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Background: Recent decades have started to realize the importance of the microbiome in cancer initiation, progression and response to therapy. Recent findings suggest that both the local and distal microbiome may influence tumor growth. Although many studies focused on the gut microbiome, the breast itself also possesses a unique microbiome which differs between healthy and breast cancer patients, and between tumor subtypes. However, it is poorly understood whether these bacteria in the gut or the breast microenvironment may interact with breast cancer cells and if that interaction alters tumor growth. Bacteria produce metabolites and other signaling molecules to alter the host’s response. We have identified and purified an exopolysaccharide (EPS) produced by the commensal bacterium Bacillus subtilis, which was shown to act directly on the host’s innate immune cells to protect against various inflammatory diseases. This study aims to investigate if, and how the bacterial product (EPS) alters proliferation and tumor growth of breast cancer cells.Methods: Cell proliferation of bulk cells was conducted on PBS or EPS treated breast cancer cells (T47D, MDA-MB-468, MDA-MB-453, HCC1428, BT549, MDA-MB-231, ZR-75-30, and MCF-7) using trypan blue exclusion and the XTT assay. Flow cytometry was used to assess apoptosis with Annexin V, and cell cycle progression with propidium iodide. Cancer stem cell survival was assessed using the mammosphere forming assay. CRISPR/Cas9-mediated knockout of the Toll-like-receptor 4 (TLR4) was used to assess the requirement of TLR4 signaling on EPS-mediated phenotypes. RNA-sequencing was performed on both EPS-sensitive and resistant breast cancer cell lines after treatment with PBS or EPS for 20 hours. T47D xenografts studies were performed in vivo using EPS pretreatment and continual treatment via i.p. injection.Results: EPS inhibited proliferation of some breast cancer cell lines (T47D, MDA-MB-468, MDA-MB-453, HCC1428) in a concentration dependent manner while having little effect on others (BT549, MDA-MB-231, ZR-75-30, MCF7). EPS acted via distinct mechanisms depending on the cell line, increasing apoptosis in MDA-MB-468 cells while inducing G0/G1 cell cycle arrest in T47D cells. Although EPS directly inhibited bulk cell proliferation, the cancer stem cell population of T47D cells was increased by EPS. EPS is known to require TLR4 signaling to modulate immune cells. CRISPR/Cas9 knockout of TLR4 demonstrated that it was not required for EPS-mediated inhibition of T47D breast cancer cells, suggesting a novel mechanism of action in this cell line. RNA-sequence analysis showed that EPS altered interferon signaling and cell cycle progression pathways. Interferon signaling activates the STAT family of transcription factors, and western blots confirmed that 3h of EPS treatment induced STAT1 phosphorylation and p21 accumulation, indicating that EPS activated this pathway. Contrary to in vitro proliferation data, in vivo results showed that EPS-treated cells formed significantly larger tumors compared to PBS controls (273mg vs 59mg). The slope of tumor growth was also significantly faster in the EPS-treated group. Conclusions: These findings demonstrated the multifacet of EPS’s effect on breast cancer cells. EPS inhibits the proliferation of certain breast cancer cells in vitro, possibly via activation of STAT1 and p21 signaling, but also increases survival of cancer stem cells and promotes more aggressive tumor growth in vivo. This highlights the complexity of the influence of commensal bacteria on breast cancer growth, which can be highly context-dependent. Deeper understanding of this complex interplay between the host-microbe-tumor will be vital to the development of novel bacterial strategies in the future. Citation Format: Mai Rachel Nguyen, Katherine L Knight, Clodia Osipo. A commensal’s exopolysaccharide inhibits breast cancer proliferation in vitro but promotes tumor growth in vivo [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 P5-12-10.

Highly solvent swollen poly(N-isopropylacrylamide-co-acrylic acid) microgels are synthesized without exogenous crosslinker, making them extremely soft and deformable. These ultralow crosslinked microgels (ULC) are incubated under controlled osmotic pressure to provide a slow (and presumably thermodynamically controlled) approach to higher packing densities. It is found that ULC microgels show stable colloidal packing over a very wide range of osmotic pressures and thus packing densities. Surprising observation of co-existence between hexagonal and square lattices is also made over the lower range of studied osmotic pressures, with microgels apparently changing shape from spheres to cubes in defects or grain boundaries. It is proposed that the unusual packing behavior observed for ULC microgels is due to the extreme softness of these particles, where deswelling causes deformation and shrinking of the particles that result in unique packing states governed by contributions to the entropy at the colloidal system, single particle and ionic levels. These observations further suggest that more detailed experimental and theoretical studies of ultra-soft microgels are required to obtain a complete understanding of their behavior in packed and confined geometries.

