Epigenetic balance ensures mechanistic control of MLL amplification and rearrangement.

MLL/KMT2A amplifications and translocations are prevalent in infant, adult, and therapy-induced leukemia. However, the molecular contributor(s) to these alterations are unclear. Here, we demonstrate that histone H3 lysine 9 mono- and di-methylation (H3K9me1/2) balance at the MLL/KMT2A locus regulates these amplifications and rearrangements. This balance is controlled by the crosstalk between lysine demethylase KDM3B and methyltransferase G9a/EHMT2. KDM3B depletion increases H3K9me1/2 levels and reduces CTCF occupancy at the MLL/KMT2A locus, in turn promoting amplification and rearrangements. Depleting CTCF is also sufficient to generate these focal alterations. Furthermore, the chemotherapy doxorubicin (Dox), which associates with therapy-induced leukemia and promotes MLL/KMT2A amplifications and rearrangements, suppresses KDM3B and CTCF protein levels. KDM3B and CTCF overexpression rescues Dox-induced MLL/KMT2A alterations. G9a inhibition in human cells or mice also suppresses MLL/KMT2A events accompanying Dox treatment. Therefore, MLL/KMT2A amplifications and rearrangements are controlled by epigenetic regulators that are tractable drug targets, which has clinical implications.

Automated microscope-independent fluorescence-guided micropipette.

Glass micropipette electrodes are commonly used to provide high resolution recordings of neurons. Although it is the gold standard for single cell recordings, it is highly dependent on the skill of the electrophysiologist. Here, we demonstrate a method of guiding micropipette electrodes to neurons by collecting fluorescence at the aperture, using an intra-electrode tapered optical fiber. The use of a tapered fiber for excitation and collection of fluorescence at the micropipette tip couples the feedback mechanism directly to the distance between the target and electrode. In this study, intra-electrode tapered optical fibers provide a targeted robotic approach to labeled neurons that is independent of microscopy.

Side-viewing photoacoustic waveguide endoscopy.

Side-viewing hollow optical waveguides allow for minimally invasive endoscopy by concentrically guiding light and sound for photoacoustic generation and detection. Here, we characterize the side-viewing photoacoustic waveguide (PWG) endoscope by scanning 7.2 µm diameter carbon fiber threads within phantom tissues and animal tissues. Photoacoustic signals are carried along the 5.5 and 10.0 cm length of the PWG with minimal attenuation. Thus, this technology enables 360°, deep-tissue photoacoustic imaging. Photoacoustic signals were identified up to 8.0 mm from the PWG imaging window in an optically clear medium. The outer diameter of this device is measured as just over 1.0 mm, with the potential to be further miniaturized due to its unique design. The PWG is an ideal candidate for a myriad of pre-clinical and clinical applications where typical photoacoustic endoscopy systems are impractical, due to their size. Presented here, is the first side-viewing photoacoustic waveguide endoscope.

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry.

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

Monitoring changes in the healthy female metabolome across the menstrual cycle using GC × GC-TOFMS.

Urinary metabolomics offers a non-invasive means of obtaining information about the system-wide biological health of a patient. Untargeted metabolomics approaches using one-dimensional gas chromatography (GC) are limited due to the chemical complexity of urine, which poorly detects co-eluting low-abundance analytes. Metabolite detection and identification can be improved by applying comprehensive two-dimensional GC, allowing for the discovery of additional viable biomarkers of disease. In this work, we applied comprehensive two-dimensional GC coupled with time-of-flight mass spectrometry (GC × GC-TOFMS) to the analysis of urine samples collected daily across 28-days from 10 healthy female subjects for a personalized approach to female reproductive health monitoring. Through this analysis, we identified 935 unique volatile metabolites. Two statistical methods, a modified T-statistic and Wilcoxon Rank Sum, were applied to analyze differences in metabolome abundance on ovulation days as compared to non-ovulation days. Four metabolites (2-pentanone, 3-penten-2-one, carbon disulfide, acetone) were identified as statistically significant by the modified T-statistic but not the Rank Sum, after a false-discovery rate of 0.1 was set using a Benjamini-Hochberg correction. Subsequent analyses by boxplot indicated that the putative volatile metabolic biomarkers for fertility are expressed in increased or decreased abundance in urine on the day of ovulation. Individual analysis of metabolome expression across 28-days revealed some subject-specific features, which suggest a potential for long-term, personalized fertility monitoring using metabolomics.

Photoacoustic Flow System for the Detection of Ovarian Circulating Tumor Cells Utilizing Copper Sulfide Nanoparticles.

The development of cell-specific photoacoustic (PA) contrast agents within systems of fluidic flow provides opportunities for the accurate detection of early stage cancer metastasis. Despite the promise of exogenous contrast agents for use in clinical settings, applications are currently limited by both material biocompatibility and target specificity. In this study, folic acid functionalized copper sulfide nanoparticles (FA-CuS NPs) are synthesized to enable ovarian-cancer-specific binding and PA detection in a custom flow system. Folate receptors, known to be overexpressed on the surface of ovarian cancer cells, have remained an ideal candidate for specific targeting through functionalization on nanoparticles and other contrast agents. In combination with copper sulfide nanoparticles' strong absorbance in the near-infrared (NIR), these FA-CuS NPs are an ideal contrast agent capable of being detected by photoacoustic flow cytometry. For the first time, this study shows a potential PA contrast agent to accurately identify ovarian circulating tumor cells in flow.

Intrauterine photoacoustic and ultrasound imaging probe.

Intrauterine photoacoustic and ultrasound imaging are probe-based imaging modalities with translational potential for use in detecting endometrial diseases. This deep-tissue imaging probe design allows for the retrofitting of commercially available endometrial sampling curettes. The imaging probe presented here has a 2.92-mm diameter and approximate length of 26 cm, which allows for entry into the human endometrial cavity, making it possible to use photoacoustic imaging and high-resolution ultrasound to characterize the uterus. We demonstrate the imaging probes' ability to provide structural information of an excised pig uterus using ultrasound imaging and detect photoacoustic signals at a radial depth of 1 cm.