A Conserved Bicycle Model for Circadian Clock Control of Membrane Excitability

SummaryCircadian clocks regulate membrane excitability in master pacemaker neurons to control daily rhythms of sleep and wake. Here, we find that two distinctly timed electrical drives collaborate to impose rhythmicity on Drosophila clock neurons. In the morning, a voltage-independent sodium conductance via the NA/NALCN ion channel depolarizes these neurons. This current is driven by the rhythmic expression of NCA localization factor-1, linking the molecular clock to ion channel function. In the evening, basal potassium currents peak to silence clock neurons. Remarkably, daily antiphase cycles of sodium and potassium currents also drive mouse clock neuron rhythms. Thus, we reveal an evolutionarily ancient strategy for the neural mechanisms that govern daily sleep and wake

Survival of the resilient: Mechano-adaptation of circulating tumor cells to fluid shear stress

During metastasis, cancer cells traverse the circulation to reach distant organs. Conventionally, this journey has been regarded as mechanically destructive to circulating tumor cells from solid tissues. We have recently shown that cancer cells from diverse tissues actively resist destruction by fluid shear stress through a mechano-adaptive RhoA-actomyosin mechanism

The Narrow Abdomen Ion Channel Complex Is Highly Stable and Persists from Development into Adult Stages to Promote Behavioral Rhythmicity

The sodium leak channel NARROW ABDOMEN (NA)/ NALCN is an important component of circadian pacemaker neuronal output. In Drosophila, rhythmic expression of the NA channel regulator Nlf-1 in a subset of adult pacemaker neurons has been proposed to contribute to circadian regulation of channel localization or activity. Here we have restricted expression of Drosophila NA channel subunits or the Nlf-1 regulator to either development or adulthood using the temperature-inducible tubulin-GAL80ts system. Surprisingly, we find that developmental expression of endogenous channel subunits and Nlf-1 is sufficient to promote robust rhythmic behavior in adults. Moreover, we find that channel complex proteins produced during development persist in the Drosophila head with little decay for at least 5–7 days in adults. In contrast, restricting either endogenous or transgenic gene expression to adult stages produces only limited amounts of the functional channel complex. These data indicate that much of the NA channel complex that functions in adult circadian neurons is normally produced during development, and that the channel complex is very stable in most neurons in the Drosophila brain. Based on these findings, we propose that circadian regulation of NA channel function in adult pacemaker neurons is mediated primarily by post-translational mechanisms that are independent of Nlf-1

Quantification of capture efficiency, purity, and single-cell isolation in the recovery of circulating melanoma cells from peripheral blood by dielectrophoresis

This paper describes a dielectrophoretic method for selection of circulating melanoma cells (CMCs), which lack reliable identifying surface antigens and are extremely rare in blood. This platform captures CMCs individually by dielectrophoresis (DEP) at an array of wireless bipolar electrodes (BPEs) aligned to overlying nanoliter-scale chambers, which isolate each cell for subsequent on-chip single-cell analysis. To determine the best conditions to employ for CMC isolation in this DEP-BPE platform, the static and dynamic dielectrophoretic response of established melanoma cell lines, melanoma cells from patient-derived xenografts (PDX) and peripheral blood mononuclear cells (PBMCs) were evaluated as a function of frequency using two established DEP platforms. Further, PBMCs derived from patients with advanced melanoma were compared with those from healthy controls. The results of this evaluation reveal that each DEP method requires a distinct frequency to achieve capture of melanoma cells and that the distribution of dielectric properties of PBMCs is more broadly varied in and among patients versus healthy controls. Based on this evaluation, we conclude that 50 kHz provides the highest capture efficiency on our DEP-BPE platform while maintaining a low rate of capture of unwanted PBMCs. We further quantified the efficiency of single-cell capture on the DEP-BPE platform and found that the efficiency diminished beyond around 25% chamber occupancy, thereby informing the minimum array size that is required. Importantly, the capture efficiency of the DEP-BPE platform for melanoma cells when using optimized conditions matched the performance predicted by our analysis. Finally, isolation of melanoma cells from contrived (spike-in) and clinical samples on our platform using optimized conditions was demonstrated. The capture and individual isolation of CMCs, confirmed by post-capture labeling, from patient-derived samples suggests the potential of this platform for clinical application.This is a pre-print of the article Chen, Han, Sommer Osman, Devon Moose, Marion Vanneste, Jared Anderson, Michael Henry, and Robbyn Anand. “Quantification of Capture Efficiency, Purity, and Single-Cell Isolation in the Recovery of Circulating Melanoma Cells from Peripheral Blood by Dielectrophoresis.” ChemRxiv, 2023. DOI: 10.26434/chemrxiv-2023-cs884. Attribution-NonCommercial 4.0 International (CC BY-NC 4.0). Copyright 2023 The Authors. Posted with permission

Generation of <i>Trp53/Pten</i> mice associated with luciferase expression.

