Faculty Bibliography

The adult periosteum serves as a niche for skeletal stem cells, and its contribution to fracture healing and bone regeneration is well documented (Colnot 2009). All bones in the skeleton possess regenerative features and share a similar histological architecture, however, their embryonic origins are diverse: the appendicular skeleton develops from the mesoderm, while the craniofacial bones, except the parietal bones, are derived from the neural crest. With notable exceptions, the embryonic origin determines their differential expression of Hox genes, transcriptional factors that vary along the cranial-caudal axis to regulate the anterior-posterior positional patterning during development (Mallo et al. 2010). Previous studies have identified a superior regenerative potential of craniofacial osteoprogenitor cells (OPCs) to appendicular OPCs (Leucht et al. 2008). Here, we employed RNAseq analysis to interrogate OPCs from four distinct anatomic locations (Fig. 1A). We hypothesized that embryonic Hox gene expression is maintained into adulthood, provides a transcriptional signature that best differentiates between craniofacial and appendicular OPCs, and that Hox gene expression regulates adult OPC fate decisions. Hierarchical cluster analysis (Fig.1B.1), PCA (Fig.1B.2) and MA plot analysis (Fig.1B.3,4) revealed that OPCs negative for Hox gene expression contain 5390 differentially expressed genes compared to Hox-positive OPCs, showing for the first time that on a transcriptional level, OPCs from the craniofacial skeleton and the appendicular skeleton present two distinct cell populations. While the embryonic function of Hox genes is well characterized (Mallo et al. 2010), their role in adult cells is not clearly defined yet. Here, we used siRNA and antisense oligonucleotide (ASOs) to knockdown epigenetic regulators of Hox expression in adult periosteal OPCs in vitro and then assessed their differentiation potential. Hox knockdown in Hox-expressing tibial OPCs resulted in a more osteogenic and less adipogenic cell fate, demonstrating that Hox gene expression is crucial for adult OPC cell fate decisions (Fig 1C). This study provides evidence that embryonic Hox gene expression is maintained into adulthood, where one of its functions is to regulate OPC cell fate decisions. Epigenetic manipulation can be utilized to target and manipulate Hox gene expression, which may provide potential therapeutic opportunities with the goal to improve fracture healing. (Figure Presented)

Binding of the gp120 envelope (Env) glycoprotein to the CD4 receptor is the first step in the HIV-1 infectious cycle. Although the CD4-binding site has been extensively characterized, the initial receptor interaction has been difficult to study because of major CD4-induced structural rearrangements. Here we used cryogenic electron microscopy (cryo-EM) to visualize the initial contact of CD4 with the HIV-1 Env trimer at 6.8-Ã… resolution. A single CD4 molecule is embraced by a quaternary HIV-1-Env surface formed by coalescence of the previously defined CD4-contact region with a second CD4-binding site (CD4-BS2) in the inner domain of a neighboring gp120 protomer. Disruption of CD4-BS2 destabilized CD4-trimer interaction and abrogated HIV-1 infectivity by preventing the acquisition of coreceptor-binding competence. A corresponding reduction in HIV-1 infectivity occurred after the mutation of CD4 residues that interact with CD4-BS2. Our results document the critical role of quaternary interactions in the initial HIV-Env-receptor contact, with implications for treatment and vaccine design.

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

Virological synapses (VS) are adhesive structures that form between infected and uninfected cells to enhance the spread of HIV-1. During T cell VS formation, viral proteins are actively recruited to the site of cell-cell contact where the viral material is efficiently translocated to target cells into heterogeneous, protease-resistant, antibody-inaccessible compartments. Using correlative light and electron microscopy (CLEM), we define the membrane topography of the virus-containing compartments (VCC) where HIV is found following VS-mediated transfer. Focused ion beam scanning electron microscopy (FIB-SEM) and serial sectioning transmission electron microscopy (SS-TEM) were used to better resolve the fluorescent Gag-containing structures within the VCC. We found that small punctate fluorescent signals correlated with single viral particles in enclosed vesicular compartments or surface-localized virus particles and that large fluorescent signals correlated with membranous Gag-containing structures with unknown pathological function. CLEM imaging revealed distinct pools of newly deposited viral proteins within endocytic and nonendocytic compartments in VS target T cells.

