bluuebunny/crossref_metadata_2025 huggingface dataset:
Germ cell nuclear factor (Gcnf/Nr6a1) plays a novel role in neural crest cell induction
Authors: Achilleos Annita , Dennis Jennie , Bhatt Shachi , Sakai Daisuke , Trainor Paul
Published: June 2021

The neural crest (NC) is a multipotent, migratory cell population that is a hallmark of vertebrate evolution and gives rise to a diverse array of tissues including peripheral nervous system and craniofacial skeleton. Formation of the NC encompasses several steps including induction and specification, epithelial to mesenchymal transition (EMT), migration and differentiation into diverse cell types. Previous work in aquatic and avian systems revealed that Wnt, BMP and FGF signaling pathways drive NC formation. However, knockout mouse models failed to recapitulate a role for these pathways in mammalian NC induction. Our approach uses a mouse mutant in germ cell nuclear factor (Gcnf)/Nr6a1, to study mammalian NC formation. Our analysis of Gcnf−/− embryos revealed a total absence of NC posterior to the first branchial arch and a great anterior reduction in cranial NC. Both phenotypes result from a failure of neural progenitors to differentiate into NC and undergo EMT. Along with our global gene expression and protein interaction analysis, we show that Gcnf regulates two key processes during NC formation: stem cell maintenance and EMT. Therefore, we hypothesize that Gcnf acts as a bimodal switch (i) repressing pluripotency genes and activating NC‐specific genes and (ii) regulating EMT during NC delamination. This is the first example of a total loss of NC in a specific domain of the neuraxis in a mammalian system.

Grant Funding Source: AAA Postdoctoral Fellowship
A simple PCR‐based strategy for estimating species‐specific contributions in chimeras and xenografts
Authors: Ealba Erin Lynn , Schneider Richard Allen
Published: June 2021

Various tissue‐engineering strategies for repair and regeneration involve using cells from different species in chimeras or xenografts. Yet a major challenge is distinguishing between donor‐versus host‐effects on gene expression. This study provides a simple molecular strategy to quantify species‐specific contributions in chimeras and xenografts using species‐specific primers for ribosomal protein L19 (RPL19). cDNA from quail, duck, chicken, mouse, and human was mixed in pairs as a dilution series, species‐specific RPL19 expression was measured, a linear regression was calculated, and the ratios of donor and host cells were estimated. To test the accuracy of the approach, quail cells were transplanted into transgenic‐GFP chick and resulting chimeras were analyzed with species‐specific primers. Fluorescent‐activated cell sorting validated the RPL19 strategy. To apply the RPL19 strategy, Runx2 expression was measured in quail‐duck chimeras. Elevated Runx2 levels correlated with higher percentages of donor cells. Finally, RPL19 primers were also shown to discriminate human‐mouse samples. Thus, this study offers a straightforward and universal strategy that enables chimeras and xenografts to be screened efficiently and rapidly prior to obtaining critical information at the molecular level. Supported by K08 DE021705 and T32 DE007306 from NIDCR to ELE.; R01 DE016402 from the NIDCR to RAS.

Grant Funding Source: NIDCR
Effect of high copy retrotransposon insertions on yeast genome stability
Authors: Lanzillotta Cristina , Scheifele Lisa Z
Published: June 2021

Retrotransposons are dispersed sequences that are able to copy themselves and reinsert throughout the genome. These mobile elements display preferences for the positions in the genome at which they insert and over 90% of retrotransposon insertions in Saccharomyces cerevisiae are within 2 kb upstream of tRNA genes. The yeast genome is comprised of only 3% retrotransposons as compared with 44% in humans; to use the yeast genome as a model to study insertion preferences in retrotransposon‐dense genomes, Retrotransposon Overdose (RO) strains were made by inserting a large number of Ty1 retrotransposons within the yeast genome. Our first goal was to analyze the genome sequences of these strains to determine the sites of Ty1 insertion. Analysis of the sequenced genomes revealed that as the number of retrotransposons increased, the number of insertions into less preferred target sites increased, including a significant number of insertions within essential genes, which may decrease either the quantity or size of transcription products. Our second goal was to analyze the targeting of Ty1 retrotransposons once all preferred sites in the genome were saturated. We have found that pairs of retrotransposons are frequently formed. Retrotransposons that are adjacent to each other in the genome are hypothesized to form fragile sites, which are sites of chromosome breakage that are associated with tumor formation in humans. RO strains contain numerous pairs of retrotransposons as well as higher order Ty1 multimers, despite the genome instability that might result from these retrotransposon arrangements.
Function of CDK8 Submodule and TRIM28 in Transcriptional Regulation at HSPA1B
Authors: Bunch Heeyoun , Calderwood Stuart
Published: June 2021

Recently, it has been reported that a majority of induced or developmental genes possesses Pol II paused at the promoter proximal site about 50–100 bp from the transcription start site (TSS). HSP70 has been well characterized for the promoter proximal pausing before induction that releases the paused Pol II for processive elongation.

