Single and coupled cavity mode sensing schemes using a diagnostic field.

Precise optical mode matching is of critical importance in experiments using squeezed-vacuum states. Automatic spatial-mode matching schemes have the potential to reduce losses and improve loss stability. However, in quantum-enhanced coupled-cavity experiments, such as gravitational-wave detectors, one must also ensure that the sub-cavities are also mode matched. We propose what we believe to be a new mode sensing scheme, which works for simple and coupled cavities. The scheme requires no moving parts, nor tuning of Gouy phases. Instead a diagnostic field tuned to the HG20/LG10 mode frequency is used. The error signals are derived to be proportional to the difference in waist position, and difference in Rayleigh ranges, between the sub-cavity eigenmodes. The two error signals are separable by 90 degrees of demodulation phase. We demonstrate reasonable error signals for a simplified Einstein Telescope optical design. This work will facilitate routine use of extremely high levels of squeezing in current and future gravitational-wave detectors.

Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies.

Nitro fatty acids (NO2-FAs) are endogenously generated lipid signaling mediators from metabolic and inflammatory reactions between conjugated diene fatty acids and nitric oxide or nitrite-derived reactive species. NO2-FAs undergo reversible Michael addition with hyperreactive protein cysteine thiolates to induce posttranslational protein modifications that can impact protein function. Herein, we report a novel mechanism of action of natural and non-natural nitroalkenes structurally similar to (E) 10-nitro-octadec-9-enoic acid (CP-6), recently de-risked by preclinical Investigational New Drug-enabling studies and Phase 1 and Phase 2 clinical trials and found to induce DNA damage in a TNBC xenograft by inhibiting homologous-recombination (HR)-mediated repair of DNA double-strand breaks (DSB). CP-6 specifically targets Cys319, essential in RAD51-controlled HR-mediated DNA DSB repair in cells. A nitroalkene library screen identified two structurally different nitroalkenes, a non-natural fatty acid [(E) 8-nitro-nonadec-7-enoic acid (CP-8)] and a dicarboxylate ester [dimethyl (E)nitro-oct-4-enedioate (CP-23)] superior to CP-6 in TNBC cells killing, synergism with three different inhibitors of the poly ADP-ribose polymerase (PARP) and γ-IR. CP-8 and CP-23 effectively inhibited γ-IR-induced RAD51 foci formation and HR in a GFP-reported assay but did not affect benign human epithelial cells or cell cycle phases. In vivo, CP-8 and CP-23's efficacies diverged as only CP-8 showed promising anticancer activities alone and combined with the PARP inhibitor talazoparib in an HR-proficient TNBC mouse model. As preliminary preclinical toxicology analysis also suggests CP-8 as safe, our data endorse CP-8 as a novel anticancer molecule for treating cancers sensitive to homologous recombination-mediated DNA repair inhibitors.

Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies.

Nitro fatty acids (NO 2 -FAs) are endogenously generated lipid signaling mediators from metabolic and inflammatory reactions between conjugated diene fatty acids and nitric oxide or nitrite-derived reactive species. NO 2 -FAs undergo reversible Michael addition with hyperreactive protein cysteine thiolates to induce posttranslational protein modifications that can impact protein function. Herein, we report a novel mechanism of action of natural and non-natural nitroalkenes structurally similar to ( E ) 10-nitro-octadec-9-enoic acid (CP-6), recently de-risked by preclinical Investigational New Drug-enabling studies and Phase 1 and Phase 2 clinical trials and found to induce DNA damage in a TNBC xenograft by inhibiting homologous-recombination (HR)-mediated repair of DNA double-strand breaks (DSB). CP-6 specifically targets Cys319, essential in RAD51-controlled HR-mediated DNA DSB repair in cells. A nitroalkene library screen identified two structurally different nitroalkenes, a non-natural fatty acid [( E ) 8-nitro- nonadec-7-enoic acid (CP-8)] and a dicarboxylate ester [dimethyl ( E )nitro-oct-4-enedioate (CP- 23)] superior to CP-6 in TNBC cells killing, synergism with three different inhibitors of the poly ADP-ribose polymerase (PARP) and γ-IR. CP-8 and CP-23 effectively inhibited γ-IR-induced RAD51 foci formation and HR in a GFP-reported assay but did not affect benign human epithelial cells or cell cycle phases. In vivo, CP-8 and CP-23's efficacies diverged as only CP-8 showed promising anticancer activities alone and combined with the PARP inhibitor talazoparib in an HR-proficient TNBC mouse model. As preliminary preclinical toxicology analysis also suggests CP-8 as safe, our data endorse CP-8 as a novel anticancer molecule for treating cancers sensitive to homologous recombination-mediated DNA repair inhibitors.

