Miniature Arcs for Synthesis of Carbon Nanotubes in Microgravity

Although many methods are available for producing single-walled carbon nanotubes (SWNTs), the conventional carbon arc process remains the most popular due to its simplicity and large production rate. In the carbon arc, SWNTs are catalytically synthesized by rapidly evaporating a graphite anode impregnated with NiN metal catalyst from which the nanotubes grow in an inert atmosphere. However, high temperatures inside the carbon arc generate strong buoyancy driven convection, and it is hypothesized that the non-uniform environment created by this flow has a large effect on the growth and morphology of the SWNTs. To study the effect of buoyancy on the arc process, a miniature carbon arc apparatus was developed to synthesize SWNTs in a microgravity environment substantially free from these strong convective flows. The reactor was operated for either 2.2 or 5 seconds during free-fall in the drop towers at the NASA Glenn Research Center. Two apparatus designs differing mainly in their production rate and power capacity were investigated. The first consisted of a miniaturized carbon arc employing a 1 mm diameter graphite anode and powered by a 0.54 F capacitor bank charged to 65 V. The second, larger apparatus employed a 4 mm diameter anode and was powered by a portable battery pack capable of providing in excess of 300 amps at 30 volts to the arc for the duration of a 5 second drop. Initial results indicated that transient heating is a very large effect in the short-duration drop tower carbon arcs, and thermal equilibrium of the arc plasma, buffer gas, and apparatus was not attained during the short microgravity periods. In addition, removal of the buoyant convection by the microgravity now allowed clear observation of large jets of evaporated carbon vapor streaming from the anode and mixing with the inert buffer gas. The initial mixing of these jets with the cold buffer gas combined with the thermal transient made it difficult to establish a uniform high temperature environment around the arc in the 2.1 to 5 second microgravity time interval, and even with a very high-powered arc, the arc region was cooler than in continuously operated arcs. Despite these difficulties, the miniature arc produced SWNTs in microgravity. However, given the large thermal transient to overcome, no dramatic difference in sample yield or composition was noted between normal gravity and q2-,andL%econd long microgravity runs

LGR5 receptor promotes cell-cell adhesion in stem cells and colon cancer cells via the IQGAP1 -Rac1 pathway

Leucine-rich repeat-containing G protein–coupled receptor 5 (LGR5) is a bona fide marker of adult stem cells in several epithelial tissues, most notably in the intestinal crypts, and is highly up-regulated in many colorectal, hepatocellular, and ovarian cancers. LGR5 activation by R-spondin (RSPO) ligands potentiates Wnt/β-catenin signaling in vitro; however, deletion of LGR5 in stem cells has little or no effect on Wnt/β-catenin signaling or cell proliferation in vivo. Remarkably, modulation of LGR5 expression has a major impact on the actin cytoskeletal structure and cell adhesion in the absence of RSPO stimulation, but the molecular mechanism is unclear. Here, we show that LGR5 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1), an effector of Rac1/CDC42 GTPases, in the regulation of actin cytoskeleton dynamics and cell–cell adhesion. Specifically, LGR5 decreased levels of IQGAP1 phosphorylation at Ser-1441/1443, leading to increased binding of Rac1 to IQGAP1 and thus higher levels of cortical F-actin and enhanced cell–cell adhesion. LGR5 ablation in colon cancer cells and crypt stem cells resulted in loss of cortical F-actin, reduced cell–cell adhesion, and disrupted localization of adhesion-associated proteins. No evidence of LGR5 coupling to any of the four major subtypes of heterotrimeric G proteins was found. These findings suggest that LGR5 primarily functions via the IQGAP1–Rac1 pathway to strengthen cell–cell adhesion in normal adult crypt stem cells and colon cancer cells

A Cell Motility Screen Reveals Role for MARCKS-Related Protein in Adherens Junction Formation and Tumorigenesis