In order to understand the promoter proximal pausing of Pol II in HSP70 transcriptional regulation, we investigated the function of CDK8 submodule (CDK8, Med12, Med13, and Cyclin C) at HSPA1B using an in vitro transcription system. The results show that CDK8 submodule activates transcription at HSPA1B and that Pol II is stably paused at around +70 at HSPA1B. The activating function of CDK8 submodule is dependent on the timing of its availability to the transcription machinery. In spite of their function as activators, CDK8 submodule and P‐TEFb are unable to relieve Pol II from the pausing site as individual. We also screened a transcription factor(s) that possibly regulates the Pol II promoter proximal pause via oligo pull‐down assays followed by mass spectrometry (MS). TRIM28 was identified to bind near the Pol II pausing site of the HSPA1B non‐template DNA. In vivo knock‐down of TRIM28 increases HSPA1B mRNA expression under the unstressed condition. TRIM28 depletion also alters the expression profile of other known pausing genes, proposing the function of TRIM28 in the Pol II promoter proximal pausing.
Regulation of CFTR gene expression by recruitment of an intestinal transcription factor network.
Authors: Kerschner Jenny , Harris Ann
Published: June 2021

We recently identified members of a transcription factor (TF) network that interact with a critical cis‐regulatory element for the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This element, located in intron 11 of the gene, 100 kb distal to the promoter with which it interacts, contains an intestine‐selective enhancer, and associates with enhancer signature proteins and tissue‐specific TFs important for regulating CFTR expression. Chromatin immunoprecipitation identified FOXA1, FOXA2, HNF1, and CDX2 as trans‐interacting factors at this and other CFTR cis‐regulatory elements. Moreover, siRNA‐mediated knockdown of CDX2 or FOXA1/A2 causes a significant reduction in endogenous CFTR transcription in intestinal cells, suggesting that these factors are important for maintaining high levels of CFTR expression in these cells. More recently we showed that local changes in chromatin structure and nucleosome positioning are triggered by the interaction of members of the TF network. Our data suggest this group of TFs may play a more global role in regulating intestinal expression of the gene. The mechanism by which they regulate CFTR expression and influence chromatin environment is under analysis.

Funding: Cystic Fibrosis Foundation; National Institutes of Health (R01HL094585 and R01HD68901); and Northwestern University Cellular and Molecular Basis of Disease Training Grant
Replication and transcription of SV40 direct distinct epigenetic signaling
Authors: Milavetz Barry , Kallestad Les , Gefroh Amanda , Woods Emily , Christensen Kendra
Published: June 2021

We have characterized how transcriptional regulatory sequences direct histone modifications during replication and transcription of Simian Virus 40. The role of DNA replication in directing histone modification was determined by analyzing the introduction of histone modifications (H3K4me1,me2,me3 and H3K9me1,me2,me3) from 24 hours to 48 hours post‐infection and following aphidicolin inhibition of DNA replication. Notably, only the introduction of H3K9me1 and H3K4me1 appeared to be totally dependent upon DNA replication. Since the repressive regulatory sequence, Site I, results in the introduction of H3K9me1 during replication, we determined its effect on transcriptional repression early in infection. Consistent with repression, we observed a reduction in SV40 minichromosomes containing RNA polymerase II from 2 hours to 8 hours post‐infection. Unexpectedly, we also observed a reduction in the presence of H3K9me1, H3K9me2, and H3K9me3 indicating that these modifications were not being introduced along with repression. We also analyzed SV40 recombinants containing either one or two copies of Site I and confirmed that the introduction of H3K9me1 was only associated with Site I during replication. Taken together these results indicate that transcriptional regulatory sequences can alter the epigenetic profile of a gene depending upon whether they function during replication or transcription.
Novel method for the scarless single‐step assembly of many small DNA sequences
Authors: Lee Roth Theodore , Milenkovic Ljiljana , Scott Matthew
Published: June 2021

Techniques in the assembly of DNA fragments have expanded rapidly, enabling complex and efficient assemblies that traditional restriction digest based assembly cannot accomplish. However, the scarless assembly of multiple small (<1kb) DNA fragments remains difficult and time consuming, especially a problem as most genetically modified proteins are constructed on this scale. Here we present a novel method using a cycled ligation reaction to assembly many small DNA fragments into a transformation ready plasmid. First, the reaction is heated to a denaturing temperature. Disposable Oligonuleotide Connectors (DOCs), oligonulceotides ~40 bp in length that are homologous to the end of one insert sequence and the beginning of the next then act as a guide, bringing the end and beginning of two insert strands together when the reaction is decreased to an annealing temperature. This allows for a ligase to ligate the two insert strands together, in a sequence exactly determined by the user with no scar sequences. The reaction is then cycled between denaturing and annealing/ligating temperatures, allowing the ligated products of early cycle to serve as a template for further ligations in later cycles. We have successfully assembled as many as 10 ~150 bp inserts into a transformable plasmid in a single reaction. DOC‐based Assembly represents a powerful new tool for the construction of complex genetic constructs.
Modulation of Signaling Proteins by Reversible Cysteine Modification
Authors: Keyes Jeremiah David , Nelson Kimberly , Parsonage Derek , Daniel Larry , Furdui Cristina , Poole Leslie
Published: June 2021

In order to elicit an appropriate response toward a stimulus, cells have developed intricate systems to control signaling pathways. These pathways are often centered on kinase cascades, which are modulated by 2nd messengers and protein‐protein interactions. Such modulation is necessary for the spatial‐temporal control of kinase signal exertion. It is through these mechanisms that a signal can induce a specific response through a core kinase cascade that is shared by other signals to elicit different responses. Recently recognized 2nd messengers are Reactive Oxygen Species (ROS) such as H2O2. These ROS oxidize cysteine residues of proteins, which has the potential to modify the surface and configuration of a protein, thereby affecting interactions with small molecules, proteins, lipids, or nucleotides. Our collaborative team recently identified that ROS are necessary for the survival and proliferative response of ovarian and prostate cancer cells to lysophosphatidic acid (LPA), a signaling lipid found in the ascites fluid of ovarian and prostate tumors. Using techniques developed by our collaborative team, I have found that key kinases and phosphatases are oxidized during LPA signaling. Additionally, I report signaling proteins that are reversibly inhibited by oxidation towards specific substrates as a result of LPA signaling.
Mitophagy as a quality control mechanism in Saccharomyces cerevisiae
Authors: Abeliovich Hagai , Zarei Mostafa , Rigbolt Kristoffer T.G. , Youle Richard J. , Dengjel Joern
Published: June 2021

