Three-dimensional Extracellular Matrix Hydrogel Environments for Embryonic Stem Cell Growth

Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of the blastocyst that can give rise to cells of the ectoderm, endoderm and mesoderm lineages. Once isolated from the blastocyst, ESCs can be cultured indefinitely in vitro in an undifferentiated state or can be induced to differentiate. In the case of mouse ESCs (mESCs), the cytokine leukemia inhibitory factor (LIF) is added to culture media to maintain pluripotency and is removed to induce differentiation. Although it is known that extracellular matrix (ECM) components influence stem cell maintenance, proliferation and differentiation, the precise effects of ECM environments on embryonic stem cell behavior have not been systematically studied. The main purpose of this thesis project was to investigate the behavior of undifferentiated mESCs cultured in different 3D hydrogel matrices and to determine whether viscoelastic and biochemical variations in the matrices differentially affect the ability of stem cells to self-renew; that is, retain their pluripotency or undifferentiated phenotype. Their behavior in 3D environments was compared to mESC behavior in traditional 2D culture. In addition, a new method of casting hydrogels in polydimethylsiloxane (PDMS) molds was developed in order to efficiently cast multiple hydrogels of varying sizes and shapes. The findings of this thesis project will benefit both the scientific and engineering community as it encourages researchers to re-evaluate the quality of standard 2D embryonic stem cell culture methods versus potentially novel and advantageous 3D hydrogel culture methods.M.S.Committee Chair: McDevitt, Todd; Committee Member: Babensee, Julia; Committee Member: Temenoff, Johnn

The relationship between mood disorder and insomnia depends on race in US veterans with epilepsy

Few data exist on race, medical/psychiatric comorbidities, and insomnia symptoms in US veterans with epilepsy. Our aims were to examine 1) whether insomnia symptom prevalence was different between Black and White veterans and 2) whether predictors of insomnia symptoms varied by race. This retrospective, cross-sectional study included veterans evaluated in an epilepsy clinic over the course of 1.5years. Individuals completed standardized assessments for epilepsy and sleep complaints. Insomnia criteria were met by 1) report of difficulty with sleep initiation, maintenance, or premature awakenings accompanied by daytime impairment or 2) sedative-hypnotic use on most days of the month. Demographics, medical/psychiatric comorbidities, and medications were determined per electronic medical record review. Hierarchical multivariable logistic regression analyses were performed to determine if race, medical/mental health comorbidities, and the potential interaction of race with each comorbid condition were associated with insomnia. Our sample consisted of 165 veterans (32% Black). The unadjusted prevalence of insomnia was not different between Black and White veterans (42% vs 39%, p=0.68). In adjusted analyses, the association between mood disorder and insomnia varied by race. Depressed White veterans had over 11-times higher predicted odds of insomnia (OR 11.4, p<0.001) than non-depressed White veterans, while depressed Black veterans had 4-times higher predicted odds of insomnia (OR 4.1, p=0.06) than non-depressed Black veterans. Although mood disorder diagnosis was associated with insomnia for both racial groups, White veterans had a stronger association between mood disorder diagnosis and insomnia than Black veterans. The relationship between mood disorder diagnosis and insomnia was stronger for White than Black veterans with epilepsy. Future studies are needed to explore mental health symptoms and psychosocial determinants of insomnia with larger samples of minority individuals with epilepsy. •The role of comorbidities and race in predicting insomnia in epilepsy is unclear.•Insomnia prevalence was similar in White and Black veterans with epilepsy.•The relationship between mood disorder and insomnia was moderated by race.•The association between mood and insomnia was stronger for Whites than Blacks

The coiled-coil of ATG16L1 is highly conserved across vertebrates.

<p>(A) Cross-species alignment of the coiled-coil (human residues M126 – A207) of ATG16L1 reveals levels of sequence identity with the human sequence of between 73% and 100%. The percentage identity listed is in comparison with the human sequence. The common names and database identifies for each sequence are listed in Materials and Methods. (B) The syntenic position of <i>Atg16L1</i> is well conserved across species. The three adjacent upstream and downstream genese to <i>Atg16L1</i> are displayed. Genes are denoted by individual blocks; yellow indicates a position on the forward strand and green a position on the reverse strand. Gene identities are as follows: ATG16L1 – autophagy related protein 16 isoform 1; SAG – S-antigen, retina and pineal gland; DGKD – diacylglycerol kinase delta; USP40 – ubiquitin specific peptidase 40; INPP5D – inositol polyphosphate-5-phosphatase; NEU2 – sialidase 2; NGEF - neuronal guanine exchange factor; TRPV2 – transient receptor potential cation channel, subfamily V, member 2; UBB – ubiquitin B; AC106876.2 – uncharacterised; 00570 (ENSMODG00000000570) – uncharacterised; 23152 (ENSMODG00000023152) – uncharacterised, BLASTp indicates a possible ortholog of glyceraldehydes 3-phosphate dehydrogenase.</p

Characterisation of the oligomeric status of the human ATG16L1 coiled-coil domain.