The EstroGene Database Reveals Diverse Temporal, Context-Dependent, and Bidirectional Estrogen Receptor Regulomes in Breast Cancer.

As one of the most successful cancer therapeutic targets, estrogen receptor-α (ER/ESR1) has been extensively studied over the past few decades. Sequencing technological advances have enabled genome-wide analysis of ER action. However, comparison of individual studies is limited by different experimental designs, and few meta-analyses are available. Here, we established the EstroGene database through unified processing of data from 246 experiments including 136 transcriptomic, cistromic, and epigenetic datasets focusing on estradiol (E2)-triggered ER activation across 19 breast cancer cell lines. A user-friendly browser (https://estrogene.org/) was generated for multiomic data visualization involving gene inquiry under user-defined experimental conditions and statistical thresholds. Notably, annotation of metadata associated with public datasets revealed a considerable lack of experimental details. Comparison of independent RNA-seq or ER ChIP-seq data with the same design showed large variability and only strong effects could be consistently detected. Temporal estrogen response metasignatures were defined, and the association of E2 response rate with temporal transcriptional factors, chromatin accessibility, and heterogeneity of ER expression was evaluated. Unexpectedly, harmonizing 146 E2-induced transcriptomic datasets uncovered a subset of genes harboring bidirectional E2 regulation, which was linked to unique transcriptional factors and highly associated with immune surveillance in the clinical setting. Furthermore, the context dependent E2 response programs were characterized in MCF7 and T47D cell lines, the two most frequently used models in the EstroGene database. Collectively, the EstroGene database provides an informative and practical resource to the cancer research community to uniformly evaluate key reproducible features of ER regulomes and unravels modes of ER signaling. SIGNIFICANCE: A resource database integrating 246 publicly available ER profiling datasets facilitates meta-analyses and identifies estrogen response temporal signatures, a bidirectional program, and model-specific biases.

EstroGene database reveals diverse temporal, context-dependent and directional estrogen receptor regulomes in breast cancer.

As one of the most successful cancer therapeutic targets, estrogen receptor-α (ER/ESR1) has been extensively studied in decade-long. Sequencing technological advances have enabled genome-wide analysis of ER action. However, reproducibility is limited by different experimental design. Here, we established the EstroGene database through centralizing 246 experiments from 136 transcriptomic, cistromic and epigenetic datasets focusing on estradiol-treated ER activation across 19 breast cancer cell lines. We generated a user-friendly browser ( https://estrogene.org/ ) for data visualization and gene inquiry under user-defined experimental conditions and statistical thresholds. Notably, documentation-based meta-analysis revealed a considerable lack of experimental details. Comparison of independent RNA-seq or ER ChIP-seq data with the same design showed large variability and only strong effects could be consistently detected. We defined temporal estrogen response metasignatures and showed the association with specific transcriptional factors, chromatin accessibility and ER heterogeneity. Unexpectedly, harmonizing 146 transcriptomic analyses uncovered a subset of E2-bidirectionally regulated genes, which linked to immune surveillance in the clinical setting. Furthermore, we defined context dependent E2 response programs in MCF7 and T47D cell lines, the two most frequently used models in the field. Collectively, the EstroGene database provides an informative resource to the cancer research community and reveals a diverse mode of ER signaling.

Loss of E-cadherin Induces IGF1R Activation and Reveals a Targetable Pathway in Invasive Lobular Breast Carcinoma.