The clearance of malfunctioning mitochondria is an important housekeeping function in respiring eukaryotic cells, and plays a role in physiological homeostasis as well as in the progression of maternally inherited late‐onset diseases and aging phenomena. Clearance of entire mitochondrial comartments is thought to occur using the endo‐lysosomal system, by a specific form of autophagic degradation called mitophagy. In standard autophagy assays, such as starvation‐induced macroautophagy, no mitophagy is observed in S. cerevisiae even at after very long periods of time. We identified conditions where, in the absence of external insult, massive mitophagy occurs in stationary phase cells. Our data suggest an active role for mitochondrial dynamics in mediating mitophagy, as opposed to a facilitating role in generating small, “eatable” mitochondrial fragments. This is consistent with the potential existence of a distillation process that utilizes mitochondrial fission and fusion to segregate defective mitochondrial components from functional components. To better understand intra‐mitochondrial segregation events during mitophagy, we carried out a SILAC‐ based proteomic screen, searching for proteins which behave differently in mutants relative to WT cells. The results allow us to test and formulate new hypotheses regarding intra‐mitochondrial protein sorting events that occur during mitophagy.
Unraveling the architecture of the A1 complex of the Nanoarchaeum equitans A1A0 ATP synthase
Authors: Mohanty Soumya , Hogue Christopher Warren Victor
Published: June 2021

ATP synthases are multimeric motors which are indispensible for cellular energetics. The archaeal ATP synthases are the oldest but are relatively understudied. Nanoarchaeum equitans is a nanosized parasitic hyperthermophile with several ancestral traits. Genomic annotation shows that the typical archaeal ATPase subunits are missing, suggesting that the N.equitans ATP synthase is either compromised or constructed partially. The annotated regulatory B subunit is shortened compared to the B subunits of other archaeal ATP synthases, which led to the speculation whether the A and B subunits are capable of forming a functional complex. This study aims at understanding the architecture and functionality of the catalytic A1 complex. The recombinant N. equitans ATPase subunits were purified, reconstituted in‐vitro and characterized using dynamic light scattering, size exclusion chromatography and Blue Native PAGE and isothermal titration calorimetry. Results show the stoichiometric association of A and the annotated smaller B subunits in the presence of nucleotides to form higher order complexes. Therefore our results suggest that the N.equitans ATP synthase might be a minimalistic, yet functional form which would provide a vital connection to the understanding of the evolution of ATP synthases.

Supported by Mechanobiology Institute, National University of Singapore (Research Grant R714–009‐007–271)
Ceramides increase mitochondrial ROS generation via altered mitochondrial dynamics in skeletal muscle
Authors: Tucker Braden J , Smith Melissa E , Bikman Benjamin T
Published: June 2021

The role of reactive oxygen species (ROS) as mediators of chronic diseases is increasingly well established. Ceramides are known to induce mitochondrial ROS production, which likely explains some of the deleterious effects of ceramide accumulation. We propose that ceramide accrual in skeletal muscle induces mitochondrial fission, which is requisite for ceramide‐induced ROS generation. We found that exogenous ceramide treatment and endogenous ceramide biosynthesis both increased mitochondrial fission and increased expression of dynamin‐related protein 1 (Drp1), which is a prime mediator of mitochondrial fission. Moreover, ceramide accrual increased H2O2 production in muscle cells. Inhibition of serine palmitoyltransferase (SPT) via pharmacological (myriocin) and genetic (SPT2 siRNA) inhibition prevented increased ROS production in palmitate‐treated cells. Also, lack of Drp1 (siRNA) prevented mitochondrial fission with ceramide treatment and completely prevented ROS generation. Altogether, these findings suggest ceramide‐induced mitochondrial fission is an important mediator of the observed ROS generation in the presence of ceramide‐rich cells. Given the multiple chronic diseases that are affected by ceramide metabolism, such as insulin resistance, these findings may lead to improved and novel therapies. This work was supported by a BYU Undergraduate Research Award (BJT) and a BYU Mentoring Environment Grant (BTB).
Weight loss and role of fat free mass in obese patients
Authors: Ippoliti Flora , Canitano Nicoletta , Businaro Rita
Published: June 2021

Study objective

We evaluated body composition analysis, to monitor obese patients in the early stages of immune‐metabolic dysregulation. The expanded activated adipocytes induce a low‐grade chronic inflammation involving an altered body composition of Fat Free Mass (FFM),mostly muscle and bone tissue leading to the onset of Medically Unexplained Symptoms (MUS).

Materials and methods

We enrolled 109 patients 79 females and 30 males all free of disease but with MUS and problems of excess weight.

We obtained parameters known as prognostic indicators for inflammatory processes by using BIA‐ACC bioimpedance device (Biotekna‐Italy), performed at the first medical examination, (time T0), and after 6 weeks of proteic, low‐caloric diet (1200 calories).

