bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibiting pathways involved in B‐cell development enhances sensitivity of B‐cell acute lymphoblastic leukemia to glucocorticoids
Authors: Pufall Miles A , Kruth Karina , Fang Mimi A , Shelton Dawne , Abu‐Halawa Ossama , Tasian Sarah K , Kampmann Martin
Published: June 2021

Glucocorticoids, such as dexamethasone (dex), are used in combination chemotherapy for leukemias and lymphomas. Dex activates the glucocorticoid receptor (GR), which in turn regulates genes that induce cell death. Although dex is a remarkably effective therapeutic, some patients are insensitive to it, and others develop resistance. From among the thousands of GR‐regulated genes, it is not known which are required to induce cell death, nor how resistance arises in most cases. To understand which GR regulated genes drive lymphoid cell death, and which signaling pathways modulate dex‐sensitivity, we took an integrated functional genomic approach. We first identified sets of dex‐regulated genes by measuring differential gene expression in a panel of childhood B‐cell acute lymphoblastic leukemia (B‐ALL) primary samples and cell lines. We then performed a next‐generation genome‐wide shRNA screen to identify genes that affect growth and sensitivity to dex in B‐ALL. By comparing regulation and effect on sensitivity we essentially validated all genes en masse in B‐ALL, and were able to show that only 10% of dex‐regulated genes contributed to cell death. These genes predominantly function in transcription regulation and B‐cell development. This screen also identified cellular pathways that modulate dex‐induced death, including the B‐cell receptor pathway (BCR), a key checkpoint in B‐cell development. By integrating the gene expression and shRNA data sets we identified a double‐negative feedback loop between GR and PI3Kδ, a lynchpin in the BCR and IL7R pathways. Inhibition of PI3Kδ attenuated phosphorylation of GR, enhanced dex‐induced gene regulation, and restored sensitivity to even highly resistant B‐ALL specimens. Thus, this work elaborates with high resolution fundamental mechanistic details of GR function, and, in doing so, identifies numerous opportunities for enhanced lymphoid‐specific combination chemotherapy.

Support or Funding Information

NIH, NSF, Carver Charitable Trust, V Foundation
BECN Homologs And ATG14 Form A Metastable Coiled‐Coil To Mediate Autophagy
Authors: Sinha Sangita , Su Minfei , Li Yue , Levine Beth , Colbert Christopher
Published: June 2021

Autophagy, an essential eukaryotic cellular homeostasis pathway, is responsible for sequestration of unwanted, damaged or harmful cytoplasmic components in vesicles called autophagosomes, followed by their lysosomal degradation, thereby enabling nutrient recycling. Autophagosome nucleation is mediated by Class III phosphatidylinositol 3‐kinase complexes that include two key autophagy proteins, a BECN (also called Beclin, ATG6 or VPS30) homolog and ATG14/BARKOR. BECN1 was amongst the first mammalian autophagy proteins to be identified1. Recently, mammals were shown to encode a second BECN paralog, BECN22. BECN2 shares 57% sequence identity with BECN1 is also involved in autophagy as well as in ligand‐induced endolysosomal degradation of several cellular GPCRs. BECN2 heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, obesity and insulin resistance2. Further, BECN2 regulates endolysosomal degradation and oncogenic signaling of GPCRs encoded by the Kaposi's Sarcoma‐associated herpesvirus3.

Here we focus on understanding the structure of the helical, coiled‐coil domain (CCD) of BECN homologs and the similarities and differences in their interaction with ATG14. X‐ray crystal structures of the CCDs of human BECN1 and BECN2 (unpublished data), show that they form similar autophagy‐inactive, anti‐parallel homodimers, which includes six and seven non‐ideal packing interactions respectively, although the BECN2 CCD homodimer is significantly more curved and has a tighter binding affinity than that of BECN1. Conversely, the CCDs of both BECN1 and BECN2 bind the ATG14 CCD to form a parallel, curved heterodimer stabilized by multiple, conserved polar interactions. Isothermal calorimetry experiments demonstrate that compared to BECN1, the BECN2 CCD forms a stronger heterodimer with the ATG14 CCD. Mutation of non‐ideal BECN1/BECN2 interface residues to more ideal pairs improves homodimer self‐association and thermal stability. Many of these BECN1 mutations knock‐out binding to ATG14, but all BECN2 CCD mutants bind ATG14, although more weakly than wild‐type. Thus, polar CCD interface residues of BECN homologs result in a metastable homodimer, facilitating dissociation; but enable better interactions with polar ATG14 residues stabilizing the BECN:ATG14 heterodimer. Further, these structure‐based mechanistic differences in BECN1 and BECN2 homodimerization and heterodimerization likely dictate competitive ATG14 recruitment within the cell.

Support or Funding Information

This work was supported by NIH grants RO3 NS090939 (S.C.S.) and R15 GM113227 (C.L.C.); a National Science Foundation grant MCB‐1413525 (S.C.S.); and NSF and North Dakota EPSCoR grant II‐1355466 doctoral dissertation awards for M.S. (PI: S.C.S.).
Role of the inter‐subunit surface interaction in the regulation of the ADP‐glucose pyrophosphorylase from Agrobacterium tumefaciens
Authors: Patel Hiralben P. , Dobrzynski Emily , Liu Dali , Ballicora Miguel A.
Published: June 2021

