bluuebunny/crossref_metadata_2025 huggingface dataset:
Selection of improved peptide ligases by yeast surface display
Authors: Tran Hai , Gray Daniel , Marks James , DeGrado William , Wells James
Published: June 2021

The ability to ligate synthetic peptides with high efficiency will be of tremendous value to the scientific community by enabling efficient labeling or assembly of proteins. Previously, we have engineered a first generation peptide ligase, called subtiligase, as a tool for semi‐protein synthesis, and for proteomics applications involving labeling N‐termini (N‐terminomics). Under conditions we use for N‐terminomics, subtiligase suffers from a low ligation efficiency. A second generation subtiligase was developed using phage display and a single catalytic turnover selection scheme. However, due to the binary nature of single turnover catalysis, it became difficult to select for enzymes with higher catalytic activities. Here we present a multiple turnover selection technology using yeast display whereby individual turnover events are accounted for by the ligation of a fluorophore‐tagged peptide. The advantages that yeast display provide are high‐copy number of enzymes and dual color fluorescence activated cell sorting for selection and normalization of multiple turnover events. This study provides a rare example of selecting for improved enzyme properties using yeast display. NIGMS 5R01GM081051
Pericytes: Gatekeepers governing neutrophil extravasation during inflammation
Authors: Wang Shijun , Cao Canhong , Chen Zhongming , Bankaitis Vytas , Tzima Eleni , Sheibani Nader , Burridge Keith
Published: June 2021

During inflammation polymorphonuclear neutrophils (PMNs) traverse venular walls, composed of the endothelium, pericyte sheath and vascular basement membrane. Compared to PMN transendothelial migration, little is known about how PMNs penetrate the latter barriers. Using mouse models and intravital microscopy, we show that migrating PMNs expand and use the low expression regions (LERs) of matrix proteins in the vascular basement membrane (BM) for their transmigration. Importantly, we demonstrate that this remodeling of LERs is accompanied by the opening of gaps between pericytes, a response that depends on PMN engagement with pericytes. Exploring how PMNs modulate pericyte behavior, we discover that direct PMN‐pericyte contacts induce relaxation rather than contraction of pericyte cytoskeletons, an unexpected response that is mediated by inhibition of the RhoA/ROCK signaling pathway in pericytes. Taking our in vitro results back into mouse models, we present evidence that pericyte relaxation contributes to the opening of the gaps between pericytes and to the enlargement of the LERs in the vascular BM, facilitating PMN extravasation. Our study demonstrates that pericytes can regulate PMN extravasation by controlling the size of pericyte gaps and thickness of LERs in venular walls. This raises the possibility that pericytes may be targeted in therapies aimed at regulating inflammation.
Monocyte activation and endothelial small G‐proteins control the route of transendothelial migration
Authors: Mikhailov Alexei V , Muller William A
Published: June 2021

Monocyte extravasation can occur across endothelial junctions (paracellular migration, PM) or through endothelial cells (transcellular migration, TCM). Both routes employ similar molecules, but factors controlling the choice of transmigration route are not known. We studied the interaction of freshly isolated human monocytes on primary human umbilical vein endothelial cells (HUVEC) as well on an established cell line (iHUVEC) using live video microscopy and 3D imaging of fixed immunostained cells. Although most endothelial cells supported PM only, some cells, especially those with morphology suggestive of increased Rho activity, allowed both TCM and PM. The monocytes that chose the TCM pathway were defective in establishing polarity and directionality of migration towards the junctions, but not in movement per se. Such monocytes were better spread, had more podosomes, and deeply invaginated the endothelial surface. We treated the endothelial cells and/or monocytes with small molecule Rac/Cdc42 and Rho activators or inhibitors. TCM can be increased by activating Rac and Cdc42 in monocytes, or by activating monocytes with chemokines. Rho activation in endothelial cells significantly increased the proportion of monocytes migrating transcellularly (~3 times). Rho inhibition had the opposite effect, and prevented the effect of Rho activation. The monocytes undergoing TCM were surrounded by a zone of high RhoA activity in the underlying endothelial cells, as revealed by a FRET biosensor. We conclude that the route of monocyte transmigration is controlled by Rho activity in the endothelial cells or by the state of activation of the monocytes. Funded by NIH R37 HL064774 to WAM
Bovine serum albumin interactions with metallic nanoparticles
Authors: Demirdji Marianne C. , Shah Meika R. , Ramon Ruth H. , Rowland Leah K. , Sabir Theodore S. , Payne Marvin A. , Perry Christopher C. , Boskovic Danilo S.
Published: June 2021

Metallic nanoparticles are considered as potential adjuvants for a growing list of pharmaceutical treatments. For such a purpose the nanoparticles would need to be introduced to tissues close to the pharmacological target. Frequently this involves an intravenous or intra‐arterial administration of the combination of the nanoparticles and the pharmacological agent. When foreign surfaces, however, come in contact with blood components they become coated with a variety of plasma proteins, including albumin. This protein binding could potentially alter the properties of the nanoparticles employed. In this study we explored the interactions of bovine serum albumin (BSA) with gold nanoparticles as a model of protein‐nanoparticle interactions. BSA (1μM final) was characterized spectrophotometrically while buffered by 20mM HEPES, 150mM NaCl, 2mM CaCl2, pH 7.40. It was characterized by a major intrinsic fluorescence excitation peak between 250nm to 310nm, and an emission peak from 290nm to 410nm. We examined the effects of nanoparticles on the intrinsic BSA fluorescence spectra. We observed a concentration dependent quenching effect on intrinsic BSA fluorescence with increasing gold nanoparticle levels. The BSA emission peaks were integrated from 310nm to 390nm and were found to be substantially quenched by increasing nanoparticle levels. Since this type of energy transfer is exquisitely dependent on the proximity of the emitting and quenching components it implies that the fluorescent protein (BSA) and the quenching surface (gold nanoparticles) had to be bound to one another.
ACAD expression and role of mitochondrial fatty acid β‐oxidation in retinal pigment epithelium
Authors: Polinati Padmini , Tyni Tiina
Published: June 2021