Invasion through the extracellular matrix (ECM) is important for wound healing, immunological responses and metastasis. We established an invasion-based cell motility screen using Boyden chambers overlaid with Matrigel to select for pro-invasive genes. By this method we identified antisense to MARCKS related protein (MRP), whose family member MARCKS is a target of miR-21, a microRNA involved in tumor growth, invasion and metastasis in multiple human cancers. We confirmed that targeted knockdown of MRP, in both EpRas mammary epithelial cells and PC3 prostate cancer cells, promoted in vitro cell migration that was blocked by trifluoperazine. Additionally, we observed increased immunofluoresence of E-cadherin, β-catenin and APC at sites of cell-cell contact in EpRas cells with MRP knockdown suggesting formation of adherens junctions. By wound healing assay we observed that reduced MRP supported collective cell migration, a type of cell movement where adherens junctions are maintained. However, destabilized adherens junctions, like those seen in EpRas cells, are frequently important for oncogenic signaling. Consequently, knockdown of MRP in EpRas caused loss of tumorigenesis in vivo, and reduced Wnt3a induced TCF reporter signaling in vitro. Together our data suggest that reducing MRP expression promotes formation of adherens junctions in EpRas cells, allowing collective cell migration, but interferes with oncogenic β-catenin signaling and tumorigenesis

Perceived preparedness of dental academic institutions to cope with the COVID-19 pandemic: a multi-country survey

Dental academic institutions are affected by COVID-19. We assessed the perceived COVID19 preparedness of these institutions and the characteristics of institutions with greater perceived preparedness. An international cross-sectional survey of dental academics was conducted from March to August 2020 to assess academics’ and institutional attributes, perceived preparedness, and availability of infection prevention and control (IPC) equipment. Principal component analysis (PCA) identified perceived preparedness components. Multilevel linear regression analysis assessed the association between perceived preparedness and fixed effect factors (academics’ and institutions’ attributes) with countries as random effect variable. Of the 1820 dental academics from 28 countries, 78.4% worked in public institutions and 75.2% reported temporary closure. PCA showed five components: clinic apparel, measures before and after patient care, institutional policies, and availability of IPC equipment. Significantly less perceived preparedness was reported in lower-middle income (LMICs) (B = −1.31, p = 0.006) and upper-middle income (UMICs) (B = −0.98, p = 0.02) countries than in high-income countries (HICs), in teaching only (B = −0.55, p < 0.0001) and in research only (B = −1.22, p = 0.003) than teaching and research institutions and in institutions receiving ≤100 patients daily than those receiving >100 patients (B = −0.38, p < 0.0001). More perceived preparedness was reported by academics with administrative roles (B = 0.59, p < 0.0001). Academics from low-income countries (LICs) and LMICs reported less availability of clinic apparel, IPC equipment, measures before patient care, and institutional policies but more measures during patient care. There was greater perceived preparedness in HICs and institutions with greater involvement in teaching, research, and patient care

Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43.

Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43

IQGAP1 Is Involved in Post-Ischemic Neovascularization by Regulating Angiogenesis and Macrophage Infiltration

Neovascularization is an important repair mechanism in response to ischemic injury and is dependent on inflammation, angiogenesis and reactive oxygen species (ROS). IQGAP1, an actin-binding scaffold protein, is a key regulator for actin cytoskeleton and motility. We previously demonstrated that IQGAP1 mediates vascular endothelial growth factor (VEGF)-induced ROS production and migration of cultured endothelial cells (ECs); however, its role in post-ischemic neovascularization is unknown.Ischemia was induced by left femoral artery ligation, which resulted in increased IQGAP1 expression in Mac3(+) macrophages and CD31(+) capillary-like ECs in ischemic legs. Mice lacking IQGAP1 exhibited a significant reduction in the post-ischemic neovascularization as evaluated by laser Doppler blood flow, capillary density and α-actin positive arterioles. Furthermore, IQGAP1(-/-) mice showed a decrease in macrophage infiltration and ROS production in ischemic muscles, leading to impaired muscle regeneration and increased necrosis and fibrosis. The numbers of bone marrow (BM)-derived cells in the peripheral blood were not affected in these knockout mice. BM transplantation revealed that IQGAP1 expressed in both BM-derived cells and tissue resident cells, such as ECs, is required for post-ischemic neovascularization. Moreover, thioglycollate-induced peritoneal macrophage recruitment and ROS production were inhibited in IQGAP1(-/-) mice. In vitro, IQGAP1(-/-) BM-derived macrophages showed inhibition of migration and adhesion capacity, which may explain the defective macrophage recruitment into the ischemic tissue in IQGAP1(-/-) mice.IQGAP1 plays a key role in post-ischemic neovascularization by regulating, not only, ECs-mediated angiogenesis but also macrophage infiltration as well as ROS production. Thus, IQGAP1 is a potential therapeutic target for inflammation- and angiogenesis-dependent ischemic cardiovascular diseases