ADP‐glucose pyrophosphorylase (ADP‐Glc PPase) is a key regulatory enzyme involves in the starch and glycogen synthesis for plants and bacteria respectively, which catalyzes the synthesis of ADP‐glucose from glucose 1‐phosphate (Glc1P) and ATP. ADP‐Glc PPase from Agrobacterium tumefaciens is allosterically regulated by fructose 6‐phosphate (Fru6P) and pyruvate (Pyr). Previously, it was observed that removal of a disulfide bridge between the α‐subunits of the potato tuber enzyme resulted in a more active enzyme [1], indicating that inter‐subunit interactions are important in the allosteric mechanism [2]. By studying the structure of the ADP‐Glc PPase enzyme of Agrobacterium tumefaciens, it comes to light that D141 is interacting with the R11 of the opposing subunit [3], which was suggested to be involved in activation by pyruvate [4]. To prove our hypothesis that D141 and R11 residues are involved in regulation, we made three single mutants: D141A, D141R, R11D, and the double mutant R11D‐D141R. D141A exhibited reduced Vmax values in the absence of activators (~4‐fold), reduced apparent affinity for Fru6P (~2.5‐fold), and lower apparent affinity for ATP (~3‐fold). In the presence of Fru6P, D141A exhibited only ~4.3% of the wild‐type activity, and affinity for ATP was not affected as well indicating a virtual insensitivity to Fru6P. When D141R was compared to wild‐type enzyme, the activity lowered ~3600‐fold. For D141R the apparent affinity for ATP was decreased in the presence of Fru6P. R11D was similarly active as the wild‐type, while it is completely insensitive to both activators Fru6P and Pyr. The double mutant R11D‐D141R was showing decreased activity (~95‐fold) in compared to wild type, and insensitive to both activators. Our results suggest that the functional role of the D141 and R11 residues is to interacting with the opposing subunit and enabling the regulation of the ADP‐Glc PPase.

Support or Funding Information

National Science Foundation
Regulation of Phosphatidic Acid Phosphatase by High Glucose in the Oleaginous Yeast Yarrowia lipolytica
Authors: Hardman Derell , Fakas Stylianos
Published: June 2021

Phosphatidic acid phosphatase (PAP) catalyzes the conversion of phosphatidic acid to diacylglycerol, which is considered the committed step in triacylglycerol (TAG) biosynthesis. TAG biosynthesis in oleaginous microorganisms like Yarrowia lipolytica is typically triggered by high glucose amounts and results in cells accumulating lipids made mostly of TAGs. The objective of this work was to study the regulation of PAP during lipid accumulation induced by high glucose amounts. We hypothesized that the induction of TAG biosynthesis under these conditions should be mediated by PAP. In this work, we grew Y. lipolytica in a high glucose media (HGM) that favors lipid accumulation and in a low glucose media (LGM) where lipid accumulation is minimal. We measured PAP activity and lipid amounts throughout the cultivation period. Our results showed that, over time, the TAG amounts increased eight‐fold in the HGM cultures, while there was no increase in the LGM cultures. PAP activity was induced during TAG accumulation in HGM, which resulted in a two‐fold increase in activity. In contrast, PAP activity was down‐regulated in the LGM cultures, and that decrease coincided with the complete consumption of glucose in the medium. These results indicate that PAP plays a role in the regulation of lipid accumulation in Y. lipolytica.

Support or Funding Information

This project was supported by the Evans‐Allen Grant no.1005735 and Capacity BuildingGrants Program Grant no.1007503 from the USDA National Institute of Food and Agriculture.
Is Food Security Influencing Emotional Health? A Study with Hispanic Immigrant Mothers
Authors: Villegas Elizabeth , Hannon Bridget , Hammons Amber , Teran‐Garcia Margarita , Wiley Angela
Published: June 2021

Introduction

Hispanic immigrants in the U.S. often face a number of negative conditions that influence their emotional health. These problems can be heightened when there are low resources such as food insecurity and pressing stressors. This study looks to understand if food insecurity and acculturation predict depression levels for Hispanic immigrant mothers. From previous literature we hypothesize that food insecurity and higher levels of stress will predict higher depression levels.

Methods

This study used the Larger Multi‐State Abriendo Caminos Intervention program data. Hispanic immigrant mothers completed surveys at pre‐post and 6 month follow up time points. Food security was measured with USDA food security short module. Depression was measured with the CESD‐10 questionnaire and stress with Cohen's perceived stress scale. Multiple regression models were used to analyze data collected at baseline for the Illinois and California cohorts (N= 100). Among our participants, 36% reported some food insecurity with 6% of those having very low food security. Furthermore 24% had depression and, compared to other Hispanic populations, our participants had high levels of stress.

Results

Higher levels of food insecurity significantly predicted maternal depression (β = 0.314, p <0 .001), regardless of acculturation level. Food insecurity explained 10% of the variance in rates of depression and in this analyses higher acculturation levels to Anglo American behaviors account for another 2% of the variance although it is not significant. In addition, when stress was included in the analyses it accounted for an additional 23% of model variance and was a significant predictor of depression (β = 0.48, p < 0.001).

Conclusion

We found that food insecurity and stress significantly predicted depression regardless of acculturation level. These psychological variables have been associated with food consumption patterns and weight‐related outcomes in other low income populations. Therefore, our findings suggest the importance of including food insecurity and psychological variables in future research to explore possible interactions with effects on nutritional and behavior outcomes.