Mitochondrial fatty acid β‐oxidation (FAO) is the main energy providing pathway in the liver, heart and skeletal muscle particularly during fasting and prolonged exercise. Among several FAO defects, only patients with 3‐hydroxy‐acyl‐CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (TFP) are affected with progressive retinopathy, a major concern and long‐term complication of such disorders. Pigmentary retinopathy causes progressive disturbances of retinal pigment epithelium (RPE) leading to accumulation of photo‐oxidized products by inhibiting the phagolysosomal degradation of the outer segment. Accumulated undigested lipids like hydroxylated fatty acids and proteins are thought to cause retinopathy in LCHAD deficiency. To decipher the pathogenetic mechanisms of retinopathy, we did incubation studies on cultured human RPE‐cells with various concentrations of palmitate and hydroxypalmitate with/without carnitine for 48 hrs. Thin cell sections under electron microscope revealed a massive accumulation of rod shaped long tubular formations that could be due to accumulation of complex oxylipids. Further immunoblotting showed robust expression of long and medium chain ACADs and HADH with scant expression of very‐long acyl‐CoA dehydrogenase. We thus conclude that other than TFP all other major FAO enzymes in the FAO pathway are expressed in human RPE cells.
The role of Parkin in the maintenance of normal mitochondrial and cellular function in skeletal muscle
Authors: Godin Richard , Piquereau Jérôme , Gouspillou Gilles , Picard Martin , Mofarrahi Mahroo , Mathew Jasmin , Hussain Sabah , Hepple Russ , Burelle Yan
Published: June 2021

The E3 ligase Parkin has been implicated in the regulation of mitophagy, a quality control process whereby defective mitochondria are sequestered in autophagosomes and delivered to lysosomes for degradation. Parkin has been mainly studied for its implication in neuronal degeneration in Parkinson but its role in other tissues remains unknown. In this study, we used a murine model of Parkin deficiency to test the hypothesis that Parkin is important for mitochondrial quality control in the mitochondria‐rich skeletal muscle, a tissue in which autophagy/mitophagy play an important regulatory role. Our results indicate that Parkin is abundant in normal skeletal muscle, and that Parkin deficiency leads to mitochondria‐related abnormalities including 1) lower respiratory rates and acceptor control ratio, 2) reduced activities of respiratory chain complexes containing mtDNA encoded subunits, 3) increased susceptibility to opening of the permeability transition pore, and 4) increased expression of Bnip3 and Bax, which respectively regulate mitophagy and apoptosis. Parkin deficiency is also associated with an increased expression of ER‐stress markers (CHOP, GRP78), and a greater proportion of enlarged muscle fibers, which suggest broader phenotypic changes within skeletal muscle. Overall, these results establish the role of Parkin in the maintenance of normal mitochondrial function in skeletal muscle.
Maternal obesity enhances ZFP423 expression and adipogenic differentiation of E14.5 fetal muscle of mice
Authors: Yang Qiyuan , Rogers Carl Jacob , Huang Yan , Zhu Mei-Jun , Du Min
Published: June 2021

Obesity including maternal obesity, is increasing rapidly, which impairs fetal skeletal muscle development. ZFP423 is the earliest marker of adipogenic commitment currently identified and the 5′‐UTR region of ZFP423 has extremely enriched CG islands. We hypothesized that maternal obesity enhanced the expression of ZFP423 and thus adipogenesis through altering DNA methylation in the ZFP423 promoter. Female mice (one month old) were fed either a control (Con) or obesogenic (OB) diet to induce obesity before pregnancy, and maintained in their respective diets during pregnancy. Muscle samples were harvested from fetal mice at E14.5, a very early stage for adipogenic differentiation. The methylation status of ZFP423 5′‐UTR region was analyzed using bisulfite sequencing. The mRNA expression of ZFP423 was higher in OB fetal muscle, which was associated with attenuated methylation in the ZFP423 promoter. The primary cultured cells prepared from OB fetal muscle demonstrated enhanced adipogenic potential. Our data show that maternal obesity enhances adipogenic differentiation of multipotent cells in fetal muscle, at least partially through altering the DNA methylation of ZFP423, a critical initiator of adipogenesis. This research is funded by NIH grant R01HD067449.
Deficiency of the DNA glycosylase OGG1 predisposes mice to metabolic disease
Authors: Sampath Harini , Vartanian Vladimir , Rollins Michael R , Lloyd R. Stephen
Published: June 2021

Exposure to oxidative stress can result in damage to DNA bases, especially to mitochondrial DNA, and is correlated with numerous human diseases. The 8‐oxoguanine DNA glycosylase, OGG1, initiates base excision repair of 8‐OH‐dG, the most common oxidative DNA lesion, and is implicated in the prevention of cancer and neurodegeneration. Here, we report a novel role for this DNA repair enzyme in the maintenance of metabolic homeostasis. Mice deficient in OGG1 (Ogg1−/−) show an increased propensity to adiposity and hepatic lipid accumulation, compared to wild‐type counterparts, when exposed to an oxidative stress in the form of a 10‐week long high‐fat diet (HFD). Glucose tolerance is significantly impaired in HFD‐fed Ogg1−/− animals, despite increased plasma insulin levels. While food intake and voluntary activity appear unchanged in Ogg1−/− mice, indirect calorimetry measurements reveal a slight increase in day‐time RER, suggesting a preference for carbohydrates as a fuel source in HFD‐fed Ogg1−/− mice. Microarray analyses of hepatic gene expression reveal a shift towards glycolysis over TCA cycle metabolism and fatty acid oxidation in Ogg1−/− mice. Collectively, these data indicate that OGG1 deficiency results in an increased susceptibility to obesity, potentially by altering mitochondrial integrity and substrate preference. Support: AHA 11POST7480004 to HS, NIH RO1 DK075974 to RSL
Hepatic MGAT1 is directly regulated by PPARγ2 and promotes fatty liver
Authors: lee yoojeong , Kim Jae-woo
Published: June 2021