<p>A) ROSA26 Lox-stop-Lox Luciferase, Pten^fl/fl male mice were crossed with Trp53^fl/fl female mice to generate offspring with ROSA26 LSL-Luc, Pten^fl/fl, and Trp53^fl/fl. Ad with a Cre recombinase expression vector was injected either subcutaneously over the right leg/flank or intramuscularly into the right quadriceps to promote somatic recombination. Somatic expression of Cre recombinase promoted expression of luciferase and homozygous knockout of <i>Pten</i> and <i>Trp53</i>. B) PCR from DNA isolated from either a tumor of Cre injected mice (Cre+) or from the tail of a non-injected mouse (Cre-) (Ctrl is a primer only control) to assay for the presence of recombination and floxed <i>Pten</i> or <i>Trp53</i>.</p

<i>Trp53/Pten</i> undifferentiated pleomorphic sarcomas demonstrate immune tolerance.

<p>A) Histology of UPS reveals an infiltrate of small mature lymphocytes (arrow), which is further confirmed by immunohistochemistry for CD45 (B, arrow). Immunohistochemistry was performed for PD-L1 (B7-H1), showing expression patterns ranging from focal/variable staining (C), to diffusely and strongly positive (D), suggesting that UPS in the <i>Trp53/PTEN</i> model are immunogenic and might evade immunosurveillance by PD-L1 upregulation.</p

<i>Trp53/Pten</i> mice injected with Ad CMV-Cre demonstrate a longitudinal increase in BLI signal.

<p>A) BLI of <i>Trp53/Pten</i> mice injected either SQ or IM with Ad CMV-Cre. Mice were injected with intraperitoneal D-luciferin and imaged using an Ami X imager. Shown are representative images of five mice from each cohort at 1 week, 9 weeks, and 17 weeks post-viral injection, demonstrating a steady increase in luciferase signal over time. B) Quantified whole-body <i>in vivo</i> BLI values as measured in photons/sec/cm²/sr are shown for individual mice for each injection group (n = 9), and compared between the two injection groups C), highlighting similar luciferase expression kinetics and values between SQ- and IM-injected animals. The difference between SQ and IM injection was statistically significant for day 1 post-injection (p = 0.045), but not thereafter. From the 2way ANOVA, overall variance due to injection method was statistically significant (p = 0.0019). In comparison to the first day post-injection, all values for both injection methods were significant (p<0.0001).</p

Trp53^fl/flPten^fl/fl mice injected with Ad CMV-Cre form soft tissue tumors with 100% penetrance.

<p>A) Photographs of representative intact SQ and IM soft tissue tumors (*). B) Primary tumor dissections highlighting the superficial nature of tumors in the SQ-injected group compared to the deep, invasive tumors in the IM group, which develop within the native quadriceps muscle and adhere firmly to the surrounding femur and musculature. C) Palpable tumor latency as defined by time to earliest manual palpation of soft tissue tumor (p = 0.3201) D) Primary tumor mass and tumor volume with corresponding BLI measurements. Tumor mass (blue squares, right y-axis) measured after surgical excision and volume (red circles, left y-axis) measured by calipers are correlated with photon flux (X-axis); Spearman <i>r</i> = 0.857 and r = 0.733, respectively. Asterisk indicates one outlying tumor that was removed from the analysis.</p

IM-injected Ad CMV-Cre results in increased systemic viral spread.

<p>A) Quantified leg-only or abdomen-only BLI values are shown for individual Trp53^fl/flPten^fl/fl mice injected either SQ or IM with Ad CMV-Cre. B) Comparisons between injection cohorts demonstrate lower non-specific signal in the abdomen with SQ injection. There was no significant difference in luciferase signal between the SQ- or IM-injected legs; however, there is a significant difference between SQ and IM abdomen injections for week 1 and beyond (p<0.05). The difference in comparison to the first day post-injection was statistically significant for both SQ (p<0.002) and IM (p<0.0001) leg injections as well as IM abdominal BLI (p<0.02). No significant difference was found for SQ abdominal BLI in comparison to day 1. C) <i>Ex vivo</i> BLI images of livers from one SQ- and one IM-injected mouse, depicting multifocal low-level recombination and luciferase expression in the livers of mice with abdominal BLI signal, suggesting systemic viral spread and infection (left panels). Histological sections of liver demonstrated extramedullary hematopoiesis, noted with arrows, but no neoplasm was identified (right panel).</p