Support or Funding Information

This project was supported by Agriculture and Food Research Initiative Competitive grantno. 2015‐60001‐23246 from the USDA National Institute of Food and Agriculture.
Infant formula feeding alters the proliferative status of neonatal mammary glands independent of estrogen signaling
Authors: Bhattacharyya Sudeepa , Mercer Kelly E , Hennings Leah , Chaudhurry Mousumi , Yeruva Laxmi , Ronis Martin , Badger Thomas
Published: June 2021

Soy infant formula contains many phytochemicals, including phytoestrogens, which are structurally similar to estradiol (E2). The mammary gland is particularly sensitive to estrogens, and there are concerns that use of soy‐based infant formulas may potentially have adverse effects on mammary tissue development. In this study, we fed female piglets a soy‐based infant formula, a dairy milk‐based infant formula, or milk formula supplemented with E2, in comparison to piglets receiving sow's breast milk from postnatal day 2–21. Interestingly, we observed a slight but significant increase in terminal end bud number in the mammary glands of the all formula fed groups compared to the sow's milk control. Microarray analysis identified 976 differentially expressed genes between mammary glands from milk formula, soy formula and milk formula supplemented with E2 vs sow fed piglets (±1.5 fold, P<0.05). Of these genes, 45% were held in common between all formula groups. E2 supplemented milk formula altered 369 genes. Milk and soy formula altered 45 and 69 genes, respectively, in comparison to the sow's milk control. Functional annotation analysis indicated up‐regulation of angiogenesis, adipocyte differentiation, and canonical Wnt signaling pathways in the formula fed groups. Real‐time RT‐PCR confirmed decreased Wnt10b mRNA expression, and increased Wnt2b, cyclin D and n‐myc expression, suggesting an heightened proliferative state in the mammary glands of formula fed piglets compared to piglets receiving sow's milk, P<0.05. Pathways unique to the soy formula group had no apparent relevance to mammary gland development and/or function. Real time RT‐PCR expression of the E2‐responsive genes progesterone receptor (Pgr) and transforming growth factor beta‐2 (Tgfb2) were significantly up‐regulated in the E2‐supplemented formula group, compared to the milk formula and soy formula groups and the sow's milk control. These data show that dietary exposure to phytoestrogens associated with soy infant formula do not elicit an estrogenic response in the developing mammary gland. However, the neonatal mammary gland is generally quiescent. Our data also suggest that infant formula feeding may initiate proliferative and cell differentiation pathways independent of estrogenic signaling.

Support or Funding Information

This work was funded by USDA‐ARS Project 6026‐5100‐010‐05S.
Proteomic Analysis of 3T3‐L1 Adipocytes Treated with Methylglyoxal; The Potential Impact of Dietary Sugar on Adipocyte Metabolism
Authors: Komanetsky Susan M , Hedrick Victoria , Sobreira Tiago Jose Paschoal , Kim Kee Hong
Published: June 2021

Rising levels of obesity in the United States (US) and in nations adopting a Western diet are of concern due to the associated risk of chronic diseases. As an endocrine organ, adipose tissue regulates whole body immunity and energy homeostasis, but during obesity, disruptions in the normal metabolism of adipocytes, such as increased oxidative stress and reduced mitochondrial function, may contribute to disease development. Diets high in added sugar, particularly sucrose and high fructose corn syrup, may induce metabolic abnormalities in adipocytes by increasing the rate of glycolysis and the accumulation of glycolytic metabolites. Methylglyoxal (MG), a highly reactive metabolite of glycolysis, damages proteins and other cellular components. MG is found at higher concentrations in the serum and tissues of individuals with diabetes and is directly involved in the pathophysiology of diabetic complications, however, MG may have negative effects on cell metabolism before the onset of disease. Recent studies using animal models suggest MG accumulates in adipose tissue during high fructose feeding and impairs insulin signaling pathways. However, the specific effects of MG in adipocytes require further investigation. The objective of this study was to characterize changes in the proteome of mature adipocytes exposed to non‐toxic levels of MG. We treated fully differentiated 3T3‐L1 adipocytes with either MG or PBS as a control for 48 hours. Cells were collected, delipidated, and proteins precipitated. After denaturation, in‐solution protein digestion was performed with trypsin and the resulting peptide fragments were separated by liquid chromatography and scanned by mass spectrometry (MS), followed by additional MS/MS scanning. MS/MS data was analyzed with MaxQuant software and proteins identified by comparison with the Uniprot mouse database. The proteome of MG‐treated adipocytes was characterized by reduced expression of both mitochondria‐related proteins and proteins involved in endogenous antioxidant pathways. In addition, MG treatment altered the expression of proteins involved in several key metabolic pathways, including oxidative phosphorylation, the citric acid cycle, and pyruvate metabolism. We conclude that MG induces changes in adipocytes which may not only contribute to oxidative stress, but which may disrupt normal metabolic pathways. Though this may represent a temporary adaptation to MG exposure, over time, repeated such exposures may make adipocytes more vulnerable to the serious metabolic abnormalities known to be associated with obesity‐related diseases.

Support or Funding Information

USDA NIFA grant #2011‐38420‐20038, Core Pilot grant Indiana Clinical and Translational Sciences Institute #UL1TR001108
The efficacy of nano‐emulsification to improve vitamin D bioaccessibility
Authors: Kadappan Alagu Selvi , Gumus Cansu E. , Bessey Amy , McClements David J. , Wood Richard J. , Liu Zhenhua
Published: June 2021

Observational, preclinical and clinical studies strongly suggest that vitamin D deficiency is associated with a wide range of diseases, including multiple forms of cancer with the colorectal cancer (CRC) in particular. Vitamin D is among those nutrients and dietary bioactive components that receive substantial attention for CRC prevention. However, after several decades of research, we still lack an efficient dietary strategy to improve vitamin D status. The prevalence of vitamin D inadequacy and deficiency is still common, up to 32%, in the United States. Using an in vitro simulated gastrointestine (GI) system, the present study examined the efficacy of oil‐in‐water nanoemulsion in improving vitamin D bioaccessibility. Comparing to the conventional emulsion of vitamin D with corn oil, the nanoemulsion significantly altered the physiochemical properties of the lipid particles. Nanoemulsion increased negative charges, as indicated by larger negative values of the ζ‐potential, across all the stages of simulated GI tract consisting of mouth, stomach and small intestine phases (p < 0.05), and the particle sizes were significantly decreased along those stages (p < 0.05). Nanoemulsion prevented the loss of cholecalciferol though the simulated GI tract and improved its content in the intestinal digesta by 3.94 folds (p < 0.05), and further improved the incorporation of vitamin D into micelles by 16.7% (p < 0.05). Taken together, the nanoemulsion improved the bioaccessibility, as evaluated by the in vitro simulated GI model, by 4.60 folds. These results provide useful information for designing nanoemulsion‐based delivery system to improve vitamin D status.

