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Optical frequency domain reflectometry measurements in internally clad single crystal sapphire fiber have received attention in recent years due to their high temperature distributed sensing potential. As work with these fibers has proceeded, there have been some inconsistencies in the results. Deeper investigation and testing has identified two critical considerations for the proper functioning of these fibers. First, users must address the propagation of multimode light along the outer surface of the fiber. By observing the far-field image of an internally clad sapphire fiber when adding index matching fluid to the outer fiber surface, we demonstrate the effects of removing the higher order modes from these fibers. The addition of index matching fluid resulted in nearly single mode performance where multimode performance was previously observed. Second, users must address the effect that coupling the fiber to the interrogator via silica based fiber has on the internally clad sapphire fibers performance. Direct fusion splicing of silica to sapphire, as has been used in the recent work with these fibers, has a mode filtering effect which can be beneficial towards the modal behavior of the fibers. However, in this paper we demonstrate that the splicing can cause a sensing failure due to little or no low order mode light, that is useful for sensing, returning to the detector. The positive results from recent years have demonstrated that optical frequency domain reflectometry sensing performance will be successful in clad sapphire fiber; but only when the considerations described herein are addressed properly.

Figure S1. Summary of HRD testing in PrECOG and combined cisplatin cohorts; Figure S2. Distribution of HRD scores in PrECOG and combined cisplatin cohorts; Figure S3. Performance of HRD in predicting response in PrECOG and combined cisplatin cohorts; Figure S4. Logistic regression models to predict response in PrECOG and combined cisplatin cohorts; Table S1. Study cohorts for threshold training set; Table S2. Summary statistics of HRD scores in BRCA1/2 deficient samples by tissue in training; Table S3. Patient clinical and demographic data; Table S4. HRD score and association with therapy response in entire cohorts; Table S5. Univariate associations between clinical variables and RCB 0/1 or HR deficiency status; Table S6. Analysis of RCB 0/1 and pCR in combined PrECOG 0105 and Cisplatin Trials cohorts (logistic regression models adjusted for cohort)

Purpose:BRCA1/2-mutated and some sporadic triple-negative breast cancers (TNBC) have DNA repair defects and are sensitive to DNA-damaging therapeutics. Recently, three independent DNA-based measures of genomic instability were developed on the basis of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST).Experimental Design: We assessed a combined homologous recombination deficiency (HRD) score, an unweighted sum of LOH, TAI, and LST scores, in three neoadjuvant TNBC trials of platinum-containing therapy. We then tested the association of HR deficiency, defined as HRD score ≥42 or BRCA1/2 mutation, with response to platinum-based therapy.Results: In a trial of neoadjuvant platinum, gemcitabine, and iniparib, HR deficiency predicted residual cancer burden score of 0 or I (RCB 0/I) and pathologic complete response (pCR; OR = 4.96, P = 0.0036; OR = 6.52, P = 0.0058). HR deficiency remained a significant predictor of RCB 0/I when adjusted for clinical variables (OR = 5.86, P = 0.012). In two other trials of neoadjuvant cisplatin therapy, HR deficiency predicted RCB 0/I and pCR (OR = 10.18, P = 0.0011; OR = 17.00, P = 0.0066). In a multivariable model of RCB 0/I, HR deficiency retained significance when clinical variables were included (OR = 12.08, P = 0.0017). When restricted to BRCA1/2 nonmutated tumors, response was higher in patients with high HRD scores: RCB 0/I P = 0.062, pCR P = 0.063 in the neoadjuvant platinum, gemcitabine, and iniparib trial; RCB 0/I P = 0.0039, pCR P = 0.018 in the neoadjuvant cisplatin trials.Conclusions: HR deficiency identifies TNBC tumors, including BRCA1/2 nonmutated tumors more likely to respond to platinum-containing therapy. Clin Cancer Res; 22(15); 3764–73. ©2016 AACR.