There are two biochemical patheways for triacylglycerol (TG) synthesis, the glycerol 3‐phosphate pathway and the MGAT pathway. MGAT pathway is merely involved in the TG biosynthesis because of the very low expression and activity of MGAT1 in normal liver. However, MGAT1 expression increased robustly in liver of ob/ob mice. We found that MGAT1 is upregulated in high fat fed B6 mice but not in C3H mice, which do not express PPARγ in liver. This indicates that MGAT1 is one of the target genes regulated by PPARγ. Moreover, the experiment using adenoviral‐PPARγ2 showed that MGAT1 mRNA level was increased by PPARγ2. Overexpression of MGAT1 dramatically increased the lipid droplets, suggesting that MGAT1 is responsible for the increased TG synthesis. Moreover, B6 mice injected with adenoviral‐MGAT1 resulted in significantly increased TG level in liver. These findings suggest that enhanced TG synthesis may be due to activated MGAT1 pathway by overexpression of MGAT1, indirectly glycerol 3‐phosphate pathway. Taken together, we concluded that PPARγ upregulates MGAT1 expression, thereby promoting alternative pathway of TG synthesis, which resulted in the hepatic steatosis.

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).
Adhesion of albumin to FDA type II soft contact lenses
Authors: Harkas Youstina , Harkas Maryam , Nichols Jessica , Cooper Danielle , Janoff Andrea , Keith Edward O.
Published: June 2021

Objective

Human tears have over 60 different proteins that deposit on contact lenses. Protein deposits on contact lenses can cause conjunctival irritation and bacterial infection. The adhesion of albumin to omafilcon A soft contact lenses was examined.

Methods

Lenses were incubated for 1 – 4 days in a 2.0 mg/dl albumin solution. The protein concentration in the incubation vials and protein deposition on the lenses was determined each day.

Results

Albumin deposited on the lenses in a steadily increasing fashion, while the albumin concentration in the incubation solution decreased, especially in the vials in which the lenses were incubated the longest.

Discussion

Human serum albumin is a single peptide chain of 585 amino acids that binds and releases a variety of hydrophobic molecules. Previous studies report that albumin deposits more on hydrophobic surfaces compared to hydrophilic surfaces. Omafilcon lenses are coated with phosphorylcholine which may reduce adhesion by the hydrophobic domains of albumin.

Conclusion

In this study, the negatively charged tear protein albumin absorbed in a linear fashion to the slightly negative omafilcon material, consistent with the findings of others that albumin deposits on contact lenses in a concentration‐dependent way with a single timescale.

Supported by the Farquhar College of Arts and Sciences and an NSU President's Faculty Research and Development Grant.
Mitochondrial transcription factor A binding to mitochondrial DNA during aging and calorie restriction
Authors: Picca Anna , Lezza Angela , Leeuwenburgh Christiaan , Tornaletti Silvia
Published: June 2021

According to the mitochondrial theory of aging mitochondria are involved with aging through endogenous production of oxidants. Recently it has become clear that in all species there is a marked decline in mitochondrial (mt) DNA, but the mechanisms are unknown. Calorie restriction (CR) is, so far, the only treatment shown to delay or avoid the onset of many age‐related features in various species. Mitochondrial transcription factor A (TFAM) is a nuclear‐encoded protein that plays a critical role in maintaining mtDNA structural integrity and functionality. Based on TFAM function in mtDNA maintenance and on the known accumulation of mtDNA oxidative DNA damage during aging we have tested whether the efficiency and/or the fidelity of TFAM binding to mtDNA changes with age in rat brain and liver tissues. We have measured TFAM binding to several functionally relevant mtDNA regions by mtDNA immunoprecipitation with TFAM policlonal antibody. We found tissue‐specific changes in TFAM binding to mtDNA as a function of aging and CR treatment. These changes vary depending on the functional relevance of the DNA region bound, suggesting that TFAM functions in mtDNA maintenance processes (i.e. in replication and transcription) may be compromised during aging.
Formulation Development of Alcohol‐Free Oil‐in‐Water Microemulsions as Potential Adjuvants and Vehicles for Vaccines
Authors: Muderhwa Jean Mulimbi
Published: June 2021

An alcohol‐free, oil‐in‐water microemulsion was formulated with mineral oil and Span80/Tween 60 (25/75, HLB 12.29) as surfactant system. Using glycerol as co‐surfactant, the amount of water solubilized depended on the surfactant system to glycerol ratio, the oil chain length, the oil content and the salinity of the water. With this system, one can formulate an isotropic, shear thinning dispersion containing 20% (w/w) of mineral oil and up to 25% (w/w) of water at surfactant to glycerol ratio of 0.50. Interestingly, the inclusion of prototypical model antigens, such as conalbumin and lysozyme, at immunizing dose of 0.025 mg/ml did not affect the formation or the stability of the microemulsions. Likewise, addition of aluminum adjuvants, such as aluminum hydroxide and aluminum phosphate, at a dose of 1 mg/ml did not have an effect either. Overall, the results suggest that these dispersions have the potential of being versatile adjuvants and carriers for vaccine in a composite adjuvant formulation and useful experimental tools with which to investigate the role of the surface in antigen recognition and adjuvanticity due to their large surface area as opposed to emulsions.
Homology Modeling of Perilipin 5
Authors: Niday Zach , Tansey John
Published: June 2021