Support or Funding Information

Supported in part by the United States Department of Agriculture (2014‐67017‐21762 and MAS00454) and UMass Honors Research Grant
Higher‐Protein Intake during Sustained Negative Energy Balance Attenuates Elevations in Resting Metabolic Rate at High Altitude (4300 m)
Authors: Derosier Allyson , Berryman Claire E. , Karl J. Philip , Wilson Marques , Young Andrew J. , Pasiakos Stefan M.
Published: June 2021

Resting metabolic rate (RMR) increases in unacclimatized lowlanders who ascend from sea level (SL) to high altitude (HA), contributing to negative energy balance and lossof total body mass. Although RMR declines progressively during HA acclimatization, in part due to the loss of metabolically active fat‐free mass(FFM), RMR remains higher than at sea level (SL) for at least the first 3 wk of residence at HA. At SL, consuming a higher‐protein diet during negative energy balance spares FFM during weight loss, which mitigates the decline in RMR that typically accompanies weight loss. While an elevated RMR aids continued weight loss at SL, this effect may be detrimental while trying to maintain weight at HA. This study examined the effects of protein intake on RMR in 17healthy male SL residents (mean ± SD, 23.4 ± 5.6 y, 81.9 ± 13.9 kg)during a 22 d period of negative energy balance at HA. At SL, volunteers were given dietary instruction to consume a weight maintenance diet containing 1.0 g protein/kg/d for 21 d. Volunteers were then transported to HA (4300 m, Pikes Peak, CO) where they lived for 22 d. On arrival at HA, volunteers were randomized to consume a controlled standard protein (1.0 g protein/kg/d, STD) or higher‐protein(2.0 g protein/kg/d, HIGH) diet designed to elicit a 40% energy deficit (30%caloric deficit, 10% increase in physical activity). Prescribed physical activity and diet were closely supervised. Body weight was measured daily anybody composition was assessed by dual‐energy x‐ray absorptiometry at the end of both SL and HA. RMR was measured on day 0 at SL and days 1, 3, 5, 7, 10, 13, and 19 at HA. RMR was 1769 ± 206 kcal/d at SL (no group differences, P>0.05)and increased to 2777 ± 538 kcal/d on day 1 at HA (P<0.01). Importantly, during HA, RMR decreased to a greater extent in HIGH compared to STD (2339 ±446 vs. 2470 ± 439 kcal/d, main effect of diet P=0.02). Total weight loss (HIGH: −7.8 ± 1.2 vs. STD: −8.0 ± 2.6 kg), FFM loss (HIGH: −3.2 ± 1.5 vs. STD: −4.0 ±3.3 kg) and fat mass loss (HIGH: −3.9 ± 0.8 vs. STD: −3.3 ± 1.8 kg)did not differ between groups (P>0.05), and changes in FFM and fat mass were not associated with RMR (P>0.05). Consuming 2.0 g protein/kg/d during sustained negative energy balance at HA minimizes elevations in RMR. Whether changes in RMR due to higher‐protein intake would result in attenuated weight loss after a more prolonged stay at HA or with a less severe energy deficit requires further research. The opinions or assertions contained herein are the private views of the author(s) and are not to be construed as official or reflecting the views of the Army or the Department of Defense.

Support or Funding Information

Supported by USAMRMC.
RELATIONSHIPS BETWEEN HEMODYNAMIC AND SYMPATHETIC NEURAL RESPONSES TO HEAD‐UP TILT DURING MODERATE DEHYDRATION IN HUMANS
Authors: Posch Alexander Morley , Luippold Adam , Mitchell Katherine , Bradbury Karleigh , Cheuvront Samuel , Kenefick Robert , Charkoudian Nisha
Published: June 2021

Dehydrationcan cause an increase in supine sympathetic nerve activity (SNA) and heart rate(HR). Reflex sympatho excitation and tachycardia are classic baroreflex responses to orthostasis, increasing peripheral resistance and cardiac output, respectively, to maintain arterial pressure during upright posture. We hypothesized that the sympathetic nervous system would have a greater contribution to blood pressure regulation during orthostasis in dehydrated people compared to conditions of euhydration. We studied 14 young healthy subjects (4 F, 10 M) divided into 2groups: euhydrated (EUH, n = 6), previously exercised in 40 ºC while maintaining hydration; and dehydrated (DEH, n = 8), previously exercised in 40 ºC who lost ~4% of body weight via sweat loss. We measured supine muscle SNA (MSNA), HR, blood pressure (BP) during a 7minute baseline period, followed by MSNA, HR and BP responses to 30 and 45º HUT(2 minutes each). Supine MSNA was not different between groups (24 ± 9 vs 28 ±11 bursts/min, P > 0.05). HR and MSNA increased in all subjects during HUT, and mean responses were not different between groups (P>0.05). Individual changes in HR (ΔHR) were directly related to changes in MSNA (ΔMSNA) during 45ºHUT in both hydration conditions (EUH: R=0.92; DEH: R=0.92), suggesting similar responsiveness in both arms of the baroreflex. The MSNA ‐ HR relationships were weaker at 30º HUT in both groups. During DEH, ΔMSNA and ΔBP were positively related(30º HUT: R = 0.57; 45º HUT: R = 0.88); this was not seen in EUH. Our data suggest that, during exercise induced dehydration, there is an augmented contribution of the sympathetic nervous system to BP regulation.