Store-operated Ca²⁺entry (SOCE) is a pathway for increasing intracellular Ca²⁺ levels regulated by stromal interaction molecule 1 (STIM1), STIM2, and the Ca²⁺ channel ORAI1. SOCE-deficient patients suffer from Calcium Release-Activated Calcium (CRAC) channelopathy characterized by immunodeficiency, autoimmunity, myopathy, and anhidrotic ectodermal dysplasia. Several mitochondrial enzymes/complexes depend on Ca²⁺ but the source of Ca²⁺ required for their function are not entirely clear. We recently showed a cell-intrinsic role of SOCE in human mitochondria (Maus M et al. Cell Metab. 2017;25(3):698- 712). MitoView Green showed reduced mitochondrial volume in fibroblasts of patients with ORAI1/STIM1 lossof- function mutations. mtDNA copy numbers and mRNAs expression of selected mitochondrial transcription factors were normal. SDS-PAGE/Western blot analysis showed reduced expression of NADH ubiquinone oxidoreductase subunit-B8, Cytochrome b-c1 complex subunit-2, Cytochrome c oxidase subunit-I, Cytochrome C, Mitochondrial porin and permeability transition pore, etc. Blue native PAGE of isolated mitochondria confirmed reduced expression of CI, CIV and supercomplex CICIII2. SOCE-deficient fibroblasts had reduced mRNA and protein expression of uncoupling protein 2, higher basal mitochondrial membrane potential (MMP) and higher numbers of damaged mitochondria as suggested by increased co-localization of mitochondria and lysosomes and increased MitoKeima reporter activity indicative of lysosomal mitophagy. Oligomycininduced ATP-synthase inhibition revealed decreased electron transport and proton pumping rates measured as MMP hyperpolarization rates and reduced superoxide production assessed by MitoSOX. Maximal O2 consumption rates in SOCE-deficient cells were decreased. Skeletal myocytes had reduced CI and CIV function in 2 out of 3 ORAI1-deficient patients. Gene expression of very long chain acyl-CoA dehydrogenase and long-chain fatty acid transporter carnitine palmitoyltransferase 1B was reduced in patient fibroblasts cultured in either high glucose medium or oleic acid (OA) medium followed by starvation in 2 mM glucose medium. Furthermore, SOCE-deficient fibroblasts were lacking a starvation-induced increase in etomoxir-sensitive mitochondrial respiration in OA medium and showed reduced rates of OA beta-oxidation when cultured in 14C-OA-medium with or without subsequent starvation. Our findings indicate an important new role of SOCE in mitochondrial function

Text classification has always been an interesting issue in the research area of natural language processing (NLP). While entering the era of big data, a good text classifier is critical to achieving NLP for scientific big data analytics. With the ever-increasing size of text data, it has posed important challenges in developing effective algorithm for text classification. Given the success of deep neural network (DNN) in analyzing big data, this article proposes a novel text classifier using DNN, in an effort to improve the computational performance of addressing big text data with hybrid outliers. Specifically, through the use of denoising autoencoder (DAE) and restricted Boltzmann machine (RBM), our proposed method, named denoising deep neural network (DDNN), is able to achieve significant improvement with better performance of antinoise and feature extraction, compared to the traditional text classification algorithms. The simulations on benchmark datasets verify the effectiveness and robustness of our proposed text classifier

BACKGROUND AIMS/OBJECTIVE:Regenerative medicine employs human mesenchymal stromal cells (MSCs) for their multi-lineage plasticity and their pro-regenerative cytokine secretome. Adipose-derived mesenchymal stromal cells (ASCs) are concentrated in fat tissue, and the ease of harvest via liposuction makes them a particularly interesting cell source. However, there are various liposuction methods, and few have been assessed regarding their impact on ASC functionality. Here we study the impact of the two most popular ultrasound-assisted liposuction (UAL) devices currently in clinical use, VASER (Solta Medical) and Lysonix 3000 (Mentor) on ASCs. METHODS:). ASC yield, viability, osteogenic and adipogenic differentiation capacity and in vivo regenerative performance were assessed. RESULTS:Both UAL samples demonstrated equivalent ASC yield and viability. VASER UAL ASCs showed higher osteogenic and adipogenic marker expression, but a comparable differentiation capacity was observed. Soft tissue healing and neovascularization were significantly enhanced via both UAL-derived ASCs in vivo, and there was no significant difference between the cell therapy groups. CONCLUSIONS:Taken together, our data suggest that UAL allows safe and efficient harvesting of the mesenchymal stromal cellular fraction of adipose tissue and that cells harvested via this approach are suitable for cell therapy and tissue engineering applications.