 sets/d, 3 d/w) for 3 weeks. The rats were divided into a group fed a post‐exercise meal early after exercise (E) or a group fed the meal 4h after exercise (L). A single bout of exercise after resistance exercise training for 3 weeks increased the bone marrow ALAD activity, plasma iron concentration and transferrin saturation (TfS). Although the plasma iron concentration and TfS were lower in the E group than in the L group after a single bout of exercise, the basal hematocrit, Hb and total iron binding capacity after 3 weeks did not differ between the groups. Therefore, resistance exercise increases the bone marrow ALAD activity, while the post‐exercise meal timing has no effect on the Hb concentration in iron deficient rats.
Cranberry bioactives promote activation and proliferation of innate immune cells
Authors: Stanilka Joy M , Rowe Cheryl A , Khoo Christina , Percival Susan S
Published: June 2021

A previous human intervention study showed that consumption of a cranberry beverage enhanced proliferation of γδ‐T cells and NK cells when they were stimulated ex vivo. To explore potential mechanisms, we asked if we could replicate this observation using in vitro PBMC cultures. Buffy coats were obtained (LifeSouth, Gainesville, Fl) and incubated with several concentrations of cranberry fraction E or F. Cell counts, proliferation and activation of γδ‐T cells, NK cells and NK‐T cells were determined before culture, at 24h and 10d by flow cytometry. In general, all three cell types required more than 24 hours for proliferation to occur. There was a dose response to both cranberry fractions. Cell activation was demonstrated by an increased expression of CD69, an early activation marker on γδ‐T cells and CD314 (NKG2D) on the NK and NK‐T cells. Activation was evident within 24h and was apparent but much lower after 10d. In vivo, only gut immune cells would come into contact with these cranberry polyphenols, however, as one of our first lines of defense; this is an important population to maintain. Supported by Ocean Spray Cranberries, Inc. and AES, UF
The lactoferrin receptor is differentially expressed across several human epithelial cell types
Authors: Lopez Sydney Alissa , Nonnecke Eric B , Lönnerdal Bo L
Published: June 2021

The cationic glycoprotein lactoferrin (Lf) exhibits an array of biological activities that contribute to host development and immunity. The lactoferrin receptor (LfR) has been detected in multiple tissues and cell types including intestinal epithelial cells and lymphocytes. The internalization and subsequent cellular activities of Lf are likely dependent upon the tissue‐specific expression patterns of the LfR and physiologic state, as suggested by dynamic fluctuations of Lf concentrations in the materno‐fetal compartment across gestation, and postnatally in the neonatal small intestine of the breastfed infant. In the present study, the effect of culture period on LfR protein expression patterns was investigated using various epithelial cell models. Human intestinal (Caco‐2), placental (BeWo), hepatocellular (HepG2), and mammary (T47D) cells were seeded at a density of 1.5×105 cells per 10 cm dish in triplicate. Cellular lysates were harvested daily beginning at day 4 and thereafter for 24 days. LfR protein expression was strongly detected in BeWo, Caco‐2, and HepG2 cells, and to a lesser extent in T47D cells. Cellular LfR expression patterns varied across time, reaching maximal levels between day 7 and 20 of culture in each cell line. However, the LfR expression profile within this range was cell type specific. These results suggest that LfR expression depends on cell type as well as degree of maturation.