Support or Funding Information

Funded by USAMRMC; author views not official US Army or DOD policy.
Comparison of Growth Supplements on the Differentiation of Adipose Derived Stem Cells from Burned and Non‐Burned Human Skin
Authors: Mangum Lauren Heard , Wrice Nicole , Larson David , Natesan Shan , Christy Robert
Published: June 2021

Severe traumatic injuries, such as large total body surface area burns, limit the availability of autologous cells for wound repair. Previous experiments from our lab have demonstrated that adipose derived stem cells (ASCs) from burned debrided skin are viable, display an immunophenotype indicative of stemness, and are capable of tri‐lineage differentiation. Traditional tissue culture utilizes undefined, animal‐derived components that present numerous complications for the utilization of ASCs in human medicine. Accordingly, there is a need for defined, xeno‐free cell culture media. Human platelet lysate (hPL) is one potential media supplement for the replacement of fetal bovine serum in stem cell culture. In this study, burned human ASCs (BH‐ASCs) and ASCs derived from abdominoplasty (HAP‐ASCs) were compared in regard to the ability to proliferate and differentiate in media supplemented with traditional 10% FBS, 10% hPL, or grown in MesenPRO RS™. ASCs were obtained from the stromal vascular fractions of subcutaneous adipose tissue from burned and non‐burned skin. Cells were cultured in DMEM with 10% FBS or 10% hPL, or in MesenPRO RS™. Cells were induced to differentiate towards an adipogenic or osteogenic lineage at an early passage utilizing traditional differentiation media supplemented with FBS or 10% hPL. DMEM or αMEM was used for adipogenic and osteogenic lineages, respectively, with the addition of standard induction agents. Adipogenic differentiation was assessed after 14 days by Oil red O staining, triglyceride content, and expression of markers of adipogenesis, including PPARγ, leptin, and adiponectin. Osteogenic differentiation was assessed by alizarin red S staining and expression of markers of ostogenesis, including BMP‐2 and RUNX2. Results indicate that adipogenic and osteogenic differentiation appears to be influenced by both the ASC source (burned vs. non‐burned skin) and by the supplement in which they are grown and differentiated. Use of MesenPRO RS™ and supplementation with hPL appears to enhance differentiation towards both lineages over standard FBS. Prior experiments in our labs have indicated that both burn and non‐burn ACSs are capable of trilineage differentiation, however there is a need to characterize these cells, particularly the BH‐ASCs in xeno‐free media, with regard to their stemness and immunosuppresive capabilites if they are to be utilized in any capacity for regenerative medicine. Ongoing research efforts in our laboratory seek to address these issues to better define the utility of BH‐ASCs as a burn wound therapeutic.

Support or Funding Information

This work was possible due to the generous funding from the US Army Medical Research and Material Command and ORISE
Transcriptomic Analysis of the Right and Left Ventricle in Normoxia and Hypoxia (a Model of Pulmonary Arterial Hypertension)
Authors: Gorr Matthew W , Sriram Krishna , Insel Paul A
Published: June 2021

The pathobiology of pulmonary arterial hypertension (PAH) involves the remodeling of the pulmonary artery within the lung characterized by vasoconstriction, hypertrophy, fibrosis and thrombosis. These factors lead to increased pulmonary vascular resistance (PVR), which, in turn, can produce progressive remodeling of the right ventricle (RV) and eventual right heart failure. Current therapy for PAH emphasizes approaches that decrease PVR, but does not work for some patients and does not specifically target the RV. This study tested the hypothesis that the RV contains a unique subset of genes expressed during remodeling that may serve as a platform to identify and develop novel treatment strategies. To test this hypothesis, we subjected Sprague Dawley rats to hypoxia (10% O2, H‐rats) for two weeks, or to normoxia (N‐rats) as a control (n=3–4/treatment). H‐rats had a significant, 2.19‐fold increase in the Fulton Index (right/[left + septum] ventricular weight): 0.458 ± 0.036 (H‐rats), 0.210 ± 0.020 (N‐rats); mean ± standard deviation, Student's t‐test p<0.0001), indicating significant RV remodeling. To examine mRNA expression, we utilized RNA sequencing (RNA‐seq) of the RV and the left ventricle (LV). Using differential expression analysis, we identified 2,430 genes with significantly (False discovery rate [FDR] <0.1 using multiple testing) altered expression in the RV of H‐rats vs. N‐rats and 504 genes with significantly altered expression in the LV between the two groups of rats. Differentially expressed genes in the RV of H‐rats vs. N‐rats included GPCRs and other widely used drug targets. In normoxia, the RV and LV differentially expressed 299 genes compared to 423 genes under hypoxic conditions. These results indicate that: 1) a subset of genes are differentially expressed in the RV compared to the LV and 2) a large change in the transcriptome occurs with hypoxia‐induced RV remodeling. These findings raise the possibility that treatments for PAH might be directed at gene products selectively expressed in RV remodeling and be able to spare the LV, thus providing a novel approach for treating a currently unmet need for patients with RV hypertrophy and failure.