No special-type breast cancer [NST; commonly known as invasive ductal carcinoma (IDC)] and invasive lobular carcinoma (ILC) are the two major histological subtypes of breast cancer with significant differences in clinicopathological and molecular characteristics. The defining pathognomonic feature of ILC is loss of cellular adhesion protein, E-cadherin (CDH1). We have previously shown that E-cadherin functions as a negative regulator of the IGF1R and propose that E-cadherin loss in ILC sensitizes cells to growth factor signaling that thus alters their sensitivity to growth factor-signaling inhibitors and their downstream activators. To investigate this potential therapeutic vulnerability, we generated CRISPR-mediated CDH1 knockout (CDH1 KO) IDC cell lines (MCF7, T47D, and ZR75.1) to uncover the mechanism by which loss of E-cadherin results in IGF pathway activation. CDH1 KO cells demonstrated enhanced invasion and migration that was further elevated in response to IGF1, serum and collagen I. CDH1 KO cells exhibited increased sensitivity to IGF resulting in elevated downstream signaling. Despite minimal differences in membranous IGF1R levels between wild-type (WT) and CDH1 KO cells, significantly higher ligand-receptor interaction was observed in the CDH1 KO cells, potentially conferring enhanced downstream signaling activation. Critically, increased sensitivity to IGF1R, PI3K, Akt, and MEK inhibitors was observed in CDH1 KO cells and ILC patient-derived organoids. IMPLICATIONS: Overall, this suggests that these targets require further exploration in ILC treatment and that CDH1 loss may be exploited as a biomarker of response for patient stratification.

Modal decomposition of complex optical fields using convolutional neural networks.

Recent studies have shown convolutional neural networks (CNNs) can be trained to perform modal decomposition using intensity images of optical fields. A fundamental limitation of these techniques is that the modal phases cannot be uniquely calculated using a single intensity image. The knowledge of modal phases is crucial for wavefront sensing, alignment, and mode matching applications. Heterodyne imaging techniques can provide images of the transverse complex amplitude and phase profiles of laser beams at high resolutions and frame rates. In this work, we train a CNN to perform modal decomposition using simulated heterodyne images, allowing the complete modal phases to be predicted. This is, to our knowledge, the first machine learning decomposition scheme to utilize complex phase information to perform modal decomposition. We compare our network with a traditional overlap integral and center-of-mass centering algorithm and show that it is both less sensitive to beam centering and on average more accurate in our simulated images.

Point absorbers in Advanced LIGO.

Small, highly absorbing points are randomly present on the surfaces of the main interferometer optics in Advanced LIGO. The resulting nanometer scale thermo-elastic deformations and substrate lenses from these micron-scale absorbers significantly reduce the sensitivity of the interferometer directly though a reduction in the power-recycling gain and indirect interactions with the feedback control system. We review the expected surface deformation from point absorbers and provide a pedagogical description of the impact on power buildup in second generation gravitational wave detectors (dual-recycled Fabry-Perot Michelson interferometers). This analysis predicts that the power-dependent reduction in interferometer performance will significantly degrade maximum stored power by up to 50% and, hence, limit GW sensitivity, but it suggests system wide corrections that can be implemented in current and future GW detectors. This is particularly pressing given that future GW detectors call for an order of magnitude more stored power than currently used in Advanced LIGO in Observing Run 3. We briefly review strategies to mitigate the effects of point absorbers in current and future GW wave detectors to maximize the success of these enterprises.

Optical lock-in camera for gravitational wave detectors.

Knowledge of the intensity and phase profiles of spectral components in a coherent optical field is critical for a wide range of high-precision optical applications. One of these is interferometric gravitational wave detectors, which rely on the optical beats between these fields for precise control of the experiment. Here we describe an optical lock-in camera and show that it can be used to record optical beats at MHz or greater frequencies with higher spatial and temporal resolution than previously possible. This improvement is achieved using a Pockels cell as a fast optical switch to transform each pixel on a sCMOS array into an optical lock-in amplifier. We demonstrate that the optical lock-in camera can record fields with 2 Mpx resolution at 10 Hz with a sensitivity of -62 dBc when averaged over 2s.

Phase Shift in an Atom Interferometer due to Spacetime Curvature across its Wave Function.

Spacetime curvature induces tidal forces on the wave function of a single quantum system. Using a dual light-pulse atom interferometer, we measure a phase shift associated with such tidal forces. The macroscopic spatial superposition state in each interferometer (extending over 16 cm) acts as a nonlocal probe of the spacetime manifold. Additionally, we utilize the dual atom interferometer as a gradiometer for precise gravitational measurements.