The next generation of ultralight dark matter (DM) direct detection experiments, which could confirm sub-eV bosons as the dominant source of DM, will feature multiple detectors operating at various terrestrial locations. As a result of the wave-like nature of ultralight DM, spatially separated detectors will each measure a unique DM phase. When the separation between experiments is comparable to the DM coherence length, the spatially-varying phase contains information beyond that which is accessible at a single detector. We introduce a formalism to extract this information, which performs interferometry directly on the DM wave. In particular, we develop a likelihood-based framework that combines data from multiple experiments to constrain directional information about the DM phase space distribution. We show that the signal in multiple detectors is subject to a daily modulation effect unique to wave-like DM. Leveraging daily modulation, we illustrate that within days of an initial discovery multiple detectors acting in unison could localize directional parameters of the DM velocity distribution such as the direction of the solar velocity to sub-degree accuracy, or the direction of a putative cold DM stream to the sub-arcminute level. We outline how to optimize the locations of multiple detectors with either resonant cavity (such as ADMX or HAYSTAC) or quasistatic (such as ABRACADABRA or DM-Radio) readouts to have maximal sensitivity to the full 3-dimensional DM velocity distribution., 25 pages, 9 figures, animation available at https://github.com/joshwfoster/DM_Interferometry, updated to published version

International audience; We present the largest exome sequencing study of autism spectrum disorder (ASD) to date (n = 35,584 total samples, 11,986 with ASD). Using an enhanced analytical framework to integrate de novo and case-control rare variation, we identify 102 risk genes at a false discovery rate of 0.1 or less. Of these genes, 49 show higher frequencies of disruptive de novo variants in individuals ascertained to have severe neurodevelopmental delay, whereas 53 show higher frequencies in individuals ascertained to have ASD; comparing ASD cases with mutations in these groups reveals phenotypic differences. Expressed early in brain development, most risk genes have roles in regulation of gene expression or neuronal communication (i.e., mutations effect neurodevelopmental and neurophysiological changes), and 13 fall within loci recurrently hit by copy number variants. In cells from the human cortex, expression of risk genes is enriched in excitatory and inhibitory neuronal lineages, consistent with multiple paths to an excitatory-inhibitory imbalance underlying ASD.

Ecological speciation, which relies heavily on selection driving the emergence of new species, has become the primary paradigm through which rapid species radiations are understood. In this way, selection, particularly ecological selection, is assumed to be the driver of most species radiations. However, in many radiations, such as the radiation of Lake Malawi’s cichlids, the assumption of selection as the driver of speciation has rarely been explicitly tested, and drift, often, has completely been ignored as potential factor. In order to understand the forces driving the divergence of Lake Malawi cichlids at the microevolutionary level, we studied the genetic and phenotypic divergence of ten allopatric populations of Maylandia zebra. We estimated effective population sizes as proxy for drift. Further, we compared neutral genetic differentiation to divergence in three phenotypic traits: body size, body shape, and melanophore counts. We found small, yet significant, population differentiation in all the studied traits across most populations. Population sizes were small rendering the potential for drift to be high. However, phenotypic differentiation exceeded neutral expectations for all traits suggesting divergent local selection. Our data suggest that natural, and potentially also sexual, selection may be the dominant force driving population differentiation in Lake Malawi’s rock-dwelling cichlids, despite the potential for drift in small populations.

Physical review / D 102(4), 043528 (1-24) (2020). doi:10.1103/PhysRevD.102.043528, We have conducted extensive lattice simulations to study the postinflation dynamics of multifield models involving nonminimal couplings. We explore the parameter dependence of preheating in these models and describe the various time scales that control such nonlinear processes as energy transfer, rescattering, and the approach to radiation domination and thermalization. In the limit of large nonminimal couplings (ξI∼100), we find that efficient transfer of energy from the inflaton condensate to radiative degrees of freedom, emergence of a radiation-dominated equation of state, and the onset of thermalization each consistently occur within Nreh≲3 e-folds after the end of inflation, largely independent of the values of the other couplings in the models. The exception is the case of negative ellipticity, in which there is a misalignment between the dominant direction in field space along which the system evolves and the larger of the nonminimal couplings ξI. In those cases, the field-space-driven parametric resonance is effectively shut off. More generally, the competition between the scalar fields’ potential and the field-space manifold structure can yield interesting phenomena such as two-stage resonances. Across many regions of parameter space, we find efficient re-scattering between the distinct fields, leading to a partial memory loss of the shape of the initial fluctuation spectrum. Despite the explosive particle production, which can lead to a quick depletion of the background energy density, the nonlinear processes do not induce any superhorizon correlations after the end of inflation in these models, which keeps predictions for cosmic microwave background observables unaffected by the late-time amplification of isocurvature fluctuations. Hence the excellent agreement between primordial observables and recent observations is preserved for this class of models, even when we consider postinflation dynamics., Published by Inst.302363, Melville, NY

