15 research outputs found
Higher-order QED effects in hadronic processes
In this presentation, we describe the computation of higher-order QED effects
relevant in hadronic collisions. In particular, we discuss the calculation of
mixed QCD-QED one-loop contributions to the Altarelli-Parisi splittings
functions, as well as the pure two-loop QED corrections. We explain how to
extend the DGLAP equations to deal with new parton distributions, emphasizing
the consequences of the novel corrections in the determination (and evolution)
of the photon distributions.Comment: 7 pages, 2 figures. Contribution to the Proceedings of the EPS-HEP
2017 Conferenc
Nanostructure Embedded Microchips for Detection, Isolation, and Characterization of Circulating Tumor Cells
ConspectusCirculating
tumor cells (CTCs) are cancer cells that break away
from either a primary tumor or a metastatic site and circulate in
the peripheral blood as the cellular origin of metastasis. With their
role as a “tumor liquid biopsy”, CTCs provide convenient
access to all disease sites, including that of the primary tumor and
the site of fatal metastases. It is conceivable that detecting and
analyzing CTCs will provide insightful information in assessing the
disease status without the flaws and limitations encountered in performing
conventional tumor biopsies. However, identifying CTCs in patient
blood samples is technically challenging due to the extremely low
abundance of CTCs among a large number of hematologic cells. To address
this unmet need, there have been significant research endeavors, especially
in the fields of chemistry, materials science, and bioengineering,
devoted to developing CTC detection, isolation, and characterization
technologies.Inspired by the nanoscale interactions observed
in the tissue microenvironment,
our research team at UCLA pioneered a unique concept of “NanoVelcro”
cell-affinity substrates, in which CTC capture agent-coated nanostructured
substrates were utilized to immobilize CTCs with high efficiency.
The working mechanism of NanoVelcro cell-affinity substrates mimics
that of Velcro: when the two fabric strips of a Velcro fastener are
pressed together, tangling between the hairy surfaces on two strips
leads to strong binding. Through continuous evolution, three generations
(gens) of NanoVelcro CTC chips have been established to achieve different
clinical utilities. The first-gen NanoVelcro chip, composed of a silicon
nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer,
was created for CTC enumeration. Side-by-side analytical validation
studies using clinical blood samples suggested that the sensitivity
of first-gen NanoVelcro chip outperforms that of FDA-approved CellSearch.
In conjunction with the use of the laser microdissection (LMD) technique,
second-gen NanoVelcro chips (i.e., NanoVelcro-LMD), based on polymer
nanosubstrates, were developed for single-CTC isolation. The individually
isolated CTCs can be subjected to single-CTC genotyping (e.g., Sanger
sequencing and next-generation sequencing, NGS) to verify the CTC’s
role as tumor liquid biopsy. Created by grafting of thermoresponsive
polymer brushes onto SiNS, third-gen NanoVelcro chips (i.e., Thermoresponsive
NanoVelcro) have demonstrated the capture and release of CTCs at 37
and 4 °C, respectively. The temperature-dependent conformational
changes of polymer brushes can effectively alter the accessibility
of the capture agent on SiNS, allowing for rapid CTC purification
with desired viability and molecular integrity.This Account
summarizes the continuous evolution of NanoVelcro
CTC assays from the emergence of the original idea all the way to
their applications in cancer research. We envision that NanoVelcro
CTC assays will lead the way for powerful and cost-efficient diagnostic
platforms for researchers to better understand underlying disease
mechanisms and for physicians to monitor real-time disease progression
Precision-Guided Nanospears for Targeted and High-Throughput Intracellular Gene Delivery
An
efficient nonviral platform for high-throughput and subcellular
precision targeted intracellular delivery of nucleic acids in cell
culture based on magnetic nanospears is reported. These magnetic nanospears
are made of Au/Ni/Si (∼5 μm in length with tip diameters
<50 nm) and fabricated by nanosphere lithography and metal deposition.
A magnet is used to direct the mechanical motion of a single nanospear,
enabling precise control of position and three-dimensional rotation.