Support or Funding Information

Supported by Department of Defense (W81XWH‐14‐1‐0372) and the National Institutes of Health (5T32HL007444‐33)
Deficiencies in RNS2‐mediated ribosomal RNA turnover cause changes in the Pentose Phosphate Pathway flux and alter cell growth in Arabidopsis
Authors: MacIntosh Gustavo C , Morriss Stephanie , Liu Xiaoyi , Bassham Diane
Published: June 2021

Enzymes belonging to the RNase T2 family are essential for normal ribosomal RNA turnover in eukaryotes. In Arabidopsis thaliana this function is performed by RNS2. The null mutant rns2‐2 has increased rRNA half‐life and constitutive autophagy. To determine the effect of defective rRNA turnover on cellular homeostasis, we used comparative transcriptome and metabolome analyses of 10 day old wild‐type and rns2‐2 seedlings to identify molecular processes affected in the mutant. Bioinformatics analyses suggested additional phenotypes that were confirmed through direct plant size measurements and microscopy.

Few genes were differentially expressed in the rns2‐2 mutant, indicating that control of autophagy in this genotype is mainly achieved at the posttranscriptional level. Among differentially expressed genes we identified transcripts related to carbon flux processes, particularly the pentose phosphate pathway, and metabolite analyses confirmed changes in the levels of PPP intermediates. We also found differentially expressed genes related to cell wall loosening and found a decrease in monosaccharide components of cell wall hemicellulose. As a potential effect of weaker cell walls, rns2‐2 plants are larger than wild‐type controls, due to larger cells and increased water content. We propose that lack of rRNA recycling in rns2‐2 cells triggers a change in carbon flux, which is redirected through the pentose phosphate pathway to produce ribose‐5‐P for de novo nucleoside synthesis. rRNA or ribosome turnover is thus essential to maintain nucleoside homeostasis in Arabidopsis.

Support or Funding Information

This work was supported by a grant from the United States National Science Foundation [MCB‐1051818] to G.C.M. and D.C.B.
3D Integrated Tissue and Organ Printing System to Produce Human Body Parts with Structural Integrity
Authors: Lee Sang Jin
Published: June 2021

Tissue engineering and regenerative medicine strategies could offer new hope for patients with serious tissue injuries or end‐stage organ failure. Scientists are now applying the principles of cell transplantation, material science, and engineering to create biological substitutes that can restore and maintain normal function in diseased or injured tissues/organs. Among various three‐dimensional (3D) biofabrication technologies developed for the field of tissue engineering, 3D printing technology is one of the most attractive and powerful methods for use in constructing a structure that mimics native tissues, and thus many researchers are focusing on development of new printing technologies and applications. The hypothesis driving development of 3D bioprinting is that by precisely placing cells in relation to each other, an environment that encourages physiologically relevant cues both to and from these cells can be created, resulting in a tissue construct with functionality. Thus, we have developed 3D integrated tissue and organ printing (ITOP) system to allow construction of biological substitutes that mimic the structure and function of native tissues or organs. This system makes it possible to precisely place various cell types and biomaterials in a single 3D architecture. Our proof of concept experiments demonstrates the capabilities of our ITOP by fabricating structures of various tissue constructs of appropriate shape and size needed for clinical use. This novel organ printing system provides a leap forward in our ability to fabricate tissue constructs that could be used in translational applications.

Support or Funding Information

This work was supported, in part, by grants from the Armed Forces Institute of Regenerative Medicine (W81XWH‐08‐2‐0032), the Telemedicine and Advanced Technology Research Center at the US Army Medical Research and Material Command (W81XWH‐07‐1‐0718) and the Defense Threat Reduction Agency (N66001‐13‐C‐2027).
Characterizing the Role of Yeast Cyclin Pcl1 in the Establishment of Pheromone Receptor Polarity
Authors: Pai Chih Yu (Chloe) , Sukumar Madhushalini , Stone David
Published: June 2021

Chemotaxis (directed cell movement) and chemotropism (directed cell growth) are vital mechanisms essential to a variety of biological processes, including cell development, cancer metastasis, angiogenesis, and axon guidance. Both chemotaxis and chemotropism depend on the ability of the cell to interpret shallow and complex chemical gradients. One of the best‐studied models of eukaryotic directional sensing is the mating response of Saccharomyces cerevisiae (budding yeast). Yeast exist as two haploid mating types, MATa and MATα, that can sense pheromone gradients, chemotrop toward the closest mating partner, and eventually fuse at their tips to form diploid cells. In vegetative cells, the G protein coupled receptor (GPCR) that binds pheromone and its associated heterotrimeric G protein are uniformly distributed on the plasma membrane. Upon receptor activation, Gβγ is released from Gα and Gβ is rapidly phosphorylated on multiple residues. Free Gβγ signals through a MAP kinase cascade to trigger cell‐cycle arrest and changes in gene expression. Pheromone‐activated receptors are globally internalized and then reappear as polarized crescents that mark the chemotropic growth site, where the cell ultimately forms its mating projection. Previous studies have shown that the receptor crescents are visible before detectable actin polarization and that the receptors can polarize without actin‐dependent directed secretion [1]. On the contrary, inhibiting receptor internalization confers a moderate defect in gradient sensing [2]. A key question raised by these observations is how does the yeast cell interpret the external signals and establish the chemotropic growth site? From a directed genetic screen, we found that Pcl1 is critical for the establishment of receptor polarity. Pcl1 is a cyclin required by the cyclin dependent kinase Pho85, which has been implicated in polarization during budding. It is known that Gβ phosphorylation is crucial to receptor polarization, and Gβ is predicted to be a substrate of Pho85‐Pcl1 [3]. Additionally, our data indicated a genetic interaction between Gβ and Pcl1. Here, we show that Pcl1 localizes to the mating projection during the mating response. Moreover, Pcl1 and Gβ interacted directly on the plasma membrane of pheromone‐treated cells, as indicated by a Bimolecular fluorescence complementation assay. Lastly, Pho85 inactivation also appears to affect receptor polarity. Taken together, these results suggest that Pho85‐Pcl1 regulates polarization of the receptor by phosphorylating Gβ.

Support or Funding Information

National Science Foundation
Gulliver Prototype Development and Deployment
Authors: Williams Claire , Berdy Brittany , Epstein Slava
Published: June 2021

The current objective of our project is to develop a technology, coined Gulliver, aimed at enhancing the study of microorganisms. This device will expand upon established in situ technology to cultivate and isolate microorganisms in one step. Our long term goal is for this device is to not only isolate and culture in one step, but also to eventually equip our device with nanosensors to allow in situ monitoring of bacterial growth and activity, and transmit this data directly to the researcher, allowing an unprecedented view into the microbial world. The Gulliver design consists of a flat mounting device with a 4 mm hole in the center to act as the growth chamber. On one side of the device, a polycarbonate membrane with 0.03μm pores is attached, allowing for diffusion of nutrients and growth signals from the environment. On the reverse side, the same membrane is affixed, however it contains an approximately 1 μm entry aperture, to allow entry of a single microbial cell into the growth chamber. Once the microorganism enters the pore, the entrance is blocked. As the organism continues to grow and divide, it is provided with nutrients from the environment through the diffusion pores, and a pure colony will develop inside the device. After a period of in situ incubation, the device is removed from the environment, the growing biomass retrieved from the growth chamber, “domesticated” and grown on a petri dish for further study. Currently, testing is underway using fluorescently labeled bacterial species to prove the concept of autonomous cultivation and isolation in on step. Devices will be incubated in mixed cultures of fluorescently labeled bacteria, and growth will be collected from each chamber. Fluorescence will be used to ensure a pure culture is obtained. Thus far, we have demonstrated an ability to reliably produce 1 μm pores in polycarbonate membrane. Furthermore, we have designed a prototype of the device consisting of the polycarbonate membrane adhered to a silicone or PTFE base. The prototype is capable of withstanding incubation in an aqueous environment, without material degradation, for up to 3 months, providing an extended period of time for environmental study of microorganisms. The overall goal of this project is to gain a fuller picture of the microbial world, allowing researchers to access novel microorganisms, study the important environmental processes they perform, and utilize the valuable bioactive compounds they produce.

Support or Funding Information

This particular segment of the project is supported by the NSF, the Alpha Fund Grant, and an Northeastern Undergraduate Research and Creative Endeavors Award.
PIP4Kγ as a potential target for Huntington's disease
Authors: Giridharan Sai Srinivas Panapakkam , Al‐Ramahi Ismael , Hasegawa Junya , Safren Nathanial , Patnaik Samarjit , Gee Amanda , Titus Steve , Zheng Wei , Ferrer Marc , Southall Noel , Barmada Sami , Botas Juan , Marugan Juan , Weisman Lois S
Published: June 2021

Phosphoinositide lipids provide spatial and temporal regulation of cell signaling and membrane traffic. The PI5P‐4‐kinase family is one of the less studied phosphoinositide lipid kinases. These enzymes catalyze the conversion of phosphatidylinositol 5‐phosphate to phosphatidylinositol 4,5‐bis phosphate. A recent study by Vicinanza et al., a suggested that depletion of the PI5P‐4‐kinase, PIP4Kγ promotes a non‐canonical autophagy pathway. Moreover, this depletion reduced the presence of huntingtin‐exon1‐polyglutamine aggregates. Expression of exon1 of huntingtin with an extended polyglutamine tract is a model for huntingtin protein aggregates. To further test the hypothesis that PIP4Kγ is a potential target to reduce huntingtin aggregates, we tested the depletion of PIP4Kγ in cell culture models that express full length mutant huntingtin (mHtt). We determined that depletion of PIP4Kγ, but not PIP4Kα or PIP4Kβ, lowers the levels of mHtt in patient fibroblasts and in immortalized HdhQ111 mouse striatal neurons. We show that PIP4Kγ‐dependent depletion of mHtt requires lysosomal function because the depletion is blocked by the lysosomal inhibitor, bafilomycin A1. Moreover, we show that depletion of PIP4Kγ increases autophagic flux, as measured by an increase in LC3‐II levels by western blot analysis and an increase in LC3‐II puncta by microscopy. Our studies suggest that PIP4kγ should be further pursued as a target for Huntington's disease.

Support or Funding Information

This work was supported by AHA postdoctoral fellowship 14POST20480137 (Sai Srinivas Panapakkam Giridharan), NIH grants R01GM050403 and NS064015 (Lois S Weisman) and by HSS funding 1R03MH084839 (Juan Marugan)
Validation of the Dietary Screening Tool in a Middle‐Aged Appalachian Population
Authors: Marra Melissa Ventura , Thuppal Sowmyanarayanan V , Bailey Regan L , Johnson Elizabeth
Published: June 2021