Recent advancements in fiber optic manufacturing, sensor design, and fiber optic interrogators have provided significant opportunities towards the development of cross-cutting fiber optic sensing solutions across the nuclear industry. The addressable harsh nuclear environment markets include Gen II, II+ and IV nuclear reactors, fusion reactors, and accelerator systems. In this work the authors present a series of developments towards the implementation of singlefiber, multipoint, temperature and pressure sensors, test results in high-temperature and high-radiation environments, cryogenic environments, material compatibility studies for sensor packaging, and future development needs to address technical challenges towards sensor commercialization.

Circulating tumor cells (CTCs), CTC heterogeneity and distinct morphological CTC subtypes predict worse survival in metastatic breast cancer (MBC)BackgroundThe presence of CTCs has been shown to be a poor prognostic factor in patients with MBC. Studies in prostate cancer have shown patients with distinct morphological subtypes of CTCs have worse outcomes than high CTC burden alone and could have treatment implications. In this study, we report a comprehensive analysis of CTC phenotypes in MBC and their impact on survival.MethodsA total of 148 blood samples from 90 patients with MBC whose disease was progressing on therapy were enrolled for CTC analysis. Blood was sent overnight to Epic Sciences, processed onto glass slides and biobanked. Replicate slides were stained with antibodies targeting the androgen receptor (AR), estrogen receptor (ER), and human epidermal growth factor receptor 2 (HER2) in addition to cytokeratin, CD45, and DAPI. Total CTCs were quantified per sample from approximately 3 mL of blood. A threshold defining positive AR, ER, and HER2 protein expression was set using cultured cancer cell lines expressing the individual proteins. CTC morphology subtypes (heterogeneity [Scher et al Cancer Res 2017], small cell/neuroendocrine-like [SC/NE; Brown et al CCR 2021] and chromosomal instability [CIN+; Schonhoft et al Cancer Res 2020]) were quantified as previously described. High heterogeneity samples (Het+) were defined as having a Shannon Index (SI; a measure of morphologic diversity) of ≥1. CIN positivity was defined as having ≥0.7 CIN+ CTC/mL. Association with these CTC subtypes and survival were evaluated with a univariate COX proportional hazard model. ResultsOver 90% of samples and patients had CTCs (Table 1). The percentage of patients with the CTC subtypes evaluated are below. Table 1. Percentage of blood samples with CTCs and CTC subtypes in MBC patients, by MBC subtype The presence of CTCs was associated with worse survival, with a median survival of 15.3 months (mos) vs 24.0 mos with no CTCs (p=0.028). The presence of AR+ or HER2+ CTCs was not associated with survival. Analysis of ER is ongoing and will be presented. High morphologic heterogeneity or the presence of the SC/NE or CIN+ CTC subtypes, predicted for a worse survival outcome (Table 2). Table 2. Overall survival of patients with and without distinct CTC subtypes CTC morphological subtypes did not correlate with each other, except CTC heterogeneity by SI which correlated weakly with SC/NE and CIN+. HER2+ CTCs strongly correlated with a higher SI while AR+ CTCs did not. AR and HER2 expression did not associate with the CIN+ or SC/NE subtypes. ConclusionsDigital pathology analysis of CTCs identifies distinct CTC morphological subtypes that predict for worse prognosis in MBC patients. To our knowledge, this is the first report of SC/NE CTCs in MBC patients and that MBC patients with SC/NE and CIN+ CTCs have worse outcomes. Longitudinal characterization of CTC subtypes across the MBC continuum could potentially identify effective therapies against these more lethal cancers. Citation Format: Joyce A O'Shaughnessy, Maren K Levin, Esther San Roman Rodriguez, James Lu, Joshua T Jones, Amanda KL Anderson, Alisa Tubbs, Joseph D Schonhoft, Rick Wenstrup. Circulating tumor cells (CTCs), CTC heterogeneity and distinct morphological CTC subtypes predict worse survival in metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-03.