We study the post-inflation dynamics of multifield models involving nonminimal couplings using lattice simulations to capture significant nonlinear effects like backreaction and rescattering. We measure the effective equation of state and typical time-scales for the onset of thermalization, which could affect the usual mapping between predictions for primordial perturbation spectra and measurements of anisotropies in the cosmic microwave background radiation. For large values of the nonminimal coupling constants, we find efficient particle production that gives rise to nearly instantaneous preheating. Moreover, the strong single-field attractor behavior that was previously identified persists until the end of preheating, thereby suppressing typical signatures of multifield models. We therefore find that predictions for primordial observables in this class of models retain a close match to the latest observations., Comment: 6 pages, 7 figures. References added and minor edits made to match version to be published in Physical Review Letters

Recently, multiple studies have shown that a unique microbiome is associated with the breast, with distinct differences in microbial compositions between healthy and patients with benign versus malignant breast cancer tissue. It is well known that bacteria can produce metabolites and other signaling molecules to alter the host's response. However, little is known about whether bacteria in the breast microenvironment interact with breast cancer cells and if that interaction changes cancer progression. We aim to investigate if, and how commensal bacteria and/or their bacterial products alter growth and metastasis of breast cancer cells. Upon initial analysis of sequencing data deposited by Heiken et al, we identified the presence of the gut commensal bacterium Bacillus subtilis in some breast tissues of patients with breast cancer. Previous studies have shown that exopolysaccharide (EPS) secreted by B. subtilis can induce tolerance in the myeloid compartment to protect the host from inflammatory diseases such as diarrhea, sepsis, and graft-versus-host-disease. We first examined the effect of EPS on breast cancer proliferation by performing in vitro proliferation assays on a diverse panel of breast cancer cell lines, including hormone receptor positive and negative lines. Results showed that EPS suppressed proliferation of multiple breast cancer cell lines (T47D, MDA-MB-468, MDA-MB-453, HCC1428) in a concentration dependent manner while other cell lines were unaffected (BT549, MDA-MB-231, ZR-75-30, MCF7). Annexin V apoptosis assay and cell cycle staining indicated that EPS acted via distinct mechanisms depending on the cell line, increasing apoptosis in MDA-MB-468 cells while inducing G0/G1 cell cycle arrest in T47D cells. In addition to proliferation by bulk tumor cells, the survival of breast cancer stem cells emerges as a significant challenge to breast cancer therapies as these stem-like cells are thought to repopulate the tumor and lead to recurrence. We tested if EPS treatment alters cancer stem cell survival using the mammosphere forming assay, a 3D culture system which specifically selects for cells with stem cell characteristics. Our preliminary mammosphere results show that pretreatment with EPS increased cancer stem cell survival in T47D and MCF7 cells. Overall, we found that EPS secreted by B. subtilis can directly suppress the proliferation of some breast cancer cell lines but enhance cancer stem cell survival. Thus, effects of B. subtilis on breast cancer cells are potentially multifaceted. Future work will focus on assessing the effect of EPS on breast tumor growth in vivo and elucidating the signaling mechanism by which EPS inhibits bulk cancer cells but stimulates cancer stem cells. Elucidating this mechanism would further our understanding of how local breast microbes may influence breast cancer growth and outcome. Citation Format: Mai Rachel Nguyen, Clodia Osipo, Katherine L. Knight. Exopolysaccharide secreted by Bacillus subtilis alters breast cancer cell growth and stemness [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6101.