The Dietary Screening Tool (DST) was developed and validated to assess nutritional risk in older adults. The purpose of this study was to assess its use in a middle‐aged sample of adults (45–64 years) in Appalachia (n=87) and evaluate its validity with multiple 24‐hour dietary recalls and selected biomarkers. Adults identified by the DST as at nutrition risk (<60 of 100 points) were significantly more likely to have had lower overall diet quality as assessed by the Healthy Eating Index (64 vs 50, p<0.001) when compared to those not at risk or potential risk. Dietary intakes of vitamin A, vitamin C, vitamin B2, vitamin B6, niacin, vitamin K and potassium were also lower among those considered at risk, despite having significantly higher energy intakes (1653 ± 106 vs 2032 ± 76 kcal/day, p=0.004). The diets of those at risk were higher in added sugars (33 g vs 23 g, P=0.005) and lower in dietary fiber (10 g vs 12 g, p<0.001) than those not at risk. The at risk group had significantly lower serum levels of 13‐cis‐β carotene (40 vs 60 nmol/L, p<0.02), trans‐ β‐carotene (395 vs 711 nmol/L, p<0.001), β‐Cryptoxanthin (137 vs 198 nmol/L, p<0.01), cis‐Lutein (90 vs 137 nmol/L, p<0.02), and trans‐lutein (292 vs 424 nmol/L, p=0.01). The DST was able to identify middle‐aged adults at nutritional risk.

Support or Funding Information

USDA Northeast Regional Project NE1439, WVU Health Disparities Mountains of Excellence Flash Funding and the WVU ADVANCE Sponsorship Program.
Developmental Changes in Resting Gamma Power from Age Three Months to Five Years Are Modulated by Infant Diet
Authors: Pivik R T , Andres A , Cleves M A , Tennal K B , Gu Y , Badger T M
Published: June 2021

Gamma band activity (30–50 Hz) is a significant EEG component related to intelligence, memory and language processes, but there is limited information regarding the early development of this activity and none considering how infant diet may influence this development. The present study examined changes in gamma power from 3 months through 5 years in 497 healthy children who were breast‐fed (BF: 168, 84 boys), or fed milk formula (MF: 169, 89 boys), or soy formula (SF: 160, 90 boys) as infants. Recordings of resting EEG (5 min; eyes open) were obtained quarterly during infancy and then yearly. We used ANOVAs adjusted for relevant growth and environmental factors (gender, head circumference, gestation, birth and visit weight, SES and mother's IQ) to compare group, age, and group by age changes in gamma EEG power from 44 homologous sites distributed over frontal, central, temporal, parietal and occipital scalp regions. Across sites and groups, gamma power was lowest at 3 months (all p < .001), and across age gamma power was consistently higher at frontal than other sites (all p < .05). From 3 to 6 months gamma power increased dramatically at all sites for all groups [all p < .001; mean increase >100% for all sites, greatest for central‐parietal regions (187% mean increase across groups)]. After 6 months, gamma power generally increased across sites until a time after which significant (p < .05) decreases became evident relative to preceding peak levels. The ages after which decreases became evident varied within and across brain regions, with site‐specific time points of change ranging from 1 to 4 years and occurring later (after 3–4 years) in anterior frontal than other regions. Temporal sites differed from others in showing sharp decreases both early (after 9 months or 1 year) and later (after 3 years). Group variations in site‐related time points of change were most evident for frontal and occipital regions. Infant diet‐related effects were evident at 3 months [BF < MF, SF (p ≤ .001) and were generally sustained across age‐‐ particularly at parietal‐occipital sites where BF < MF differences (p < .05) were most prominent. These findings provide new information regarding the regional development of EEG gamma activity from infancy to the onset of middle childhood, and show that this development is modified by infant diet. Previous reports of negative associations between resting gamma and measures of language skills and intelligence suggest potential developmental advantages for breastfed infants.

Support or Funding Information

Funded by USDA‐ARS Project 6026‐51000‐010‐05S
Construction of Comprehensive Flavonoid Analysis Tool by Using UV‐vis Spectra Library, In‐house Database, and Chemometrics
Authors: Zhang Mengliang , Sun Jianghao , Harnly James , Betz Joseph M. , Chen Pei
Published: June 2021

Liquid Chromatography and mass spectrometry methods, especially ultra‐high performance liquid chromatography coupled with high resolution accurate mass‐mass spectrometry (UHPLC‐HRAM‐MS), have become the best methods for flavonoid identification and quantification. However, processing acquired UHPLC‐HRAM‐MS data for flavonoid analysis is very challenging and highly expertise‐dependent because of the complexity of the physical and chemical properties of the flavonoids. An expert data analysis program, FlavonQ, has been developed to facilitate this process. The program first categorizes the flavonoids using a chemometric model based on the UV‐Vis spectra library compiled for 146 flavonoid reference standards. A novel stepwise classification strategy is used that provides data representation in each step as optimized by a projected distance resolution (PDR) method. The stepwise classification strategy significantly improves the performance of the classifiers which results in more accurate and reliable classification results. An in‐house flavonoid database which contains 5686 previously reported flavonoids is used for identification of flavonoids. FlavonQ was validated by analyzing data from samples with spiked flavonoid mixed standards and plant samples including blueberry, mizuna, purple mustard, red cabbage, and red mustard green extract. Accuracies for identification for all samples were above 88%. FlavonQ greatly facilitates the identification and quantitation of flavonoids from UHPLC‐HRAM‐MS data. The process is automated, saving tremendous resources, and allowing less‐experienced people to perform data analysis on flavonoids with reasonable results.

Support or Funding Information

This research is supported by the Agricultural Research Service of the U.S. Department of Agriculture, an Interagency Agreement with the Office of Dietary Supplements at the National Institutes of Health (Y01 OD001298‐01). The John A. Milner Fellowship program by USDA Beltsville Human Nutrition Research Center and the NIH Office of Dietary Supplements is acknowledged for the support to Dr. Mengliang Zhang. We thank to Dr. Peter de B. Harrington for providing his codes on chemometric models.