The focus of this work is to model the creation of a cladding, which is internal to a sapphire optical fiber, by irradiating a sapphire fiber, that is surrounded by an annulus of Li-6 enriched lithium carbonate powder, in a nuclear reactor. Such a fiber has been created using the Ohio State University Research Reactor. The 6Li(n,α)3H reaction creates high energy tritons and alpha particles that irradiate the fiber simultaneously to a depth of 24 microns, along the entire periphery of the sapphire fiber. The irradiation slightly reduces the index of refraction in the fiber’s periphery, thus creating a refractive cladding within the fiber. The Monte Carlo radiation transport code MCNP was used in combination with SRIM/TRIM to predict the modification of the fiber that results from the irradiation. Our analysis predicts that whether it is displacement damage or the density of implanted ions that is responsible for the modification of η, or a combination of both, the irradiation yields a graded index in the fiber, with η decreasing monotonically from the value of the native sapphire 24 micrometers from the surface of the fiber to a minimum value at the surface of the fiber.

The authors have developed a single-mode sapphire sensor for distributed temperature and flow measurement to address the extreme environments encountered in energy applications. The sensor is designed to also detect and localize fouling and deposits that accumulate on its surface over time. Optical frequency-domain reflectometry (OFDR) and spectral back scatter analysis are employed with the single mode sapphire fiber to yield these distributed measurements. Temperature accuracy was on the order of 5°C for measurements ranging from room temperature to over 1000°C. Spatial resolution of 11 mm was attained and enabled visualization of the temperature gradients along a fiber passing through a furnace. The effects of cooling flow were characterized for steady operation, with the intent to leverage this data in future work to infer velocity profiles of high temperature flows. Dynamic cooling flow tests showed that the presence of simulated deposits on the outside of the sensor resulted in slower time response in the vicinity of the deposit. This technique could be used to determine the presence and location of deposits along the length of the sensor assembly. The newly developed sensor will be applicable to fossil energy production, nuclear energy production, and gas turbine engines or generators.

Cell cycle analysis typically relies on fixed time-point measurements of cells in particular phases of the cell cycle. The cell cycle, however, is a dynamic process whose subtle shifts are lost by fixed time-point methods. Live-cell fluorescent biosensors and time-lapse microscopy allows the collection of temporal information about real time cell cycle progression and arrest. Using two genetically-encoded biosensors, we measured the precision of the G(1), S, G(2) and M cell cycle phase durations in different cell types and identified a bimodal G(1) phase duration in a fibroblast cell line that is not present in the other cell types. Using a cell line model for neuronal differentiation, we demonstrated that NGF-induced neurite extension occurs independently of NGF-induced cell cycle G(1) phase arrest. Thus, we have begun to use cell cycle fluorescent biosensors to examine the proliferation of cell populations at the resolution of individual cells and neuronal differentiation as a dynamic process of parallel cell cycle arrest and neurite outgrowth.

Precise timing of mitosis is essential for high-fidelity cell duplication. However, temporal measurements of the mitotic clock have been challenging. Here we present a fluorescent mitosis biosensor that monitors the time between nuclear envelope breakdown (NEB) and re-formation using parallel total internal reflection fluorescence (TIRF) microscopy. By tracking tens to hundreds of mitotic events per experiment, we found that the mitotic clock of unsynchronized rat basophilic leukemia cells has a marked precision with 80% of cells completing mitosis in 32 +/- 6 min. This assay further allowed us to observe delays in mitotic timing at Taxol concentrations 100 times lower than previous minimal effective doses, explaining why Taxol is clinically active at low concentrations. Inactivation of the spindle checkpoint by targeting the regulator Mad2 with RNAi consistently shortened mitosis, providing direct evidence that the internal mitotic timing mechanism is much faster in cells that lack the checkpoint.

The ability of ribozymes to cleave specific transcripts and repair defective RNAs in the test tube has engendered speculation about their potential clinical utility. Therapeutic development has been hindered by an inability to evaluate and optimize the efficiency of RNA catalysis in vivo. We describe an experimental system that has allowed us to assess and enhance the efficiency with which a trans-splicing group I ribozyme reacts with a targeted RNA in mammalian cells. These results demonstrate that the ribozyme can convert up to 49% of a specific substrate RNA to product in the cellular environment and that the efficiency of this reaction is apparently a function of the ribozyme's ability to find and bind to the substrate RNA in the cell. These observations suggest that trans-splicing ribozymes may become useful reagents to repair a therapeutically significant fraction of mutant RNAs associated with a variety of genetic diseases.