We present the largest exome sequencing study of autism spectrum disorder (ASD) to date (n=35,584 total samples, 11,986 with ASD). Using an enhanced Bayesian framework to integrate de novo and case-control rare variation, we identify 102 risk genes at a false discovery rate ≤ 0.1. Of these genes, 49 show higher frequencies of disruptive de novo variants in individuals ascertained for severe neurodevelopmental delay, while 53 show higher frequencies in individuals ascertained for ASD; comparing ASD cases with mutations in these groups reveals phenotypic differences. Expressed early in brain development, most of the risk genes have roles in regulation of gene expression or neuronal communication (i.e., mutations effect neurodevelopmental and neurophysiological changes), and 13 fall within loci recurrently hit by copy number variants. In human cortex single-cell gene expression data, expression of risk genes is enriched in both excitatory and inhibitory neuronal lineages, consistent with multiple paths to an excitatory/inhibitory imbalance underlying ASD.

SummaryWe present the largest exome sequencing study of autism spectrum disorder (ASD) to date (n=35,584 total samples, 11,986 with ASD). Using an enhanced Bayesian framework to integrate de novo and case-control rare variation, we identify 102 risk genes at a false discovery rate ≤ 0.1. Of these genes, 49 show higher frequencies of disruptive de novo variants in individuals ascertained for severe neurodevelopmental delay, while 53 show higher frequencies in individuals ascertained for ASD; comparing ASD cases with mutations in these groups reveals phenotypic differences. Expressed early in brain development, most of the risk genes have roles in regulation of gene expression or neuronal communication (i.e., mutations effect neurodevelopmental and neurophysiological changes), and 13 fall within loci recurrently hit by copy number variants. In human cortex single-cell gene expression data, expression of risk genes is enriched in both excitatory and inhibitory neuronal lineages, consistent with multiple paths to an excitatory/inhibitory imbalance underlying ASD.

Autism spectrum disorder (ASD) is a group of complex, neurological disorders that affect early cognitive, social, and verbal development. Our understanding of ASD has vastly improved with advances in genomic sequencing technology and genetic models that have identified >800 loci with variants that increase susceptibility to ASD. Although these findings have confirmed its high heritability, the underlying mechanisms by which these genes produce the ASD phenotypes have not been defined. Current efforts have begun to "functionalize" many of these variants and envisage how these susceptibility factors converge at key biochemical and biophysical pathways. In this review, we discuss recent work on intracellular calcium signaling in ASD, including our own work, which begins to suggest it as a compelling candidate mechanism in the pathophysiology of autism and a potential therapeutic target. We consider how known variants in the calcium signaling genomic architecture of ASD may exert their deleterious effects along pathways particularly involving organelle dysfunction including the endoplasmic reticulum (ER), a major calcium store, and the mitochondria, a major calcium ion buffer, and theorize how many of these pathways intersect.

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders without any defined uniting pathophysiology. Ca2+ signaling is emerging as a potential node in the genetic architecture of the disorder. We previously reported decreased inositol trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum in several rare monogenic syndromes highly comorbid with autism – fragile X and tuberous sclerosis types 1 and 2 syndromes. We now extend those findings to a cohort of subjects with sporadic ASD without any known mutations. We developed and applied a high throughput Fluorometric Imaging Plate Reader (FLIPR) assay to monitor agonist-evoked Ca2+ signals in human primary skin fibroblasts. Our results indicate that IP3 -mediated Ca2+ release from the endoplasmic reticulum in response to activation of purinergic receptors is significantly depressed in subjects with sporadic as well as rare syndromic forms of ASD. We propose that deficits in IP3-mediated Ca2+ signaling represent a convergent hub function shared across the spectrum of autistic disorders – whether caused by rare highly penetrant mutations or sporadic forms – and holds promise as a biomarker for diagnosis and novel drug discovery.

The Chinese market provides numerous opportunities for growth and development if approached properly. In this paper, we examine the regulatory aspects of advertising in China. The history of these regulations is reviewed and the cultural foundations of advertising regulations are highlighted. Current advertising regulations are critically reviewed, with an emphasis on strategic implications to help marketers cope with the regulatory milieu.