These nanospears were further functionalized with enhanced green fluorescent
protein (eGFP)-expression plasmids via a layer-by-layer approach before
release from the underlying silicon substrate. Plasmid functionalized
nanospears are guided magnetically to approach target adherent U87
glioblastoma cells, penetrating the cell membrane to enable intracellular
delivery of the plasmid cargo. After 24 h, the target cell expresses
green fluorescence indicating successful transfection. This nanospear-mediated
transfection is readily scalable for the simultaneous manipulation
of multiple cells using a rotating magnet. Cell viability >90%
and
transfection rates >80% were achieved, which exceed conventional
nonviral
intracellular methods. This approach is compatible with good manufacturing
practices, circumventing barriers to the translation and clinical
deployment of emerging cellular therapies
Minimal cellular input required for mutant allele detection by dPCR.
<p>HPAF-II cells and Jurkat cells were laser-microdissected to obtain exact number of cells. 1 and 10 HPAF-II cells and 200 Jurkat cells were laser-microdissected. Mixtures of 1 HPAF-II cells in 200 Jurkat cells and 10 HPAF-II cells in 200 Jurkat cells were prepared and DNA extracted. (A) dPCR was successful using DNA extracted this 210-cell mixture. Mutant <i>KRAS</i> alleles from the 10 HPAF-II cells were accurately detected. (B) dPCR detected the presence of mutant <i>KRAS</i> allele in a single HPAF-II cell in the mixture of 1 HPAF-II cell and 200 Jurkat cells.</p
dPCR <i>KRAS</i> mutation analysis in FFPE EUS-FNA pancreas biopsy specimens.
<p>dPCR <i>KRAS</i> mutation analysis in FFPE EUS-FNA pancreas biopsy specimens.</p
Workflow for the same-day <i>KRAS</i> mutation analysis in fresh EUS-FNA biopsy specimens using dPCR.
<p>EUS-FNA biopsy specimens are processed for DNA extraction within half an hour from the time of biopsy. Next, the extracted DNA is prepared for analysis with dPCR. The entire thermocycling protocol requires approximately 3 hours. Following completion of the PCR step, the dPCR chip is read and analyzed using the chip-reader. Output data are inspected manually for quality check and <i>KRAS</i> mutation status is determined.</p
Cross-reactivity of allele-specific TaqMan probes for <i>KRAS</i> mutation subtypes.
<p>(A) DNA extracted from HPAF-II (G12D <i>KRAS</i> mutation; heterozygous with 3:1 mutant allele specific amplification), CFPAC (G12V <i>KRAS</i> mutation: heterozygous with 1:1 mutant to WT alleles), MDAMB-231 (G<b>13</b>D <i>KRAS</i> mutation) and Jurkat cells (WT <i>KRAS</i>) were analyzed by dPCR using the <i>KRAS</i>_520 probe that is specific for G12V mutant subtype. Presence of the FAM fluorescence signal, which is indicative of mutant <i>KRAS</i> allele, was seen only in the dPCR reaction that used DNA extracted from CFPAC cells. (B) The same groups of DNA were analyzed using the <i>KRAS</i>_521 probe that is specific for G12D mutation subtype. As expected, FAM signal was seen only in the dPCR reaction that used DNA extracted from HPAF-II cells.</p
<i>KRAS</i> mutation analysis in fresh (non-fixed) EUS-FNA biopsy specimens.
<p><i>KRAS</i> mutation analysis in fresh (non-fixed) EUS-FNA biopsy specimens.</p
Comparison of <i>KRAS</i> mutation analysis in FFPE EUS-FNA by dPCR and surgically resected tissue by Sanger sequencing<sup>a</sup>.
<p>Comparison of <i>KRAS</i> mutation analysis in FFPE EUS-FNA by dPCR and surgically resected tissue by Sanger sequencing<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170897#t002fn001" target="_blank"><sup>a</sup></a>.</p
FFPE EUS-FNA specimens shown to have more than one <i>KRAS</i> mutations.
<p>dPCR <i>KRAS</i> analysis in FNA specimens were compared to Sanger sequencing done in matching surgically resected specimens. Since Sanger sequencing is known to have limited sensitivity, these surgical tissues were further analyzed by dPCR, which showed concordance with findings noted in FNA specimens. Mutant allele fractions for the mutant <i>KRAS</i> subtypes not seen in Sanger sequencing were all well below the theoretical limit of detection by Sanger sequencing.</p