Developing in vitro models of human ductal carcinoma in situ from primary tissue explants.

Because there are currently no reliable predictors for progression of ductal carcinoma in situ (DCIS) to invasive disease, nearly all patients receive comprehensive therapy, leading to over-treatment in many cases. Few in vitro models for studying DCIS progression have been developed. We report here the successful culture and expansion of primary DCIS from surgical specimens using a conditional reprogramming protocol. Patients with percutaneous core-needle biopsy demonstrating DCIS were enrolled in a tissue banking protocol after informed consent was received. Fresh tissue was taken from lumpectomy or mastectomy specimens, mechanically and enzymatically dissociated, cultured in medium conditioned by irradiated mouse fibroblasts and supplemented with rho-associated protein kinase (ROCK) inhibitor, and characterized by immunocytochemistry. Out of 33 DCIS cases, 58% (19) were expanded for up to 2 months in culture, and 42% (14) were frozen immediately after mechanical dissociation for future growth. The cultures are almost exclusively composed of cytokeratin 8- and EpCAM-positive luminal and cytokeratin 14-, cytokeratin 5-, and p63-positive basal mammary epithelial cells, suggesting maintenance of heterogeneity in vitro. Furthermore, as assessed by luminal and basal marker expression, these cells retain their cellular identities both in the "conditionally reprogrammed" proliferative state and after conditioned media and ROCK inhibitor withdrawal. When grown to 100 % confluency, the cultures organize into luminal and basal layers as well as luminal compartments surrounded by basal cells. Primary cultures of DCIS derived directly from patient tissues can be generated and may serve as in vitro models for the study of DCIS.

Molecular drivers of lobular carcinoma in situ.

Lobular carcinoma in situ (LCIS) is considered to be a risk factor for the development of invasive breast carcinoma, but it may also be a non-obligate precursor to invasive lobular carcinoma (ILC). Many LCIS lesions do not progress to ILC, and the molecular changes that are necessary for progression from LCIS to ILC are poorly understood. Disruption in the E-cadherin complex is the hallmark of lobular lesions, but other signaling molecules, such as PIK3CA and c-src, are consistently altered in LCIS. This review focuses on the molecular drivers of lobular carcinoma, a more complete understanding of which may give perspective on which LCIS lesions progress, and which will not, thus having immense clinical implications.

Assessment of Cr(VI)-induced cytotoxicity and genotoxicity using high content analysis.

Oral exposure to high concentrations of hexavalent chromium [Cr(VI)] induces intestinal redox changes, villus cytotoxicity, crypt hyperplasia, and intestinal tumors in mice. To assess the effects of Cr(VI) in a cell model relevant to the intestine, undifferentiated (proliferating) and differentiated (confluent) Caco-2 cells were treated with Cr(VI), hydrogen peroxide or rotenone for 2-24 hours. DNA damage was then assessed by nuclear staining intensity of 8-hydroxydeoxyguanosine (8-OHdG) and phosphorylated histone variant H2AX (γ-H2AX) measured by high content analysis methods. In undifferentiated Caco-2, all three chemicals increased 8-OHdG and γ-H2AX staining at cytotoxic concentrations, whereas only 8-OHdG was elevated at non-cytotoxic concentrations at 24 hr. Differentiated Caco-2 were more resistant to cytotoxicity and DNA damage than undifferentiated cells, and there were no changes in apoptotic markers p53 or annexin-V. However, Cr(VI) induced a dose-dependent translocation of the unfolded protein response transcription factor ATF6 into the nucleus. Micronucleus (MN) formation was assessed in CHO-K1 and A549 cell lines. Cr(VI) increased MN frequency in CHO-K1 only at highly cytotoxic concentrations. Relative to the positive control Mitomycin-C, Cr(VI) only slightly increased MN frequency in A549 at mildly cytotoxic concentrations. The results demonstrate that Cr(VI) genotoxicity correlates with cytotoxic concentrations, and that H2AX phosphorylation occurs at higher concentrations than oxidative DNA damage in proliferating Caco-2 cells. The findings suggest that in vitro genotoxicity of Cr(VI) is primarily oxidative in nature at low concentrations. Implications for in vivo intestinal toxicity of Cr(VI) will be discussed.