Simultaneous siRNA Targeting of Src and Downstream Signaling Molecules Inhibit Tumor Formation and Metastasis of a Human Model Breast Cancer Cell Line

Src and signaling molecules downstream of Src, including signal transducer and activator of transcription 3 (Stat3) and cMyc, have been implicated in the development, maintenance and/or progression of several types of human cancers, including breast cancer. Here we report the ability of siRNA-mediated Src knock-down alone, and simultaneous knock-down of Src and Stat3 and/or cMyc to inhibit the neoplastic phenotype of a highly metastatic human model breast cancer cell line, MDA-MB-435S, a widely used model for breast cancer research.Src and its downstream signaling partners were specifically targeted and knocked-down using siRNA. Changes in the growth properties of the cultured cancer cells/tumors were documented using assays that included anchorage-dependent and -independent (in soft agar) cell growth, apoptosis, and both primary and metastatic tumor growth in the mouse tumor model. siRNA-mediated Src knock-down alone, and simultaneous knock-down of Src and Stat3 and/or cMyc inhibited the neoplastic phenotype of a highly metastatic human model breast cancer cell line, MDA-MB-435S. This knock-down resulted in reduced growth in monolayer and soft agar cultures, and a reduced ability to form primary tumors in NOD/SCID mice. In addition, direct intra-tumoral injection of siRNAs targeting these signaling molecules resulted in a substantial inhibition of tumor metastases as well as of primary tumor growth. Simultaneous knock-down of Src and Stat3, and/or Myc exhibited the greatest effects resulting in substantial inhibition of primary tumor growth and metastasis.These findings demonstrate the effectiveness of simultaneous targeting of Src and the downstream signaling partners Stat3 and/or cMyc to inhibit the growth and oncogenic properties of a human cancer cell line. This knowledge may be very useful in the development of future therapeutic approaches involving targeting of specific genes products involved in tumor growth and metastasis

Prognostic Significance and Gene Expression Profiles of p53 Mutations in Microsatellite-Stable Stage III Colorectal Adenocarcinomas

Although the prognostic value of p53 abnormalities in Stage III microsatellite stable (MSS) colorectal cancers (CRCs) is known, the gene expression profiles specific to the p53 status in the MSS background are not known. Therefore, the current investigation has focused on identification and validation of the gene expression profiles associated with p53 mutant phenotypes in MSS Stage III CRCs. Genomic DNA extracted from 135 formalin-fixed paraffin-embedded tissues, was analyzed for microsatellite instability (MSI) and p53 mutations. Further, mRNA samples extracted from five p53-mutant and five p53-wild-type MSS-CRC snap-frozen tissues were profiled for differential gene expression by Affymetrix Human Genome U133 Plus 2.0 arrays. Differentially expressed genes were further validated by the high-throughput quantitative nuclease protection assay (qNPA), and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and by immunohistochemistry (IHC). Survival rates were estimated by Kaplan-Meier and Cox regression analyses. A higher incidence of p53 mutations was found in MSS (58%) than in MSI (30%) phenotypes. Both univariate (log-rank, P = 0.025) and multivariate (hazard ratio, 2.52; 95% confidence interval, 1.25–5.08) analyses have demonstrated that patients with MSS-p53 mutant phenotypes had poor CRC-specific survival when compared to MSS-p53 wild-type phenotypes. Gene expression analyses identified 84 differentially expressed genes. Of 49 down-regulated genes, LPAR6, PDLIM3, and PLAT, and, of 35 up-regulated genes, TRIM29, FUT3, IQGAP3, and SLC6A8 were confirmed by qNPA, qRT-PCR, and IHC platforms. p53 mutations are associated with poor survival of patients with Stage III MSS CRCs and p53-mutant and wild-type phenotypes have distinct gene expression profiles that might be helpful in identifying aggressive subsets