Results

All patients decreased the BMI but 3 subgroups showed: a)75.23% loss of Fat Mass but also FFM b)17.43% loss of FM with preservation or even increase FFM c)7.34% loss of FFM alone. The main components with a cumulative variance of 94% were only 3 which shows that the analyzed data are statistically highly consistent. The 1st principal component is the FFM correlated closely with BMR, TBW and TBCa, to a lesser extent with Gly, TBK and TBprotein.

The 2nd component is AT, FM, AAT with particular relevance to the visceral fat.

The 3rd component is the stress with inflammation.

Conclusion

The number of parameters provided by the analysis of body composition after appropriate low‐calorie diet, have allowed us to evaluate, also in the presence of a decrease in BMI, the real changes in FM and FFM in relation to all the other variables and the essential role of the preservation of FFM during weight loss.
Significant weight gain in mice exposed to the pollutant benzo[a]pyrene is associated with modifications in hepatic lipoprotein receptors and lipid status
Authors: Yen Frances T , Layeghkhavidaki Hamed , Lanhers Marie‐Claire , Akbar Samina , Grova Nathalie , Appenzeller Brice , Feidt Cyril , Corbier Catherine
Published: June 2021

Although recent studies have demonstrated a link between environmental contaminants exposure and obesity, the molecular mechanisms remain to be determined. Reduced expression of the hepatic lipolysis stimulated lipoprotein receptor (LSR), which is important for chylomicron clearance, is associated with increased body fat mass in mice. Since the polycylic aromatic hydrocarbon benzo[a]pyrene (B[a]P) is associated with weight gain in mice, the objective of this study was to determine if B[a]P modified hepatic LSR and lipid status. In 11‐wk old male C57BL/6RJ mice treated with B[a]P (0.5 mg/kg, i.p. every 48h, 15 days), the increased weight gain was associated with increased plasma triglycerides and cholesterol, and increased liver cholesterol content as compared to control mice. LDL‐receptor protein was decreased, and the positive correlation between hepatic LSR and LDL‐receptor protein in control animals was absent in the B[a]P‐treated mice, pointing towards a dysregulation of hepatic lipid homeostasis in the mice exposed to this pollutant. Hepa1–6 cells treated with 0.001 mM B[a]P also led to decreased LSR and LDL‐receptor protein, as well as modified LSR activity as lipoprotein receptor. We propose that dysregulation of lipid homeostasis in the liver may contribute towards B[a]P‐induced weight gain in mice.
Investigating the import of folded proteins into peroxisomes
Authors: Larkin Angelyn , Rapoport Tom A.
Published: June 2021

Peroxisomes are essential organelles that play a critical role in a variety of metabolic pathways, such as the oxidation of fatty acids and the biosynthesis of certain amino acids and steroids. In addition, these organelles segregate toxic reactive oxygen species, including hydrogen peroxide and superoxide anions, from the rest of the cell to prevent damage to proteins and nucleic acids associated with many diseases. To support these functions, the peroxisome must recognize and transport over 100 distinct proteins from the cytoplasm into the peroxisomal matrix. Recent studies have established that in contrast to several other cellular organelles, which import proteins as unfolded polypeptides, peroxisomes are capable of transporting folded and even oligomeric proteins across its membrane. Although nearly twenty proteins have been implicated in this translocation process, the mechanistic details of import are still unclear. Herein, we present our work which addresses the initial docking stage of peroxisomal protein import using a combination of biochemical and biophysical methods, and outline ongoing studies to address the precise mechanism by which folded proteins are translocated across the peroxisomal membrane.
Characterization of mutations in small heat shock proteins that influence substrate binding
Authors: McMenimen Kathryn , Caminiti Courtney , Liu Pei , Kamalova Aichurok , Weber Caroline
Published: June 2021

The small heat shock proteins (sHsp), ubiquitous cellular homeostasis machinery, are oligomeric chaperone proteins. sHsps play an important role in maintaining cell function and survival under stress conditions such as high temperatures. By binding to non‐native proteins in an ATP‐independent manner, sHsps effectively prevent harmful aggregation of denatured proteins, promote proper protein folding, or facilitate protein degradation. Defects in sHsps can result in abnormal accumulation of proteins or aberrant protein folding, which lead to many pathologies such as cataract, tumor and neurodegenerative diseases including Parkinson's disease and Alzheimer's disease.

Structural studies have demonstrated that sHsps contain a highly conserved α‐crystallin domain flanked by variable N‐terminal and C‐terminal regions. They usually exist in large oligomers and are capable of adopting different oligomeric states in response to different conditions. Therefore, because of their dynamic structures, the specific mechanisms of how sHsp bind to substrate and function are still not well understood. Nonetheless, it is suggested that the variable N‐terminal region of the protein contributes to substrate binding and oligomerization.

We have made several mutations in the sHsps to characterize regions that influence oligomerization and make contacts with substrate proteins. Our studies suggest that the hydrophobic regions of the N‐terminal domain are important for substrate‐binding. Additionally, mutations in the a‐crystallin domain also contribute to the dynamics of protein oligomerization and sHsp‐client interactions.
Quantitative Profiling of Cysteine S‐nitrosylation Susceptibility in Cancer
Authors: Zhou Yani , Convertier Shalise , Weerapana Eranthie
Published: June 2021

We aimed to quantitatively measure the susceptibility of cysteine S‐nitrosylation (SNO), the transfer of a NO moiety to cysteine (Cys) on protein. SNO represents a form of redox modulation and plays an important role in diverse cellular processes. Recent developments of chemical methods to detect SNO have made it possible to identify SNO‐Cys sites. However, these methods do not monitor the extent of Cys to undergo nitrosylation in a complex proteome.