<p>(A) Size exclusion chromatography of CCD3. CCD3 – red trace; Blue Dextran – blue trace; Molecular size markers – black trace (mass labelled in kDa). (B) Native-PAGE analysis of CCD3. 1 µg of CCD3 was analysed by Native-PAGE both before (lane 1) and after (lane 2) freeze/thawing at −80°C. M – NativeMark™ protein standards (Life Tehnologies). (C) Nanospray Electrospray Ionisation Mass Spectroscopy (ESI-MS) analysis of CCD3 under conditions that preserve non-covalent interactions. CCD3 dimers are detected in the low m/z range corresponding to charge states of +11 to +7. There is no evidence of any additional species of CCD3. (D) Analytical ultracentrifugation sedimentation velocity data. Interference optical signal distributions of CCD3FH at time intervals of 320 s at a rotor speed of 50,000 rpm and a temperature of 20°C, with systematic noise subtracted. The residuals are from the fit with the hybrid discrete/continuous model. Component sedimentation coefficient distributions showed a dominant population of the dimeric species, with a uniform frictional ratio of Fk,w = 1.73. The final r.m.s.d. was 0.006.</p

Summary of human ATG16L1 construct expression in <i>E. coli</i> Rosetta™ 2 cells.

<p>Footnote: All constructs possessed a C-terminal FLAG-6His epitope tag. Abbreviations: 6His – 6× Histidine; GST – Glutathione-S-transferase; 6His-NusA – 6× Histidine N utilisation substance protein A; 6His-MBP – 6× Histidine Maltose binding protein.</p

The coiled-coil of human ATG16L1 is alpha helical.

<p>(A) PSIPRED prediction of the secondary structure of the minimal coiled-coil domain (residues M126 – A207; CCD3) of human ATG16L1. Sequencing numbering corresponds to the human protein, alpha helices are marked as pink cylinders and the confidence level of the prediction for each residue is provided by the blue bars. (B) Circular dichroism analysis of CCD3. Mean residual ellipicity (θ) plotted against wavelength (nm) indicates a helical protein.</p

Organisation of ATG16L1.

<p>(A) Domain organisation of yeast ATG16 and human ATG16L1. ATG5 binding motifs are coloured green (yeast) and pink (human); the coiled-coil domain (CCD) is cyan (yeast) and blue (human); the human WD40 repeats are yellow. The position of the Crohn's Disease susceptibility polymorphism T300A is marked on human ATG16L1. (B) Structure of the yeast and human ATG5 binding motifs and the yeast ATG16 CCD. Colours as in panel (A). (C) Expression constructs used in this study outlining the boundaries of human ATG16L1. FL – full length ATG16L1, CCD1 – coiled-coil domain construct 1, CCD2 – coiled-coil domain construct 2.</p

[catalog] Africa explores : 20th century African art /

Publ. in conjunction with an exhibition, The center for African art, New York, 199

Developing the Neurology Diversity Officer A Roadmap for Academic Neurology Departments

Academic neurology departments must confront the challenges of developing a diverse workforce, reducing inequity and discrimination within academia, and providing neurologic care for an increasingly diverse society. A neurology diversity officer should have a specific role and associated title within a neurology department as well as a mandate to focus their efforts on issues of equity, diversity, and inclusion that affect staff, trainees, and faculty. This role is expansive and works across departmental missions, but it has many challenges related to structural intolerance and cultural gaps. In this review, we describe the many challenges that diversity officers face and how they might confront them. We delineate the role and duties of the neurology diversity officer and provide a guide to departmental leaders on how to assess qualifications and evaluate progress. Finally, we describe the elements necessary for success. A neurology diversity officer should have the financial, administrative, and emotional support of leadership in order for them to carry out their mission and to truly have a positive influence

Hsp70 forms antiparallel dimers stabilized by post-translational modifications to position clients for transfer to Hsp90

Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90