Harjus Birk

The human genome produces thousands of long noncoding RNAs (lncRNAs)-transcripts >200 nucleotides long that do not encode proteins. Although critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. We developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in seven diverse cell lines, including six transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth-modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type-specific manner. These data underscore the functional importance and cell type specificity of many lncRNAs.

The potentiostatic electrochemical template synthesis of nanowires (Ni, Co, Cu, Au, and polyporrole) in polycarbonate track-etched membranes with nominal pore diameters dN between 10 and 200 nm is studied. Along the wire the cross section is found to vary: the wire diameter, which is argued to directly reflect the pore diameter, is observed (for all deposits) to be substantially larger in the middle than at both ends. Therefore, the pores are not cylindrical with constant cross-section, in general, but appear to be “cigarlike”. Inside the membrane, the pores are wider by up to a factor 3. Comparing the potentiostatically measured current-time characteristics obtained during wire growth for different pore dimensions, a pore-size dependence of the diffusion coefficient D for the metal ions is found: D = 2.5, 1.5, and 0.7 × 10-6 cm2/s for dN = 80, 30, and 10 nm, respectively.

Electrical current fluctuations through tunnel junctions are studied with a scanning-tunneling microscope. For single-tunnel junctions classical Poisson shot noise is observed, indicative for uncorrelated tunneling of electrons. For double-barrier tunnel junctions, formed by a nanoparticle between tip and surface, the shot noise is observed to be suppressed below the Poisson value. For strongly asymmetric junctions, where a Coulomb staircase is observed in the current-voltage characteristic, the shot-noise suppression is periodic in the applied voltage. This originates from correlations in the transfer of electrons imposed by single-electron charging effects.

We have demonstrated that a gold scanning tunneling microscope (STM) tip can be used as a miniature solid-state emission source for directly depositing nanometer-size gold structures. The process has been demonstrated in ultrahigh vacuum on gold substrates, and in air on gold and platinum substrates. Studies made in air suggest that the process is fast, repeatable, and field-induced. The emission mechanism is believed to be field evaporation of tip atoms, which is enhanced by the close proximity of the substrate. The technique has been used to write several thousand features with no apparent degradation of the tip’s ability to write. Elevated and room temperature studies show the written structures to be stable over periods of weeks, in contrast to some previous STM measurements of gold self-diffusion.

BACKGROUND: Long non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients. RESULTS: We use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy. CONCLUSIONS: These studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy.