TRAIL-induced cytokine production via NFKB2 pathway promotes neutrophil chemotaxis and immune suppression in triple negative breast cancers.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic that induces apoptosis in cancer cells while sparing the non-malignant cells in preclinical models. However, its efficacy in clinical trials has been limited, suggesting unknown modulatory mechanisms responsible for the lack of TRAIL activity in patients. Here, we hypothesized that TRAIL treatment elicits transcriptional changes in triple negative breast cancer (TNBC) cells that alter the immune milieu. To test this, we performed an RNAseq analysis of MDA-MB-231 cells treated with TRAIL, followed by validation in additional TNBC cell lines. TRAIL significantly induces expression of multiple cytokines such as CXCLs 1, 2, 3, 8,11 and IL-6, which are known to modify neutrophil function. Mechanistically, the induction of these cytokines was predominantly mediated by death receptor 5, caspase 8 (but not caspase 8 enzymatic activity), and the non-canonical NFKB2 pathway. The cytokines produced by the TRAIL-treated TNBC cells enhanced chemotaxis of healthy human donor isolated neutrophils. In vivo , TRAIL treated TNBC murine xenograft tumors showed activation of the NFKB2 pathway, elevated production of CXCLs and IL-6, and increased neutrophil recruitment into the tumors. Moreover, donor isolated neutrophils preincubated in supernatants from TRAIL-treated TNBC cells exhibited impaired cytotoxic effect against TNBC cells. Transcriptomic analysis of neutrophils incubated with either TRAIL alone or supernatant of TRAIL-treated TNBC cells revealed increased expression of inflammatory cytokines, immune modulatory genes, immune checkpoint genes, and genes implicated in delayed neutrophil apoptosis. Functional studies with these neutrophils confirmed their suppressive effect on T cell proliferation and an increase in Treg suppressive phenotype. Collectively, our study demonstrates a novel role of TRAIL-induced NFKB2-dependent cytokine production that promotes neutrophil chemotaxis and immune suppression.

Tongue orthotopic xenografts to study fusion-negative rhabdomyosarcoma invasion and metastasis in live animals.

PAX3/7 fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Limited understanding of advanced FN-RMS is partially attributed to the absence of sequential invasion and dissemination events and the challenge in studying cell behavior, using, for example, non-invasive intravital microscopy (IVM), in currently used xenograft models. Here, we developed an orthotopic tongue xenograft model of FN-RMS to study cell behavior and the molecular basis of invasion and metastasis using IVM. FN-RMS cells are retained in the tongue and invade locally into muscle mysial spaces and vascular lumen, with evidence of hematogenous dissemination to the lungs and lymphatic dissemination to lymph nodes. Using IVM of tongue xenografts reveals shifts in cellular phenotype, migration to blood and lymphatic vessels, and lymphatic intravasation. Insight from this model into tumor invasion and metastasis at the tissue, cellular, and subcellular level can guide new therapeutic avenues for advanced FN-RMS.

Fusion-negative rhabdomyosarcoma orthotopic tongue xenografts for study of invasion, intravasation and metastasis in live animals.

PAX3/7 Fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Towards achieving a more complete understanding of advanced FN-RMS, we developed an orthotopic tongue xenograft model for studies of molecular basis of FN-RMS invasion and metastasis. The behavior of FN-RMS cells injected into murine tongue was examined using in vivo bioluminescence imaging, non-invasive intravital microscopy (IVM), and histopathology and compared to the prevailing hindlimb intramuscular and subcutaneous xenografts. FN-RMS cells were retained in the tongue and invaded locally into muscle mysial spaces and vascular lumen. While evidence of hematogenous dissemination to the lungs occurred in tongue and intramuscular xenografts, evidence of local invasion and lymphatic dissemination to lymph nodes only occurred in tongue xenografts. IVM and RNA-seq of tongue xenografts reveal shifts in cellular phenotype and differentiation state in tongue xenografts. IVM also shows homing to blood and lymphatic vessels, lymphatic intravasation, and dynamic membrane protrusions. Based on these findings, the tongue orthotopic xenograft of FN-RMS is a valuable model for tumor progression studies at the tissue, cellular and subcellular levels providing insight into kinetics and molecular bases of tumor invasion and metastasis and, hence, new therapeutic avenues for advanced FN-RMS.