We utilized activity‐based protein profiling to identify nitrosylated Cys and to measure the susceptibility of Cys SNO in cell lysates after treatment with NO donor. A gel based dose‐dependent experiment revealed that Cys residues on different proteins demonstrated diverse susceptibilities to SNO. Isotopic tandem orthogonal proteolysis‐activity‐based protein profiling (isoTOP‐ABPP) method was applied to further quantitatively monitor the SNO susceptibility. Several novel Cys SNO targets were identified and verified by overexpression in mammalian cells. One of these is the serine/threonine‐protein kinase 1 (AKT1) which plays a critical role in many different signaling pathways, and might contribute to tumor progression. Our study showed that one Cys on AKT1 is highly susceptible to SNO. We are exploring structural motifs that govern this selectivity, and further investigate the role of nitrosylation in modulating AKT1 activity in vivo.
Investigating the targeting and regulation of Ded1, a DEAD‐box ATPase, in returning repressed mRNAs to translation
Authors: Pollard Beth , Hilliker Angela
Published: June 2021

Translation regulation is important, especially during stress, in neurons, and in early development, where many mRNAs are stored in translationally repressed states. These repressed mRNAs can be returned to translation, but this process is poorly understood. We have previously shown evidence that the RNA helicase, Ded1, performs two sequential functions to return a repressed mRNA back to translation in an ATP‐dependent manner. First, Ded1 promotes the formation of a “pre‐48S mRNP” that includes the initiation factors eIF4F and Pab1. Then, Ded1 hydrolyzes ATP, allowing the mRNA to become translated. We hypothesize that Ded1 acts as an ATP‐dependent switch to return mRNAs back to the translating pool.

We are investigating how Ded1 forms this pre‐48S intermediate. Ded1's ability to form this repressed mRNP in vitro depends on the presence of a polyA tail. Additionally, Ded1 increases the recruitment of Pab1 to the m7G cap. We are currently investigating the relationship between Pab1 and Ded1 in formation of this repression complex.
The role of mTORC1 and mTORC2 in UVB‐induced
Authors: Lewis Martha , Shantz Lisa
Published: June 2021

Mammalian target of rapamycin (mTOR) plays an important role in cell proliferation and survival. mTOR has been shown to exist in at least two complexes, mTORC1 and mTORC2. mTORC1 is inhibited by rapamycin, contains the mTOR catalytic subunit as well as the proteins RAPTOR and mLST8. It shows that mTORC1 phosphorylates p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). mTORC2 is known to be the kinase responsible for AKT phosphorylation on Serine 473 (S473). Based on previous research, we hypothesize that mTOR is a useful target in the prevention and treatment of nonmelanoma skin cancer. The goal of this project is to expand those results using wild‐type (WT) and RICTOR‐null (Rictor−/−) mouse embryo fibroblasts (MEFs). Since phosphorylation of AKT on Threonine 308 (T308) has been reported to be facilitated by S473 phosphorylation,

Aim

is to determine the phosphorylation state of AKT T308 in WT and Rictor−/− cells, both with and without UVB treatment and to examine the effect of Torin‐2, which inhibits both complex on proliferation in WT and Rictor−/− MEFs.

RESULTS

Western blot analysis demonstrated that UVB exposure induces AKT phosphorylation at T308 in both cells suggesting that the absence of mTORC2 signaling does not prevent T308 phosphorylation in response to UVB. MTS analysis showed that mTORC1 inhibition with rapamycin had no significant effect on cell proliferation. Treatment of WT and Rictor−/− cells with Torin‐2 decreased proliferation in a dose‐dependent manner. Since TORC2 activity is absent in MEFs lacking Rictor, this result suggests the presence of a rapamycin‐insensitive mTORC1 activity that may play a role in cell proliferation.
Regulation of β‐catenin localization by retinoblastoma protein in osteoblasts
Authors: Otaño‐Rivera Victor Emmanuel , Vázquez‐Rivera Viviana , Noel Richard , Santiago‐Cardona Pedro
Published: June 2021

pRb is a tumor suppressor that plays an important role in the coactivation of the osteoblast‐specific transcription factor Runx2. It has been shown that osteosarcomas are characterized by high rates of inactivation of both pRb and Wnt. The aim of this study is to determine whether pRb is capable of affecting Wnt signaling by regulating β‐catenin localization, hypothesizing that pRb activity is necessary for Wnt activation. We performed immunocytology (IC) for β‐catenin in cultured pRb‐expressing and pRb‐deficient MC3T3 cell lines in order to assess β‐catenin localization. In addition, a β‐catenin immunohistochemical (IHC) analysis was done on normal bone and osteosarcoma tissues. IC qualitative results showed an increase in nuclear β‐catenin localization in the presence of a functional pRb. Our IHC confirmed a correlation between pRb and β‐catenin localization. Interestingly, in osteosarcoma tissues we observed a correlation (Spearman r = 0.9878), in which pRb's localization determines β‐catenin's localization. Importantly, we observed that cells with cytoplasmic pRb in osteosarcoma tissues also had cytoplasmic β‐catenin localization, again strengthening the idea that a functional pRb is needed to activate Wnt. We concluded that pRb promotes β‐catenin nuclear re‐localization, showing that pRb plays an important role in the regulation of Wnt signaling.
Adenovirus 36 as a Risk Factor for Clinical Overweight/Obesity
Authors: Voss Jameson , Burnett Daniel , Olsen Cara , Haverkos Harry , Atkinson Richard
Published: June 2021

We evaluated Adenovirus 36 (Ad36) serostatus and follow‐up body mass index (BMI), waist circumference (WC), and clinical records among Air Force (AF) recruits.