We estimated the effective population sizes (Ne ) and tested for short-term temporal demographic stability of populations of two Lake Malawi cichlids: Maylandia benetos, a micro-endemic, and Maylandia zebra, a widespread species found across the lake. We sampled a total of 351 individuals, genotyped them at 13 microsatellite loci and sequenced their mitochondrial D-loop to estimate genetic diversity, population structure, demographic history and effective population sizes. At the microsatellite loci, genetic diversity was high in all populations. Yet, genetic diversity was relatively low for the sequence data. Microsatellites yielded mean Ne estimates of 481 individuals (±99 SD) for M. benetos and between 597 (±106.3 SD) and 1524 (±483.9 SD) individuals for local populations of M. zebra. The microsatellite data indicated no deviations from mutation-drift equilibrium. Maylandia zebra was further found to be in migration-drift equilibrium. Temporal fluctuations in allele frequencies were limited across the sampling period for both species. Bayesian Skyline analyses suggested a recent expansion of M. zebra populations in line with lake-level fluctuations, whereas the demographic history of M. benetos could only be estimated for the very recent past. Divergence time estimates placed the origin of M. benetos within the last 100 ka after the refilling of the lake and suggested that it split off the sympatric M. zebra population. Overall, our data indicate that micro-endemics and populations in less favourable habitats have smaller Ne , indicating that drift may play an important role driving their divergence. Yet, despite small population sizes, high genetic variation can be maintained.

The neurofibromatosis 2 tumor suppressor gene product merlin has strong sequence identity to the ezrin-radixin-moesin (ERM) family over its approximately 300-residue N-terminal domain. ERM proteins are membrane cytoskeletal linkers that are negatively regulated by an intramolecular association between domains known as NH(2)- and COOH-ERM association domains (N- and C-ERMADs) that mask sites for binding membrane-associated proteins, such as EBP50 and E3KARP, and F-actin. Here we show that merlin has self-association regions analogous to the N- and C-ERMADs. Moreover, the N-/C-ERMAD interaction in merlin is relatively weak and dynamic, and this property is reflected by the ability of full-length recombinant merlin to form homo-oligomers. Remarkably, the merlin C-ERMAD has a higher affinity for the N-ERMAD of ezrin than the N-ERMAD of merlin. Both the ezrin and merlin N-ERMAD bind EBP50. This interaction with the ezrin N-ERMAD can be inhibited by the presence of the ezrin C-ERMAD, whereas interaction with the merlin N-ERMAD is not inhibited by either C-ERMAD. E3KARP binds tightly to the ezrin N-ERMAD but has little affinity for the merlin N-ERMAD. The implications of these associations and the hierarchies of binding for the function and regulation of merlin and ERM proteins are discussed.

▪ Abstract The ezrin-radixin-moesin (ERM) family of proteins have emerged as key regulatory molecules in linking F-actin to specific membrane proteins, especially in cell surface structures. Merlin, the product of the NF2 tumor suppressor gene, has sequence similarity to ERM proteins and binds to some of the same membrane proteins, but lacks a C-terminal F-actin binding site. In this review we discuss how ERM proteins and merlin are negatively regulated by an intramolecular association between their N- and C-terminal domains. Activation of at least ERM proteins can be accomplished by C-terminal phosphorylation in the presence of PIP2. We also discuss membrane proteins to which ERM and merlin bind, including those making an indirect linkage through the PDZ-containing adaptor molecules EBP50 and E3KARP. Finally, the function of these proteins in cortical structure, endocytic traffic, signal transduction, and growth control is discussed.

In research animals, the immune adjuvant activity of silicone in eliciting antibodies to associated protein antigens is now well established. In humans, the immune adjuvant activity of silicone remains controversial. Clusters of data from various research groups are beginning to define the boundary conditions for future large epidemiological studies. In that spirit, this manuscript reports pilot clinical data from a serological study conducted 3 years ago and reported previously in this journal. Sera from a self-selected symptomatic population of patients with breast implants were assayed for elevated concentrations of antibodies showing binding avidity to silicone surface associated antigens [anti-SSAA(x)]. In that study of 249 patients, two distinct statistically significant subgroups were identified on the basis of the serological assay alone: patients without any elevated anti-SSAA(x) (negative) and patients with elevated anti-SSAA(x) (positive) (p < 0.001). In this study, a clinical survey returned by 226 of those patients was correlated with the previously acquired serological findings. The 11 most common clinical complaints reported by the 199 anti-SSAA(x) negative patients and by the 27 anti-SSAA(x) positive were compared and their frequencies analyzed. The 199 anti-SSAA(x) negative patients, as a group, showed a lower frequency of a variety of signs and symptoms compared to the 27 anti-SSAA(x) positive patients. Statistically significant differences were seen in three of the symptoms: fever, foot pain, and sleep disturbance (p < 0.05). In addition, the syndrome of fever in the absence of local chest pain was a significantly associated with anti-SSAA(x) positivity (p < 0.001). © 1995 John Wiley & Sons, Inc.