Label-Free Techniques for Probing Biomolecular Condensates.

Biomolecular condensates play important roles in a wide array of fundamental biological processes, such as cellular compartmentalization, cellular regulation, and other biochemical reactions. Since their discovery and first observations, an extensive and expansive library of tools has been developed to investigate various aspects and properties, encompassing structural and compositional information, material properties, and their evolution throughout the life cycle from formation to eventual dissolution. This Review presents an overview of the expanded set of tools and methods that researchers use to probe the properties of biomolecular condensates across diverse scales of length, concentration, stiffness, and time.

Label-Free Imaging of DNA Interactions with 2D Materials.

Two-dimensional (2D) materials offer potential as substrates for biosensing devices, as their properties can be engineered to tune interactions between the surface and biomolecules. Yet, not many methods can measure these interactions in a liquid environment without introducing labeling agents such as fluorophores. In this work, we harness interferometric scattering (iSCAT) microscopy, a label-free imaging technique, to investigate the interactions of single molecules of long dsDNA with 2D materials.

SMC complexes can traverse physical roadblocks bigger than their ring size.

Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use in vitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin.

CENP-B-mediated DNA loops regulate activity and stability of human centromeres.

Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins.

FIB-milled plasmonic nanoapertures allow for long trapping times of individual proteins.

We have developed a fabrication methodology for label-free optical trapping of individual nanobeads and proteins in inverted-bowtie-shaped plasmonic gold nanopores. Arrays of these nanoapertures can be reliably produced using focused ion beam (FIB) milling with gap sizes of 10-20 nm, single-nanometer variation, and with a remarkable stability that allows for repeated use. We employ an optical readout where the presence of the protein entering the trap is marked by an increase in the transmission of light through the nanoaperture from the shift of the plasmonic resonance.

DNA sequence-directed cooperation between nucleoid-associated proteins.

Nucleoid-associated proteins (NAPs) are a class of highly abundant DNA-binding proteins in bacteria and archaea. While both the composition and relative abundance of the NAPs change during the bacterial growth cycle, surprisingly little is known about their crosstalk in mutually binding and stabilizing higher-order nucleoprotein complexes in the bacterial chromosome. Here, we use atomic force microscopy and solid-state nanopores to investigate long-range nucleoprotein structures formed by the binding of two major NAPs, FIS and H-NS, to DNA molecules with distinct binding site arrangements.

Translocation of DNA through Ultrathin Nanoslits.

2D nanoslit devices, where two crystals with atomically flat surfaces are separated by only a few nanometers, have attracted considerable attention because their tunable control over the confinement allows for the discovery of unusual transport behavior of gas, water, and ions. Here, the passage of double-stranded DNA molecules is studied through nanoslits fabricated from exfoliated 2D materials, such as graphene or hexagonal boron nitride, and the DNA polymer behavior is examined in this tight confinement. Two types of events are observed in the ionic current: long current blockades that signal DNA translocation and short spikes where DNA enters the slits but withdraws.

Detection of CRISPR-dCas9 on DNA with Solid-State Nanopores.

Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore experiments that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing.

Double Barrel Nanopores as a New Tool for Controlling Single-Molecule Transport.

The ability to control the motion of single biomolecules is key to improving a wide range of biophysical and diagnostic applications. Solid-state nanopores are a promising tool capable of solving this task. However, molecular control and the possibility of slow readouts of long polymer molecules are still limited due to fast analyte transport and low signal-to-noise ratios.

Lithography-based fabrication of nanopore arrays in freestanding SiN and graphene membranes.

We report a simple and scalable technique for the fabrication of nanopore arrays on freestanding SiN and graphene membranes based on electron-beam lithography and reactive ion etching. By controlling the dose of the single-shot electron-beam exposure, circular nanopores of any size down to 16 nm in diameter can be fabricated in both materials at high accuracy and precision. We demonstrate the sensing capabilities of these nanopores by translocating dsDNA through pores fabricated using this method, and find signal-to-noise characteristics on par with transmission-electron-microscope-drilled nanopores.

SieveSifter: a web-based tool for visualizing the sieve analyses of HIV-1 vaccine efficacy trials.

Summary: Analysis of HIV-1 virions from participants infected in a randomized controlled preventive HIV-1 vaccine efficacy trial can help elucidate mechanisms of partial protection. By comparing the genetic sequence of viruses from vaccine and placebo recipients to the sequence of the vaccine itself, a technique called 'sieve analysis', one can identify functional specificities of vaccine-induced immune responses. We have created an interactive web-based visualization and data access tool for exploring the results of sieve analyses performed on four major preventive HIV-1 vaccine efficacy trials: (i) the HIV Vaccine Trial Network (HVTN) 502/Step trial, (ii) the RV144/Thai trial, (iii) the HVTN 503/Phambili trial and (iv) the HVTN 505 trial.

Early discharge in the bariatric population does not increase post-discharge resource utilization.

Introduction: There is a trend toward shorter-stay bariatric surgery. However, reducing LOS may increase complications and post-discharge resource utilization. Our goal was to compare outcomes before and after implementation of short-stay bariatric surgery.

Dynamic imaging of Au-nanoparticles via scanning electron microscopy in a graphene wet cell.

High resolution nanoscale imaging in liquid environments is crucial for studying molecular interactions in biological and chemical systems. In particular, electron microscopy is the gold-standard tool for nanoscale imaging, but its high-vacuum requirements make application to in-liquid samples extremely challenging. Here we present a new graphene based wet cell device where high resolution scanning electron microscope (SEM) and energy dispersive x-rays (EDX) analysis can be performed directly inside a liquid environment.

Surfactant-activated microgels: a new pathway to rheology modification.

Alkali swellable microgels are widely used to control rheology of formulated products containing surfactants. However, formulations based on these pH-responsive polymers show undesirably large changes in yield stress in a range of pH close to the pKa of the acid group. Analysis of the behavior of a cross-linked copolymer of ethyl acrylate and methacrylic acid in the nonionized form (at pH below the pKa of methacrylic acid) in the presence of sodium dodecyl sulfate shows surfactant-mediated swelling (an increase in particle diameter by over 2.

Kinase pathway dependence in primary human leukemias determined by rapid inhibitor screening.

Kinases are dysregulated in most cancers, but the frequency of specific kinase mutations is low, indicating a complex etiology in kinase dysregulation. Here, we report a strategy to rapidly identify functionally important kinase targets, irrespective of the etiology of kinase pathway dysregulation, ultimately enabling a correlation of patient genetic profiles to clinically effective kinase inhibitors. Our methodology assessed the sensitivity of primary leukemia patient samples to a panel of 66 small-molecule kinase inhibitors over 3 days.

MET receptor sequence variants R970C and T992I lack transforming capacity.

High-throughput sequencing promises to accelerate the discovery of sequence variants, but distinguishing oncogenic mutations from irrelevant "passenger" mutations remains a major challenge. Here we present an analysis of two sequence variants of the MET receptor (hepatocyte growth factor receptor) R970C and T992I (also designated R988C and T1010I). Previous reports indicated that these sequence variants are transforming and contribute to oncogenesis.

Mathematics, critical literacy, and youth participatory action research.

This article examines mathematics education as both the site and object of transformation for a youth PAR project in which students researched and evaluated their urban high school in Oakland, California. These youth researchers were trained as part of a sociology course as well as a mathematics class designed to both remediate gaps in math preparation and accelerate students into higher-order math literacy. This study differs from and extends other studies that describe mathematics as a tool for social critique.