Male enlistees (n=500) were followed from time of entry into AF beginning in 1995 to 2012 or separation, for 2915 person‐years. Numerically randomized selection occurred within four BMI quartiles (ranging from 20–30 kg/m2), stratified by year of entry. The exposure, Ad36 antibody, was assessed by ELISA from banked serum.

The primary outcome, follow‐up BMI, was similar by serostatus. This contrasted with secondary outcomes. The hazard of clinical overweight/obesity showed interaction (p=0.007) between baseline BMI and serostatus. At a baseline BMI of 22.5, survey weighted cox proportional hazards showed the seropositive had a 4.0 fold increased hazard of diagnosis of clinical overweight/obesity (p=0.003) as compared with seronegative, controlling for baseline BMI. The maximum WC was higher among seropositive.

Ad36 serostatus is associated with increased hazard of overweight/obesity diagnosis among lean, male, AF recruits.

The opinions are those of authors, not the DoD or USUHS. IDCRP funded the study and Obetech, LLC provided lab testing.
Depot differences in gene expression in response to dexamethasone in human adipose tissue
Authors: Pickering Richard Taylor , Lee Mi‐Jeong , Fried Susan K
Published: June 2021

Hypercortisolemia leads to a preferential fat deposition in visceral adipose tissues. We previously noted that the glucocorticoid receptor agonist dexamethasone (Dex, 25 nM) regulated many metabolic and inflammatory gene pathways in human omental (Om) and abdominal subcutaneous (Sc) adipose tissues. To determine whether differential sensitivity to Dex contributes to depot differences in global gene expression, fragments of Sc and Om adipose tissues from severely obese females (n=3) were cultured in the presence of 7nM insulin plus 0 (control), 1, 10, or 1000 nM Dex for 7 days. 1 and 10 nM Dex regulated, respectively, 88 and 414 genes in Om, and 173 and 594 genes in Sc by 2‐fold or more. Thus, almost 2‐fold more genes were regulated by Dex in Sc than Om at low Dex. We noted and confirmed by qPCR that INHBA, a gene known to inhibit adipogenesis, was ~6‐fold higher in Om and was suppressed in both, but remained higher in Om at Dex 1 and 10 nM. In primary cultured predipocytes (n=3), INHBA was similar in confluent preadipocytes, but was less suppressed when a standard differentiation cocktail containing Dex was added. At day 14 of differentiation, INHBA expression was ~50‐fold higher in Om cultures in parallel to poorer differentiation. Overall, our data suggest that differential sensitivity to physiologically relevant concentrations of Dex can contribute to depot differences in adipocyte differentiation and function.
Factors Affecting a Healthy Diet for the Individual Who Is Homeless
Authors: Ekvall shirley maxine , Ekvall valli
Published: June 2021

The individual who is homeless is one of the most vulnerable populations. This study examines two means of potentially improving thenutritional status of this person. The nutritional quality of the diets often is inadequate for this individual who is homeless due to the food offered (which depends on the donations and donors) as well as the knowledge of the staff and the food choices made and consumed by this individual. Nutrition education can be used to affect these pivotal decisions. Missions depend on donors for 80–90% of their food supply;thus the donor is key for nutrition education. Other education‐related factors include: the knowledge base of individual's receiving education, the staff, the individual who is homeless's attitude towards nutrition, and the concrete examples and application of this when educating. Foods found in short supply were milk, fresh fruits and vegetables, cheese, eggs, and fish. Whole grains were not lacking, but often not consumed. Increasing food temperature, decreasing sweets, and increasing variety were needed. Dietitians can play a key role in connecting shelters with food sources, educating shelter staff and the individual who is homeless, and providing educational materials to the donors.
Replacing beverages with Vita Coco coconut water can decrease dietary intake of sugars and increase vitamin C, calcium, magnesium and potassium intakes by adults aged 19 years and older: What We Eat in America (WWEIA/NHANES 2007–2010)
Authors: Keast Debra R , Kapica Cathy M , Hoerr Sharon M
Published: June 2021

The nutritional impact of replacing beverages with coconut water was assessed by modeling dietary data from WWEIA/NHANES 2007–2010. Nutrient intake from beverages consumed by adults aged 19+ y (n=11,481) were determined using combination codes to classify beverages and account for additions. Categories included milk & milk drinks; fruit/vegetable juices; carbonated soft drinks (CSD) & fruit/sports/energy drinks; coffee/tea & misc. beverages; water; and alcohol. Vita Coco nutrient composition analyses (n=155) were averaged and used to determine energy, fat, carbohydrate (CHO), sugars, protein, vitamin C, calcium (Ca), phosphorus (P), magnesium (Mg), iron (Fe), sodium (Na) and potassium (K) intakes if Vita Coco replaced CSD & fruit/sports/energy drinks. Modeled nutrient intakes from replaced beverages were all significantly different than baseline. Energy (− 36 kcal), total fat (−0.1 g), CHO (−12 g) and total sugars (−14 g) were reduced (p<0.01). Protein (+2 g), vitamin C (+186 mg), Ca (+49 mg), P (+12 mg), Mg (+53 mg), Fe (+0.4 mg), Na (+120 mg) and K (+805 mg) were increased (p<0.01). These beverage changes had an impact on total dietary intakes by increasing vitamin C, Mg and K (p<0.01), Na and calcium (p<0.05); CHO and sugars intakes and the Na/K ratio were lower (p<0.01) than baseline. Replacing non‐nutritional beverages with Vita Coco coconut water can improve the diets of adults by reducing sugars, and increasing key nutrients such as Ca, Mg and K. (Research funded by Vita Coco coconut water.)
Food access, social safety nets, and social stigma among low‐income families in Massachusetts
Authors: Ghavami Sarvnaz Modarresi , Peterman Jerusha Nelson , Anliker Jean
Published: June 2021