Kinome-wide functional genomics screen reveals a novel mechanism of TNFα-induced nuclear accumulation of the HIF-1α transcription factor in cancer cells.

Hypoxia-inducible factor-1 (HIF-1) and its most important subunit, HIF-1α, plays a central role in tumor progression by regulating genes involved in cancer cell survival, proliferation and metastasis. HIF-1α activity is associated with nuclear accumulation of the transcription factor and regulated by several mechanisms including modulation of protein stability and degradation. Among recent advances are the discoveries that inflammation-induced cytokines and growth factors affect protein accumulation of HIF-1α under normoxia conditions. TNFα, a major pro-inflammatory cytokine that promotes tumorigenesis is known as a stimulator of HIF-1α activity. To improve our understanding of TNFα-mediated regulation of HIF-1α nuclear accumulation we screened a kinase-specific siRNA library using a cell imaging-based HIF-1α-eGFP chimera reporter assay. Interestingly, this systematic analysis determined that depletion of kinases involved in conventional TNFα signaling (IKK/NFκB and JNK pathways) has no detrimental effect on HIF-1α accumulation. On the other hand, depletion of PRKAR2B, ADCK2, TRPM7, and TRIB2 significantly decreases the effect of TNFα on HIF-1α stability in osteosarcoma and prostate cancer cell lines. These newly discovered regulators conveyed their activity through a non-conventional RELB-depended NFκB signaling pathway and regulation of superoxide activity. Taken together our data allow us to conclude that TNFα uses a distinct and complex signaling mechanism to induce accumulation of HIF-1α in cancer cells. In summary, our results illuminate a novel mechanism through which cancer initiation and progression may be promoted by inflammatory cytokines, highlighting new potential avenues for fighting this disease.

The small nucleolar ribonucleoprotein (snoRNP) database.

Small nucleolar ribonucleoproteins (snoRNPs) are widely studied and characterized as guide RNAs for sequence-specific 2'-O-ribose methylation and psuedouridylation of ribosomal RNAs. In addition, snoRNAs have also been shown to interact with some tRNAs and direct alternative splicing in mRNA biogenesis. Recent advances in bioinformatics have resulted in new algorithms able to rapidly identify noncoding RNAs generally and snoRNAs specifically in genomic and metagenomic sequences, resulting in a rapid increase in the number and diversity of identified snoRNA sequences. The snoRNP database is a web-based collection of snoRNA and snoRNA-associated protein sequences from a wide range of species. The database currently contains 8994 snoRNA sequences from Bacteria, Archaea, and Eukaryotes and 589 snoRNA-associated protein sequences. The snoRNP database can be found at: http://evolveathome.com/snoRNA/snoRNA.php.

Identification, coassembly, and activity of gamma-aminobutyric acid receptor subunits in renal proximal tubular cells.

Although the properties and functions of GABA(A) receptors in the mammalian central nervous system have been well studied, the presence and significance of GABA(A) receptors in non-neural tissues are less clear. The goal of this study was to examine the expression of GABA(A) receptor alpha(1), alpha(2), alpha(4), alpha(5), beta(1), gamma(1), gamma(2), and delta subunits in the kidney and to determine whether these subunits coassemble to form an active renal epithelial cell GABA(A) receptor. Using reverse transcriptase products from RNA isolated from rat and rabbit kidney cortex and brain or cerebellum through polymerase chain reaction (PCR) and sequencing of the PCR products, we revealed that rat kidney cortex contained the alpha(1), alpha(5), beta(1), gamma(1), and gamma(2) subunits and that they were similar to the neuronal subunits. Sequencing of the PCR products revealed that the rabbit kidney cortex contained the alpha(1) and gamma(2) subunits and that they were similar to their neuronal counterparts. Immunoprecipitation and immunoblot studies using GABA(A) receptor subunit-specific antibodies and detergent-solubilized rat kidney cortex membranes identified a GABA(A) receptor complex containing alpha(5), beta(1), and gamma(1). Isolated rat renal proximal tubular cells exhibited GABA-mediated, picrotoxin-sensitive (36)Cl(-) uptake. These studies demonstrate the presence of numerous GABA(A) receptor subunits in the kidneys of two species, the assembly of the subunits into at least one novel receptor complex, and an active GABA(A) receptor in renal proximal tubular cells.