Intravital Subcellular Microscopy of the Mammary Gland.

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676 or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs ≥ 0.7 µm. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 µm or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

High Salinity and High Temperature Stable Colloidal Silica Nanoparticles with Wettability Alteration Ability for EOR Applications.

The stability of nanoparticles at reservoir conditions is a key for a successful application of nanofluids for any oilfield operations, e.g., enhanced oil recovery (EOR). It has, however, remained a challenge to stabilize nanoparticles under high salinity and high temperature conditions for longer duration (at least months). In this work, we report surface modification of commercial silica nanoparticles by combination of zwitterionic and hydrophilic silanes to improve its stability under high salinity and high temperature conditions. To evaluate thermal stability, static and accelerated stability analyses methods were employed to predict the long-term thermal stability of the nanoparticles in pH range of 4-7. The contact angle measurements were performed on aged sandstone and carbonate rock surfaces to evaluate the ability of the nanoparticles to alter the wettability of the rock surfaces. The results of static stability analysis showed excellent thermal stability in 3.5% NaCl brine and synthetic seawater (SSW) at 60 °C for 1 month. The accelerated stability analysis predicted that the modified nanoparticles could remain stable for at least 6 months. The results of contact angle measurements on neutral-wet Berea, Bentheimer, and Austin Chalk showed that the modified nanoparticles were able to adsorb on these rock surfaces and altered wettability to water-wet. A larger change in contact angle for carbonate surface than in sandstone surface showed that these particles could be more effective in carbonate reservoirs or reservoirs with high carbonate content and help improve oil recovery.

Collective cancer cell invasion requires RNA accumulation at the invasive front.

Localization of RNAs at protrusive regions of cells is important for single-cell migration on two-dimensional surfaces. Protrusion-enriched RNAs encode factors linked to cancer progression, such as the RAB13 GTPase and the NET1 guanine nucleotide exchange factor, and are regulated by the tumor-suppressor protein APC. However, tumor cells in vivo often do not move as single cells but rather utilize collective modes of invasion and dissemination. Here, we developed an inducible system of three-dimensional (3D) collective invasion to study the behavior and importance of protrusion-enriched RNAs. We find that, strikingly, both the RAB13 and NET1 RNAs are enriched specifically at the invasive front of leader cells in invasive cell strands. This localization requires microtubules and coincides with sites of high laminin concentration. Indeed, laminin association and integrin engagement are required for RNA accumulation at the invasive front. Importantly, perturbing RNA accumulation reduces collective 3D invasion. Examination of in vivo tumors reveals a similar localization of the RAB13 and NET1 RNAs at potential invasive sites, suggesting that this mechanism could provide a targeting opportunity for interfering with collective cancer cell invasion.

Experimental Investigation of Stability of Silica Nanoparticles at Reservoir Conditions for Enhanced Oil-Recovery Applications.

To be effective enhanced oil-recovery (EOR) agents, nanoparticles must be stable and be transported through a reservoir. However, the stability of a nanoparticle suspension at reservoir salinity and temperature is still a challenge and how it is affected by reservoir rocks and crude oils is not well understood. In this work, for the first time, the effect of several nanoparticle treatment approaches on the stability of silica nanoparticles at reservoir conditions (in the presence of reservoir rock and crude oil) was investigated for EOR applications. The stability of nanoparticle suspensions was screened in test tubes at 70 °C and 3.8 wt. % NaCl in the presence of reservoir rock and crude oil. Fumed silica nanoparticles in suspension with hydrochloric acid (HCl), polymer-modified fumed nanoparticles and amide-functionalized silica colloidal nanoparticles were studied. The size and pH of nanoparticle suspension in contact with rock samples were measured to determine the mechanism for stabilization or destabilization of nanoparticles. A turbidity scanner was used to quantify the stability of the nanoparticle suspension. Results showed that both HCl and polymer surface modification can improve nanoparticle stability under synthetic seawater salinity and 70 °C. Suspensions of polymer-modified nanoparticles were stable for months. It was found that pH is a key parameter influencing nanoparticle stability. Rock samples containing carbonate minerals destabilized unmodified nanoparticles. Crude oil had limited effect on nanoparticle stability. Some components of crude oil migrated into the aqueous phase consisting of amide-functionalized silica colloidal nanoparticles suspension. Nanoparticles modification or/and stabilizer are necessary for nanoparticle EOR application.