Social safety nets like SNAP, WIC, and fuel assistance can alleviate food insecurity and improve household resources. They may not prevent health consequences of food insecurity unless they promote purchase and consumption of healthful foods. Social stigma around use of benefits can prevent vulnerable families from utilizing benefits, which may further complicate food insecurity. We analyzed focus group transcripts (6 focus groups, 21 participants) with low‐income, largely food insecure individuals in Massachusetts to assess use of social safety nets and related social stigma. We used directed content analysis to examine pre‐identified themes and to identify new themes. Pre‐ identified themes included the use of SNAP and WIC to extend resources and stigmatization of the use of social safety nets by the members of the community. New themes were utilization of fuel assistance to free up resources, saving SNAP for special and healthy foods, stigmatization of low cost foods by children, stigma within the community for resource allocation of benefit users, and self‐stigmatization. Different stigma associated with the use of social benefits and purchase of low cost foods might prevent low‐income individuals from utilizing. In this context, allocation of certain benefits, such as SNAP, to purchase low quality foods might lead to health complication in the future.

Funding source: USDA‐CSREES #H994
Cellular Damage Evaluation caused by Dental Bleaching Products – a pilot study
Authors: Marto Carlos Miguel , Laranjo Mafalda , Abrantes Ana Margarida , Lopes João Casalta , Gonçalves Ana Cristina , Ribeiro Ana Bela Sarmento , Cabrita António Silvério , Ferreira Manuel Marques , Botelho Maria Filomena , Carrilho Eunice
Published: June 2021

Esthetic demands makes dental bleaching an increasingly requested therapy, however this is not a risk‐free procedure due to the cytotoxicity of whitening products. This work evaluates possible cytotoxic effect on cells caused by whitening products.

We performed an in vitro study with human fibroblasts cell cultures, exposed at different concentrations of hydrogen peroxide and a commercially available bleaching product, with and without photo activation. Evaluation was made at 45 minutes and 24, 48 and 72 hours. Flow cytometry was also performed. The Mann‐Whitney test was used for statistical analysis.

Recovery in cell viability is observed after 45min with the commercial product and only after 48 hours with hydrogen peroxide. Also, the application of hydrogen peroxide causes an increase in cell death by apoptosis, in production of intracellular peroxides and a decrease in mitochondrial membrane potential. The activating light alone causes an increase in cell death by apoptosis. These data present a p < 0.05 and a range of 95%.

Decrease of cell viability was reversibly in all situations. Hydrogen peroxide and activating light have a cytotoxic effect on cell cultures, increasing cell death, unlike the commercial product tested, so we can hypothesize some protective effect by other components from commercial bleaching product. This results may be correlated with clinical findings namely hypersensitivity.
Application of Image‐Guided Coring as a new technique for targeting breast tumor tissue in molecular pathology
Authors: Montaser‐Kouhsari Laleh , Christensen Stephen , Knoblauch Nicholas W , Baker Gabrielle , Beck Andrew H
Published: June 2021

Background

There are several techniques available to sample tissue from archival formalin‐fixed paraffin‐embedded (FFPE) tissue blocks. However, current techniques are either extremely labor intensive or lack precision for accurately sampling small lesions. Herein, we explain a new image‐guided coring (IGC) technique to conquer these limitations for molecular analyses.

Design

The H&E slides were scanned by the whole slide digital panoramic scanner (3D‐Histech). Then, a pathologist selects regions of interest from these digital images using the Pannoramic Viewer Software package (3D‐Histech). This annotated slide is overlaid onto digital image of the FFPE block via TMA‐Master software (3D‐Histech) and a computer‐guided robotic arm is used to sample cores from the targeted regions of the FFPE tissue block. This selectively acquired tissue is then used for molecular analyses.

Results

We applied IGC to 770 FFPE breast samples from the Nurses’ Health Study including 389 invasive carcinomas and 381 normal terminal duct lobular unit samples. The median concentration of extracted RNA in tumor and normal samples was 127 ng/ml and 23.31 ng/ml, respectively. A high success rate of adequate RNA extraction (20 ng/mL) from tumor and normal breast tissue via IGC was obtained (88.9% and 55.4%, respectively).