High content cell-based genetic and small molecule library screens are powerful strategies in drug discovery and investigations of disease mechanisms. We report that primary cells derived from a transgenic mouse model expressing a fluorescence mitosis biosensor provide unambiguous phenotype readouts without the need for transfection or immunocytochemistry. Phenotype profiles of cell cycle disruption and of apoptosis are easily detectable at a single time point selected from time-lapse live fluorescence microscopy. Most importantly, this transgenic mouse model may be crossed with cancer mouse models to derive biosensor-expressing primary cancer cells for use in high content screening strategies targeting discovery of tumor-specific chemotherapeutic compounds.

A survey of 1,804 human dicer-generated signaling siRNAs using automated quantitative imaging identified the phosphatidylinositol-3,4,5-trisphosphate-mTOR signaling pathway as a primary regulator of iron-transferrin uptake., Background Iron uptake via endocytosis of iron-transferrin-transferrin receptor complexes is a rate-limiting step for cell growth, viability and proliferation in tumor cells as well as non-transformed cells such as activated lymphocytes. Signaling pathways that regulate transferrin uptake have not yet been identified. Results We surveyed the human signaling proteome for regulators that increase or decrease transferrin uptake by screening 1,804 dicer-generated signaling small interfering RNAs using automated quantitative imaging. In addition to known transport proteins, we identified 11 signaling proteins that included a striking signature set for the phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3)-target of rapamycin (mTOR) signaling pathway. We show that the PI3K-mTOR signaling pathway is a positive regulator of transferrin uptake that increases the number of transferrin receptors per endocytic vesicle without affecting endocytosis or recycling rates. Conclusion Our study identifies the PtdIns(3,4,5)P3-mTOR signaling pathway as a new regulator of iron-transferrin uptake and serves as a proof-of-concept that targeted RNA interference screens of the signaling proteome provide a powerful and unbiased approach to discover or rank signaling pathways that regulate a particular cell function.

Summary The mitotic spindle assembles into a bipolar, microtubule-based protein machine during prometaphase. One proposed mechanism for this process is "search-and-capture," in which dynamically unstable microtubules (MTs) search space to capture chromosomes [1]. Although existing theoretical estimates [2, 3] suggest that dynamic instability is efficient enough to allow capture within characteristic mitotic timescales, they are limited in scope and do not address the capture times for realistic numbers of chromosomes. Here we used mathematical modeling to explore this issue. We show that without any bias toward the chromosomes, search-and-capture is not efficient enough to explain the typical observed duration of prometaphase. We further analyze search-and-capture in the presence of a spatial gradient of a stabilizing factor [4–6] that biases MT dynamics toward the chromosomes. We show theoretically that such biased search-and-capture is efficient enough to account for chromosome capture. We also show that additional factors must contribute to accelerate the spindle assembly for cells with large nuclear volumes. We discuss the possibility that a RanGTP gradient introduces a spatial bias into microtubule dynamics and thus improves the efficiency of search-and-capture as a mechanism for spindle assembly.

In mammalian cells, genetic instructions are usually revised by RNA splicing before they are translated to proteins. Here we demonstrate that a trans-splicing group I ribozyme can be employed to intentionally modify the sequence of targeted transcripts in tissue culture cells. By analyzing the ribozyme reaction products, we demonstrate that targeted trans-splicing can proceed in murine fibroblasts with high fidelity, providing direct evidence that ribozymes function as anticipated in a therapeutically relevant setting. Trans-splicing is not very specific however, and the ribozyme reacted with and tagged a variety of cellular transcripts with its 3' exon sequence. RNA tagging provides a unique approach to study RNA catalysis in mammalian cells. Such analysis should facilitate the logical development of safe, therapeutic ribozymes that can repair mutant RNAs associated with a variety of inherited diseases.

Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.