The perilipin family of lipid droplet proteins consists of five members. The most recently discovered protein, perilipin 5, also referred to as OXPAT, is primarily found in oxidative tissue and is believed to participate in lipid storage and trafficking within cells. While the protein structures of other proteins in the family, such as TIP47, have been examined, the structure of OXPAT is still unknown; as such, the goal of this project is to obtain data that will help determine its structure. A 200 amino acid segment of the carboxy‐terminus, stretching from residues 206–409, was analyzed using a series of threading programs: SWISS‐MODEL, M4T model, mod web, and I‐TASSER. Predicted structures are indicative of OXPAT being approximately 80% α‐helical for the analyzed segment. Associated QMEAN4 scores ranged from 0.50–0.83, indicating the homological models are reliable predictions. Ongoing experiments include crosslinking and protease protection assays, and X‐ray crystallography to confirm the predicted structure. Recent findings indicate that the interaction of OXPAT with the lipase ATGL and its co‐lipase CGI‐58 occurs in the carboxy‐terminus of OXPAT, which substantiates the need for further structural characterization.
O‐GlcNAc, A Novel Paradigm for Regulating Stress‐Induced Signal Transduction Pathways
Authors: Sengupta Srona , Boronina Tatiana , Paruchuri Venkata , Cole Robert , Zachara Natasha E
Published: June 2021

The modification of nucleocytoplasmic proteins by O‐linked β‐Nacetylglucosamine (O‐GlcNAc) is a novel post‐translational modification that regulates the cell's ability to sense and signal cellular stress. The mechanisms by which O‐GlcNAc regulates protein function, promoting cell survival, are unknown. To determine if O‐GlcNAc regulates stress‐induced signaling pathways, promoting cell survival, we have defined the basal and stress‐induced phosphoproteome of the cytoplasmic and nuclear fractions of O‐GlcNAc transferase (OGT) wild‐type and null cells. More than 700 phosphorylation sites were identified in the nuclear fraction, of which 65% showed altered phosphorylation when O‐GlcNAc levels were lowered. Analysis of these data suggests that O‐GlcNAc regulates signaling downstream of the ATM and GSK3β kinases, which both play key roles in cell survival decisions. Using orthologous approaches we have demonstrated that O‐GlcNAc appears to regulate both the expression and autophosphorylation of ATM kinase, such that lowering O‐GlcNAc levels appears to enhance phosphorylation of ATM substrates (p53, H2AX, Chk2). These data suggest that one mechanism by which O‐GlcNAc protects cells is by regulating the ATM kinase. Grant support: American Heart Association (NEZ, SDG #0930162N), NIH (NEZ, R21 #HL108003), NIH (NEZ, P01 #HL107153‐01), a PURA award (SS), ASBMB UAN award (SS).
Nonenzymatic and Enzymatic Functions of the Skp1 αGalactosyltransferase in Dictyostelium Oxygen‐Sensing
Authors: Schafer Christopher M. , Zhang Dongmei , West Christopher M.
Published: June 2021

In the social amoeba Dictyostelium discoideum the SCF E3 adaptor protein Skp1 is modified by a prolyl hydroxylase and three glycosyltransferases. Aberrant expression of these enzyme proteins has been shown to affect development of Dictyostelium in response to environmental O2 levels. The final enzyme in the pathway, AgtA (α3‐galactosyltransferase), modifies the core trisaccharide with 2 αGal residues. AgtA is unique being the only enzyme possessing a non‐catalytic domain. The C‐terminal domain contains 7 WD40‐repeats predicted to fold into a β‐propeller. in vitro activity studies comparing Skp1 glycoforms and free sugars implicate this domain in binding to Skp1 and allosteric modulation of the AgtA active site. Furthermore, biochemical and cell biological studies with single and double enzyme knockouts and overexpression strains suggest the WD40 domain may mediate a more complex role played by AgtA in the Skp1‐modification pathway via interference with other Skp1‐interacting proteins. From these data is emerging a model in which non‐enzymatic functions of AgtA serve to regulate the modification status and activity of Skp1 in a fashion unique among glycosyltransferases, with corresponding impact on O2‐sensing in Dictyostelium and possibly other protists that share pathway genes. (Supported by NIH grants R01‐GM37539 & R01‐GM84383)
Strategies to enhance T cell reconstitution after severe immunodepletion
Authors: Martinez Mariela Raquel , Dudakov Jarrod , van den Brink Marcel
Published: June 2021

Allogeneic hematopoietic stem cell transplant (AHSCT) is one of curative therapies for patients with hematological malignancies. Among its major complications is post‐transplant immune deficiency. This is devastating to the T cell compartment, leading to higher treatment‐associated morbidity and mortality. Strategies to promote rapid and effective reconstitution of T cell immunity improve the survival of recipients of HSCT. Our previous studies have shown that precursor T cells (preT) generated ex vivo on the OP9‐DL1 culture system can enhance T cell reconstitution in AHSCT recipients. However, the homing and seeding of preT to the thymus is relatively inefficient (only 1 out of every 10,000 iv injected). In this study, we examined strategies to improve the effectiveness of preT treatment. Direct administration of lineage‐depleted BM into the thymus can have significant benefits for T cell reconstitution. Small changes in the purification strategy for seeding OP9‐DL1 cultures (based on CD150 expression) can have significant impacts on the generation and output of preT. These findings lay the foundation for enhancing the utility of preT as a potential clinical therapy.
Determining intracellular concentrations of flavonoids in MDA‐MB‐231 cells using mass spectrometry
Authors: Pham Anh , Yadegarynia Sina , White Brandon
Published: June 2021