Conclusion

IGC is a robust new approach to obtain samples from FFPE blocks in large‐scale projects in molecular oncologic pathology. We applied this new method to accurately sample breast cancer and normal breast tissue from FFPE samples with high success rates. This method may prove generally useful for clinical molecular diagnostics as well as translational tissue‐based biomedical research.
Cytological changes in the placenta after early hgh dose intravaginal progesterone
Authors: Shanklin Douglas R. , Shokouh‐Amiri Mohammad
Published: June 2021

Intravaginal progesterone in the first trimester after IVF and in twins, has mixed results (BJOG 120:50, 2013), as has veterinary progesterone for apparent low levels (J.Zoo.Wildlife Med. 43:296, 2012). Early placental progesterone and receptors have been studied without reports on placental cytostructure. A 40 year old g 2 p 0 passed a 9 week blighted ovum four months prior to the current conception. No background health issues for either. At 6 weeks menstrual age low normal progesterone (9.21 ng/ml) prompted 200 mg vaginal insert progesterone daily for 18 days, then 400 mg daily for 10 days, a total of 7.6 g. Pregnancy was normal to loss of fetal movement at 27 weeks confirmed by sonography. Induction: 831 g fetus and 178 g placenta. Ponderal index and placental:fetal ratio below 27 week means. Placenta grossly ordinary, 14.3 × 11.7 × 1.8 cm; eccentric 3 vessel cord. Microscopic: significant villus dysplasia with many trophoblastic intrusions; scant formation of tertiary villi; reduced paracapillary migration; many persistent cytotrophoblastic columns‐islands, some arising from ramuli chorii; and extensive persistent and marked subsyncytial cytotrophoblast. The latter cells were enlarged with clear cytoplasm. Placental remodeling, and of decidual vessels by extravillous trophoblast, begins about 8 weeks. Herewith a case of first impression on changes from intensive progesterone in the first trimester.
Complement MAC expression in heart failure
Authors: Rietdorf Emily , Chow Sheryl , O'Barr Stephen
Published: June 2021

Complement is expressed during acute myocardial infarction (AMI), but little is known of its involvement in the development of heart failure (HF).

Methods

Rats were induced with HF by occlusion of the LAD coronary artery, and then sacrificed four weeks later. Controls were given a sham operation with the same procedure minus the occlusion. Hearts were excised then homogenized in RIPA buffer. Western blots were run with C7 (Quidel) and C5b‐9 (Hycult) antibodies at 1:1000, secondary antibody at 1:5000, blocked with TBST + 5% milk.

Results

Eight control samples vs. eight HF samples were evaluated. Both C7 and C5b‐9 levels in the HF samples were significantly increased in comparison with the controls.

Conclusions

These results demonstrate that complement is activated in our HF model, but more studies are needed to determine its specific role in HF pathology. Financial support was provided by the Drew Collaborative Grant # 12304P (SLC), GCRC LaBioMed #00543–38F (SLC), NIH R21DK070003–01A1, and Western University of Health Sciences MSPS Graduate Program (ER).
Mapping determinants in the gp41 CT of HIV‐1 Env that mediate resistance to antibody neutralization during cell‐cell infection.
Authors: Gray Essanna , Durham Natasha , Chen Benjamin
Published: June 2021

Human Immunodeficiency Virus Type 1 (HIV‐1) uses its surface envelope glycoprotein (Env) to mediate entry into CD4+ T‐cells. During direct T‐cell to T‐cell infection mediated by the virological synapse (VS), Env initiates cell‐cell adhesion in a CD4 dependent manner. Entry of viral capsids into a target cell resulting from this fusion can be blocked by neutralizing antibodies. However VS‐mediated infection is more resistant to neutralization compared to cell‐free infection. The transmembrane region of Env, gp41 has a cytoplasmic tail (CT) made up of 151 amino acids that has been shown to aid in the fusion process and play a role in HIV‐1's resistance to neutralizing antibodies during cell‐cell infection by regulating the exposure of neutralizing epitopes on the cell surface. Complete truncation of the gp41 CT makes VS‐mediated infection more sensitive to antibody neutralization. We hypothesize that specific structural features in the CT regulate HIV‐1 neutralization and certain amino acids correspond to the exposure of neutralizing epitopes. To map these putative conformation altering motifs, we constructed a series of C‐terminal truncations in the gp41 CT. Infectivity and neutralization assays are being performed to evaluate how these truncations affect HIV‐1's infectivity and its resistance to neutralizing antibodies during cell free and cell‐cell infection.
Pre‐TM4 residues of GluN2 subunits modulate the function of recombinant N‐methyl‐D‐aspartate (NMDA) receptors
Authors: Hughes Benjamin , Woodward John
Published: June 2021

The N‐methyl‐D‐aspartate (NMDA) receptor is a ligand‐gated ionotropic receptor activated by the neurostransmitter glutamate and is essential to fast excitatory neurotransmission. NMDARs are heteromeric tetramers composed of obligate GluN1 subunits as well as GluN2 or GluN3 subunits. Four distinct types of GluN2 subunits (GluN2A‐D) are known and impart divergent receptor properties including changes in agonist affinity and channel gating. In the present study, we identified a sequence of amino acids in the pre‐TM4 region of GluN2B that affects ligand affinity and the effectiveness of ethanol to inhibit the channel. This sequence, that immediately precedes the fourth transmembrane domain, is highly conserved amongst GluN2 subunits. Glycine substitutions at discrete locations within this sequence produce a significant leftward shift of the glutamate concentration‐response curve and currents display a significantly longer decay constant. Furthermore, glycine substitutions in this region also increase the sensitivity of the receptor to ethanol inhibition. Ongoing studies are focused on determining how this region, distinct from the S1 and S2 ligand‐binding domain, affects the sensitivity of the receptor to agonists and antagonists.

Supported by grants T32 AA007474 (B.A.H) and R37 AA009986 (J.J.W.).