Spermatogenesis-Specific Features of the Meiotic Program in Caenorhabditis elegans

In most sexually reproducing organisms, the fundamental process of meiosis is implemented concurrently with two differentiation programs that occur at different rates and generate distinct cell types, sperm and oocytes. However, little is known about how the meiotic program is influenced by such contrasting developmental programs. Here we present a detailed timeline of late meiotic prophase during spermatogenesis in Caenorhabditis elegans using cytological and molecular landmarks to interrelate changes in chromosome dynamics with germ cell cellularization, spindle formation, and cell cycle transitions. This analysis expands our understanding C. elegans spermatogenesis, as it identifies multiple spermatogenesis-specific features of the meiotic program and provides a framework for comparative studies. Post-pachytene chromatin of spermatocytes is distinct from that of oocytes in both composition and morphology. Strikingly, C. elegans spermatogenesis includes a previously undescribed karyosome stage, a common but poorly understood feature of meiosis in many organisms. We find that karyosome formation, in which chromosomes form a constricted mass within an intact nuclear envelope, follows desynapsis, involves a global down-regulation of transcription, and may support the sequential activation of multiple kinases that prepare spermatocytes for meiotic divisions. In spermatocytes, the presence of centrioles alters both the relative timing of meiotic spindle assembly and its ultimate structure. These microtubule differences are accompanied by differences in kinetochores, which connect microtubules to chromosomes. The sperm-specific features of meiosis revealed here illuminate how the underlying molecular machinery required for meiosis is differentially regulated in each sex

Perceived Preparedness of Dental Academic Institutions to Cope with the COVID-19 Pandemic: A Multi-Country Survey

Dental academic institutions are affected by COVID-19. We assessed the perceived COVID-19 preparedness of these institutions and the characteristics of institutions with greater perceived preparedness. An international cross-sectional survey of dental academics was conducted from March to August 2020 to assess academics’ and institutional attributes, perceived preparedness, and availability of infection prevention and control (IPC) equipment. Principal component analysis (PCA) identified perceived preparedness components. Multilevel linear regression analysis assessed the association between perceived preparedness and fixed effect factors (academics’ and institutions’ attributes) with countries as random effect variable. Of the 1820 dental academics from 28 countries, 78.4% worked in public institutions and 75.2% reported temporary closure. PCA showed five components: clinic apparel, measures before and after patient care, institutional policies, and availability of IPC equipment. Significantly less perceived preparedness was reported in lower-middle income (LMICs) (B = −1.31, p = 0.006) and upper-middle income (UMICs) (B = −0.98, p = 0.02) countries than in high-income countries (HICs), in teaching only (B = −0.55, p < 0.0001) and in research only (B = −1.22, p = 0.003) than teaching and research institutions and in institutions receiving ≤100 patients daily than those receiving >100 patients (B = −0.38, p < 0.0001). More perceived preparedness was reported by academics with administrative roles (B = 0.59, p < 0.0001). Academics from low-income countries (LICs) and LMICs reported less availability of clinic apparel, IPC equipment, measures before patient care, and institutional policies but more measures during patient care. There was greater perceived preparedness in HICs and institutions with greater involvement in teaching, research, and patient care