Flavonoids are polyphenolic compounds widely distributed in fruits, vegetables and beverages. In recent years, flavonoids have been found to be beneficial to human health due to their anti‐oxidant and anti‐tumor activities. Our lab has investigated the ability of flavonoids to induce cytotoxicity in human breast cancer cell lines. We have discovered that some compounds can induce cytotoxicity very readily whereas others are not. We hypothesize that this difference is attributed to the compounds ability to transverse the cell membrane and enter the cytosol. Therefore, we have used HPLC‐Mass Spectrometry (LC‐MS) analysis to quantitate the amount of flavonoids that are able to enter into MDA‐MB‐231 cells, a human breast cancer cell line. We tested kaempferol, quercetin, myricetin, daidzein, and chrysin. MDA‐MB‐231 cells were plated, and treated with 100μM flavonoids for 24 and 48 hours. After treatment, cells were counted and extracted using 40% methanol. Flavonoids from extracts were characterized and quantitated by LC‐MS using an Agilent QTOF6520 tandem mass spectrometer and an Agilent 1100 HPLC. Our results indicate as intracellular flavonoid concentration increase, cellular cytotoxicity also increases. We could not detect quercetin and myricetin in cell extracts. However, quercetin was found to be a moderate inducer of cytoxicity. We find that quercetin dimerizes over time in cell medium only in the presence of serum suggesting an enzymatic mechanism. The quercetin dimer could not be detected in cellular extracts suggesting that it could not cross the membrane.
Assessment of appendicular lean mass in older adults
Authors: Ellis Amy , Gower Barbara
Published: June 2021

Objective

Dual energy x‐ray absorptiometery (DXA) is a robust method to quantify appendicular lean mass (aLM), but cost and instrumentation limit the use of DXA beyond research purposes. Bioelectric impedance analysis (BIA) is a portable, inexpensive method to estimate fat‐free mass. In 1994, Organ et al introduced a simple BIA protocol to assess aLM from BIA [1]. The objective of this study was to compare estimates of aLM by this BIA protocol to the reference standard of DXA among healthy older adults.

Methods

Based on median body mass index (BMI; 27.4), 35 healthy men and women, ages 65–87, were categorized as low BMI (<27.4) or high BMI (≥27.4). Fat‐free mass of the upper and lower extremities was measured by DXA and BIA. Arm and leg lean mass from each method was summed for a composite of aLM.

Results

Although BIA aLM was correlated with DXA aLM (p<0.001), the mean percentage error between BIA and DXA was greater for men vs women (21.4% vs 4.0%) and among high BMI vs low BMI groups (15.8% vs 0.7%). Paired t‐tests confirmed that BIA underestimated aLM for men and those in the high BMI group (p<0.001).

Conclusions

Caution is warranted in the application of this segmental BIA protocol for older adults. These data suggest that estimates of aLM from BIA are significantly underestimated among men and individuals with BMI ≥ 27.

This work was supported by 1F31AT005384‐01 and UAB Center for Aging.
Metal transport, subcellular localization, and tissue distribution of Zip8, a Zip14 homologue
Authors: Wang Chia-Yu , Jenkitkasemwong Supak , Sparkman Brian K. , Shawki Ali , Mackenzie Bryan , Knutson Mitchell
Published: June 2021

We have previously demonstrated that Zip14, a member of the ZIP family of metal‐ion transport proteins, can transport zinc and iron into cells. The aim of the present study was to investigate metal transport, subcellular localization, and tissue distribution of Zip8, the most closely related protein to Zip14. Similar to Zip14, we found that transient transfection of HEK 293T cells with Zip8 cDNA enhanced the cellular uptake of iron and zinc, and that Zip8 is detectable at the cell surface as a transmembrane glycoprotein. Expression of Zip8 in RNA‐injected Xenopus oocytes stimulated between 15 and 150‐fold over control the uptake of 109Cd2+, 57Co2+, 55Fe2+, and 65Zn2+, but not 64Cu (I or II). Zip8‐mediated transport of 55Fe2+ (K0.5 ≈ 0.5 μM) was maximal at pH ≈ 7.5. Quantitative RT‐PCR analysis of RNA from various human tissues revealed that Zip8 is most abundantly expressed in lung and placenta, whereas Zip14 is most abundant in liver and heart. We conclude that, although Zip8 and Zip14 have similar metal transport profiles, their distinct tissue distributions suggest that they serve nonredundant functions in metal metabolism. Funded by NIH.

Grant Funding Source: NIH
Quercetin exerts anti‐adipogenic effects through modulation of 3T3‐L1 preadipocyte proliferation and differentiation
Authors: Swick Jennifer , Lee Ok Hwan , Kim Young-Cheul
Published: June 2021

Obesity is a strong risk factor for the development of diabetes, cardiovascular disease, and cancer. At the cellular level, obesity is characterized by differentiation of preadipocytes into an excessive number of adipocytes; a process tightly regulated by cell cycle events and adipogenic transcription regulators. We investigate the mechanisms by which quercetin, the most commonly consumed dietary flavonoid, regulates adipogenesis. 3T3‐L1 preadipocytes were differentiated with or without quercetin treatment. The changes in lipid accumulation, and gene expression related to cell cycle and adipogenesis were assessed. Quercetin between 10–100μM dose‐dependently inhibited mRNA expression of PPARγ and C/EBPα. Quercetin at 50μM also significantly inhibited PPARγ protein expression, suggesting the inhibitory effects at both transcriptional and translational levels. Cells treated with 50μM quercetin between 0 and 36 hrs, the early phase of mitotic clonal expansion (MCE), exhibited a significant decrease in lipid accumulation. However, there was no inhibitory effect in the cells treated on days 2 and 3. Furthermore, quercetin at 50μM suppressed mRNA expression of Cyclin A, a key regulator of cell cycle entry into S phase between 12 and 24 hrs. These results suggest quercetin suppresses adipogenesis by inhibiting the early MCE phase required for induction of adipogenic factors and terminal differentiation.
Anthocyanin Metabolites in the Mice Fed Purple‐fleshed Anthocyanin‐enriched Sweetpotato
Authors: chen Tzu-yu , xu jianteng , Lim Soyoung , Carey Edward , Katz Ben , Griffin Jason , John Tomich , Wang Weiqun
Published: June 2021