Small heat shock protein Hsp27 is required for proper heart tube formation.

The small heat shock protein Hsp27 has been shown to be involved in a diverse array of cellular processes, including cellular stress response, protein chaperone activity, regulation of cellular glutathione levels, apoptotic signaling, and regulation of actin polymerization and stability. Furthermore, mutation within Hsp27 has been associated with the human congenital neuropathy Charcot-Marie Tooth (CMT) disease. Hsp27 is known to be expressed in developing embryonic tissues; however, little has been done to determine the endogenous requirement for Hsp27 in developing embryos. In this study, we show that depletion of XHSP27 protein results in a failure of cardiac progenitor fusion resulting in cardia bifida. Furthermore, we demonstrate a concomitant disorganization of actin filament organization and defects in myofibril assembly. Moreover, these defects are not associated with alterations in specification or differentiation. We have thus demonstrated a critical requirement for XHSP27 in developing cardiac and skeletal muscle tissues.

TBX5 is required for embryonic cardiac cell cycle progression.

Despite the critical importance of TBX5 in normal development and disease, relatively little is known about the mechanisms by which TBX5 functions in the embryonic heart. Our present studies demonstrate that TBX5 is necessary to control the length of the embryonic cardiac cell cycle, with depletion of TBX5 leading to cardiac cell cycle arrest in late G(1)- or early S-phase. Blocking cell cycle progression by TBX5 depletion leads to a decrease in cardiac cell number, an alteration in the timing of the cardiac differentiation program, defects in cardiac sarcomere formation, and ultimately, to cardiac programmed cell death. In these studies we have also established that terminally differentiated cardiomyocytes retain the capacity to undergo cell division. We further show that TBX5 is sufficient to determine the length of the embryonic cardiac cell cycle and the timing of the cardiac differentiation program. Thus, these studies establish a role for TBX5 in regulating the progression of the cardiac cell cycle.

Xtn3 is a developmentally expressed cardiac and skeletal muscle-specific novex-3 titin isoform.

Titin proteins play an essential role in maintaining muscle function and structure. Recent work has implicated the involvement of the novex-3 titin isoform in sarcomere restructuring and disease. Unlike avian and mammalian systems, Xenopus laevis myogenesis is characterized by a wave of primary myogenesis followed by apoptosis of the primary muscles and formation of new muscles by secondary myogenesis. We show here that the Xenopus laevis novex-3 titin isoform (Xtn3) is developmentally expressed throughout the somites, heart, and primary muscles of the developing embryo. Downregulation of Xtn3 expression at tadpole stages appears to coincide with the change in myofiber composition from solely embryonic "fast" fiber types to myofibers containing both "fast" and "slow" fiber types. We demonstrate that Xtn3 is expressed early in the presomitic mesoderm and remains expressed in the somites, ventral myoblasts, and developing jaw muscles through late tailbud stage. Furthermore, we show that Xtn3 is expressed in the cardiac primordia prior to linear heart tube formation and remains expressed in the heart until tadpole stage, at which point it is downregulated in the heart except in discrete patches of cardiac cells. Finally, we demonstrate that Xtn3 transcripts are detectable in adult heart and muscle tissues.

Tbx5 and Tbx20 act synergistically to control vertebrate heart morphogenesis.

Members of the T-box family of proteins play a fundamental role in patterning the developing vertebrate heart; however, the precise cellular requirements for any one family member and the mechanism by which individual T-box genes function remains largely unknown. In this study, we have investigated the cellular and molecular relationship between two T-box genes, Tbx5 and Tbx20. We demonstrate that blocking Tbx5 or Tbx20 produces phenotypes that display a high degree of similarity, as judged by overall gross morphology, molecular marker analysis and cardiac physiology, implying that the two genes are required for and have non-redundant functions in early heart development. In addition, we demonstrate that although co-expressed, Tbx5 and Tbx20 are not dependent on the expression of one another, but rather have a synergistic role during early heart development. Consistent with this proposal, we show that TBX5 and TBX20 can physically interact and map the interaction domains, and we show a cellular interaction for the two proteins in cardiac development, thus providing the first evidence for direct interaction between members of the T-box gene family.