Anthocyanins are plant pigments that are epidemiologically associated with reduced chronic diseases including cancer. We previously selected a purple‐fleshed sweetpotato P40 clone that enriched a high content of anthocyanins up to 7.5 mg/g dry matter. We demonstrated that dietary P40 might prevent colorectal cancer in azoxymethane‐induced murine model. This study is to detect the potential anthocyanin metabolites in this animal model. CF‐1 mice were fed 10~30% of P40 containing 143~430 mg peonidin‐3‐glucoside equivalence/kg B.W. for 6 wks and then anthocyanin metabolites were analyzed by LC/MS and MALDI. Although anthocyanin metabolites in both plasma and liver tissues were not detectable 15‐hrs after the last feeding, four anthocyanin metabolites were identified in the feces, including cyaniding‐3,5‐diglucoside, cyaniding‐3‐p‐hydroxybenzoylsophroside‐5‐glucoside, cyaniding‐3‐sophoroside‐5‐glucoside, and peonidin‐3‐p‐hydroxybenzoylsophroside‐5‐glucoside. Better understanding of anthocyanin metabolism and bioavailability may lead the development of a functional purple‐fleshed anthocyanin‐enriched sweetpotato for health benefits.
Salty taste acuity in relation to zinc nutritional status and αENaC A663T gene polymorphism among Korean young adults
Authors: Noh Hwa Young , Paik Hee Young , Chung Jayong
Published: June 2021

Taste perception for salt affects the dietary salt intake, of which in excess is a major public health concern. Zn status and genetic factors have been associated with altered taste perception. In this study, we examined the association between zinc status, α‐epithelial sodium channel (αENaC) A663T genotype and salty taste acuity in 207 free‐living healthy young adults (20–29yr). Salty taste acuity were determined by sensory test. Dietary Zn intake data was collected by one 24‐h recall and two‐day dietary record. Serum Zn was measured by atomic absorption spectrometry. αENaC genotype was identified by the PCR‐restriction fragment length polymorphism method. We found that available Zn intake had positive association with the salty taste acuity only in female (P=0.02). Serum Zn had no effect on salty taste perception in both gender groups. When subjects were stratified according to αENaC genotype, only in females with AA genotype, available Zn intake had negative association with salty taste threshold (β= 0.833, P=0.02). In conclusion, our data shows that Zn intake affects the salty taste acuity in young females. Further, the effect of Zn intake on salty taste acuity is differed by αENaC genotypes, suggesting dietary guideline to reduce Na intake may depends on αENaC genotypes and gender of target group. This study was supported by National Research Foundation (KFR‐2008‐313‐C00978).
The Role of Autophagy in Radiation Induced Salivary Gland Dysfunction
Authors: Morgan Maria
Published: June 2021

Radiation of the head and neck region for cancer treatment causes salivary gland dysfunction. Autophagy is a “nutrient sensing” mechanism within cells that is upregulated during times of stress that is a cell survival mechanism, it may prove beneficial in salivary gland function. The objective is to determine the role of autophagy in salivary gland function following radiation. We use Aqp5‐Cre mice that allow us to specifically impair autophagy in the salivary acinar cell. Autophagy deficient mice have higher levels of cell death after radiation. This shows that autophagy may be necessary for acinar cell survival after radiation. DRAM increased after radiation in a p53 dependent manner. There is no DRAM expression in autophagy deficient mice showing DRAM may be correlated with induction of autophagy. This is critical because it is unknown whether DRAM acts in induction of autophagy or apoptosis. Current studies are focusing on salivary gland function of autophagy deficient mice after radiation. This knowledge will help us prepare therapies for those suffering from side effects of radiotherapy and greatly improve their quality of life while decreasing their financial burden.

Grant Funding Source : RC1DE020335
Effect of high fat diet on inflammatory markers and bone morphological microstructure in growing male mice
Authors: Kim Misung , Na Woori , Kim Hanlla , Park Eunhye , Lee Hyun-A , Kim Ojin , Sohn Cheongmin
Published: June 2021

This study examined the change in inflammation makers and morphological microstructure of bones in mice fed on high‐fat diet. Four‐week‐old C57BL/6J male mice were divided into 2 groups: the control group was fed 10% kcal fat diet, and the high‐fat diet group was fed 45% kcal fat diet for 12 weeks. Inflammation markers were measured by using ELISA kits and microcomputed tomography (micro‐CT) was used to measure the morphological microstructure of the femoral bone. Leptin and IL‐6 levels were significantly higher in the high‐fat diet group (p < 0.01). Oosteocalcin was not significantly different between groups; however type I collagen C‐telopeptide was significantly higher in the high‐fat diet group (p < 0.01). The thickness of bone trabecula was significantly reduced in the high‐fat diet group as compared to the control group (p < 0.05), and the gap within the bone trabecula was significantly wider in the high‐fat diet group (p < 0.05). A positive correlation (p < 0.05) was observed between IL‐6 levels and the gap within the bone trabecula. These results showed that obesity induction in mice by feeding high‐fat diet during the growth phase resulted in a change in the bone microstructure and showed that bone microstructure was correlated with inflammation index. Therefore, restriction of excessive fat intake may be required to suppress inflammation reactions and promote normal bone formation.
Patterns and predictors of growth in HIV‐exposed 24–48‐weekold Malawians receiving lipid‐based nutrient supplements as a breastmilk replacement: results of the BAN study
Authors: Flax Valerie L. , Bentley Margaret E. , Chasela Charles , Kayira Dumbani , Hudgens Michael G. , Kacheche Kopekani G. , Chavula Charity , Jamieson Denise J. , van der Horst Charles , Adair Linda S.
Published: June 2021

Lipid‐based nutrient supplements (LNS) could be used to limit morbidity and maintain growth in HIV‐exposed infants weaned early. This study examined growth in 1617 HIV‐exposed infants weaned from exclusive breastfeeding at 24–28 wks and provided with LNS as a breastmilk replacement to 48 wks. Weight, length, and BMI at 6 time points were estimated in sex‐stratified longitudinal random effects models controlling for birth measurement and maternal age, height, parity and education. Predictors included season and current and lagged fever, diarrhea and cough. From 24–48 wks, weight and length Z‐scores declined steadily (mean WAZ −0.42 to −0.76 and LAZ −0.93 to −1.56), BMI Z‐score remained >0 (mean 0.17), and proportion stunted increased (15.8% to 34.2%). Fever and diarrhea increased from 24–32 wks then leveled off; cough was stable. In boys, negative associations were observed with weight (season, fever and diarrhea), length (diarrhea), and BMI (fever and diarrhea). In girls, cough was also negatively associated with weight and BMI; diarrhea was not associated with length. To put these results in context, despite adverse effects of morbidity, HIV‐exposed infants given LNS as a breastmilk replacement had comparable growth to infants in the Malawi Demographic and Health Surveys. Support: CDC (U48 DP000059 01), Bill & Melinda Gates Foundation (OPP53107), Carolina Pop Ctr (NICHD 5 R24 HD050924).
Fluorescence Compared to Brightfield Detection of CD35 in Lymphoid Follicles of the Monkey to Assess Immunomodulatory Treatment Effect
Authors: Brodbeck Jens , Chu Felix , Edick Melissa Gonzales , Rangell Linda , Bheddah Sheila , Cain Gary
Published: June 2021

Aims

Lymphoid follicles of secondary lymphoid organs are dynamic structures inducible by immunization and/or inflammatory stimulation. Follicular dendritic cells (FDC) are specialized antigen‐presenting cells exclusive to the germinal centers (GC) of lymphoid follicles. Within the GC, FDC's form a reticular network that sustains the architecture of lymphoid follicles and interact with the GC B cells via several mechanisms including complement receptors such as CD35. The expression of CD35 in lymphoid follicles is therefore a measure of the efficacy of immunomodulatory (IMM) treatment strategies.

Methods

CD35 in formalin‐fixed paraffin‐embedded spleen sections of cynologous monkeys was detected with a mouse monoclonal antibody and visualized using a polyclonal secondary antibody coupled to either horse‐radish peroxidase for DAB precipitation or the fluorophore Alexa647. Digital brightfield and fluorescence images (20x) were acquired on a Nanozoomer 2HT (Hamamatsu) and Ariol SL‐50 (Genetix) whole slide scanner respectively. Images were quantified using Definiens Developer software.

Results

Fluorescence significantly increased the dynamic range of CD35 compared to chromogen. Fluorescence signal intensities ranged from 0 – 256 compared to 0 – 0.8 using chromogen. Both detection methods showed a small but significant decrease in follicular CD35 following IMM treatment without resolving dose effects. However the increased dynamic range of fluorescent CD35 allowed a robust distinction between follicles containing germinal centers and those without. Thus a clear dose effect after IMM treatment could be resolved using fluorescence, but not chromogen for CD35 detection.
The influence of alternative housing on dairy calf innate immune measures after weaning
Authors: Hulbert Lindsey Eve , Calvo Michelle S. , Klasing Kirk C. , Mitloehner Frank M.
Published: June 2021

Although bovine are considered a gregarious species, dairy calves are typically housed individually to reduce disease transmission, particularly when they experience stressors, such as weaning. Objective was to determine innate immune responses during weaning of individually (I; n = 18) vs. group (G; n = 51; pens of 3) housed calves. Weaning was initialized on d 53, (pm milk replacer bottle was removed) and was finalized on d 64 of age (am bottle was removed). Peripheral whole blood (WB) was sampled before the am feeding on 53, 57, 64, 67, and 71 d of age. In all calves, cortisol increased (P < 0.01) after weaning. I‐calf whole blood killing (WBK) of E. coli percent increased (P < 0.01) at 64 d, but G‐calf WBK percent did not change after weaning began. Although G‐calves had greater (P < 0.05) percent peripheral monocytes, they had less TNF‐á secretion from LPS‐simulated WB than I‐calves (P < 0.05) at age 53 d. The G calves’ peripheral neutrophils increased (P < 0.05) at age 57 and 67 d, while I‐calf neutrophils did not change after weaning began. G‐calf peripheral neutrophil phagocytosis and oxidative burst responses to a heat‐killed E. coli were suppressed compared to I‐calf neutrophil function (P < 0.05). Weaning was stressful for all calves; however, the G‐calf cellular innate immune function was dampened, suggesting that rearing calves in groups of 3 after 4 d of age may not be beneficial to their health.
The inhibitory effect of sulforaphane on the expression of VCAM‐1 in vascular smooth muscle cells
Authors: KIM JI YUN , Rhee Dong-Kwon , Pyo Suhkneung
Published: June 2021

Atherosclerosis is a chronic inflammatory disease of the large arteries. Since inflammation involves an upregulation of various adhesion molecules, inhibiting the expression of adhesion molecules can be a critical therapeutic target of inflammatory disease. Sulforaphane has been demonstrated to have a variety of biological effects including anti‐inflammatory activity; however, its molecular mechanisms are poorly understood. Here we investigate the effect of sulforaphane on the expression of the vascular cell adhesion molecule(VCAM‐1) induced by TNF‐α in cultured vascular smooth muscle cells. Cell adhesion assay and western blot analysis revealed that preincubation of VSMCs for 2 h with sulforaphane (1–5μg/ml) dose‐dependently inhibited TNF‐α‐induced adhesion of THP‐1 monocytic cells and protein expression of vascular cell adhesion molecule‐1 (VCAM‐1). In addition, flow cytometry analysis showed that sulforaphane attenuated TNF‐α induced production of intracellular reactive oxygen species (ROS). The present data also demonstrated that sulforaphane inhibited the phosphorylation of p38, ERK, JNK and the activation of NF‐κB and AP‐1 induced by TNF‐α. Overall, our results suggest that sulforaphane inhibits the adhesive capacity of VSMCs and downregulates the TNF‐α‐mediated induction of VCAM‐1 in VSMCs by inhibiting the MAPK/NF‐κB/AP‐1 signaling pathway and ROS production.
Contributions of spinal noradrenergic mechanisms to duloxetine and morphine antinociception
Authors: Meske Diana , Xie Jennifer Y. , Oyarzo Janice , Badghisi Hamid , Kuester Robert K. , Ossipov Michael H. , Porreca Frank
Published: June 2021

Development of novel therapies for acute and chronic pain states has been challenging. Experimental evidence suggests that activation of descending pain facilitatory systems promote abnormal pain. Protection against pain may result, in part, by spinopetal pain inhibitory systems, which act partly by norepinephrine (NE) acting at spinal α2‐noradrenergic receptors. Drugs that activate descending inhibition, or that mimic enhanced spinal α2‐noradrenergic tone, are effective against neuropathic pain. The antinociceptive effects of morphine are blocked by spinal α2 receptor antagonists. These observations suggest that the antinociceptive actions of morphine result, in part, by engagement of descending inhibition and spinal NE release; however, few studies have directly demonstrated this effect. Here, male Sprague‐ Dawley rats with spinal nerve ligation (SNL) or sham surgery received morphine (10 mg/kg, i.p.) or the NE/5‐HT reuptake inhibitor duloxetine (30 mg/kg, i.p.). Cerebrospinal fluid (CSF) was extracted under anesthesia. NE levels were quantified by HPLC coupled to an electrochemical detector. Duloxetine produced a significant and time‐related increased CSF NE levels in both sham and SNL rats. Surprisingly, morphine given at an antinociceptive dose failed to increase CSF NE levels suggesting that morphine effects may be too low to be detected in this study or that effects may occur indirectly and through local circuits. Supported by R01DA 012656.
An active extract of Ulmus pumila inhibits adipogenesis by inducing cell cycle arrest in 3T3L1 cells
Authors: Ghosh Chiranjit , Chung Ha-Yull , Nandre Rahul M , Kim In-Sook , Yang Seung Hak , Hwang Seong-Gu
Published: June 2021

Obesity and its associated metabolic disorders has become a major obstacle in improving the average life span. In this regard therapeutic approach using natural compounds are currently receiving much attention. Herbal compounds rich in triterpenes have the ability to regulate glucose and lipid metabolism. We here investigated the anti‐obesity ability of methanol extract of Ulmus pumila (UP) using cell culture systems. UP extract contained at least four different triterpenoids. The 3T3‐L1 cell viability was dose dependently decreased by UP (p < 0.05) by increasing cell accumulation in G1 phase while reducing in S and G2/M phase of cell cycle. UP treatment significantly decreased GPDH activity (p < 0.05) and intracellular lipid accumulation. Western blot analysis showed that the induction of PPARγ and C/EBPα was markedly attenuated by UP treatment at their transcriptional messenger RNA levels. Similarly, UP significantly decreased the expression of adiponectin and NF‐kB in a dose‐dependent manner (p < 0.05). These findings suggest that UP can be a promising therapeutic agent for obviating obesity and its related metabolic disorders.
Distinct generation, pharmacology, and distribution of sphingosine 1‐phosphate and dihydro‐sphingosine 1‐phosphate in human neural progenitor cells
Authors: Callihan Phillip , Zitomer Nicholas C. , Stoeling Michael V. , Kennedy Perry C. , Lynch Kevin R , Riley Ronald T. , Hooks Shelley B.
Published: June 2021

Studies suggest a crucial role for Sphingosine 1‐phosphate (S1P) and its receptors in nervous system development. Dihydrosphingosine 1‐phosphate (dhS1P), a reduced form of S1P, is an agonist at S1P receptors, but the biology of dhS1P has not been widely studied. The mycotoxin fumonisin B1 (FB1) is a potent inhibitor of ceramide synthases and causes selective accumulation of dihydrosphingosine and dhS1P. Maternal exposure to FB1 correlates with the development of neural tube defects in which the neural epithelial progenitor cells of the developing brain fail to fuse. We hypothesize that the altered balance of S1P and dhS1P in neural epithelial cells contributes to the developmental effects of FB1. We defined the effect of FB1 exposure on levels of sphingosine and dh‐sphingosine and their receptor active 1‐phosphates in human neural progenitor (hES‐NEP) cells, and the relative activity of dhS1P and S1P in these cells. We found that dhS1P is a more potent stimulator of inhibition of cAMP and Smad phosphorylation than is S1P, and this difference in apparent potency may be due, in part, to more persistent presence of extracellular dhS1P applied to hES‐NEP cells rather than a higher activity at S1P receptors. This study establishes hES‐NEP cells as a useful human in vitro model system to study FB1 toxicity and the molecular pharmacology of sphingolipid signaling.

Research supported by: NIH & UGA Research Foundation