Dynamic polyhedral actomyosin lattices remodel micron-scale curved membranes during exocytosis in live mice.

Actomyosin networks, the cell's major force production machineries, remodel cellular membranes during myriad dynamic processes1,2 by assembling into various architectures with distinct force generation properties3,4. While linear and branched actomyosin architectures are well characterized in cell-culture and cell-free systems3, it is not known how actin and myosin networks form and function to remodel membranes in complex three-dimensional mammalian tissues. Here, we use four-dimensional spinning-disc confocal microscopy with image deconvolution to acquire macromolecular-scale detail of dynamic actomyosin networks in exocrine glands of live mice. We address how actin and myosin organize around large membrane-bound secretory vesicles and generate the forces required to complete exocytosis5-7. We find that actin and non-muscle myosin II (NMII) assemble into previously undescribed polyhedral-like lattices around the vesicle membrane. The NMII lattice comprises bipolar minifilaments8-10 as well as non-canonical three-legged configurations. Using photobleaching and pharmacological perturbations in vivo, we show that actomyosin contractility and actin polymerization together push on the underlying vesicle membrane to overcome the energy barrier and complete exocytosis7. Our imaging approach thus unveils a force-generating actomyosin lattice that regulates secretion in the exocrine organs of live animals.

Formation of Icephobic Surface with Micron-Scaled Hydrophobic Heterogeneity on Polyurethane Aerospace Coating.

Development of an anti-icing surface on a desired industrial coating patch/object has been the persistent challenge to several industries, such as aviation and wind power. For this aim, performing surface modification to implement the icephobic property on existing commercial coatings is important for practical applications. This work accomplishes an icephobic coating overlying a PPG aerospace polyurethane coating. It manifests a clear capability to delay the formation of frost as well as to reduce the adhesion strength of ice. This icephobic coating is sustained by a unique hydrophobic heterogeneity in the micron-scale of segregation, which is realized through solution casting of a specific copolymer consisting of random rigid and soft segments, namely poly(methyl methacrylate) and poly(lauryl methacrylate-2-hydroxy-3-(1-amino dodecyl)propyl methacrylate), respectively. A wrinkled pattern developed over the coating is observed because of the diverse traits between these two segments. Besides, the OH/NH groups of the soft segment are crosslinked by a diisocyanate monomer upon drying and curing to strengthen the coating. More importantly, integration of a small dose of paraffin wax into the copolymer induces a spread of soft microdomains on the winkled pattern surface. It is hypothesized that these dual heterogeneous assemblies are responsible for the icephobicity since they instigate distinct interactions with condensed water droplets. Lastly, the thermoelectric cooling (Peltier effect) and the adhesion strength of ice on the typical coatings were assessed. This investigation also includes examination on the icephobic durability of coating, which is enhanced when a small amount of polyethylene oligomer is incorporated into the coating.

Shaping nanofiltration channels in a carbonaceous membrane via controlling the pyrolysis atmosphere.

This work investigates the effect of atmosphere on pyrolysis of a polymer matrix (precursor) for directing its transformation towards more disordered graphene species and smaller graphitic nanograins. These two structural characteristics are crucial to the generation of nano-channels (NCs) pertinent to nanofiltration (NF). Two measures are explored hereby to conduct the study: varying the pyrolysis atmosphere and implementing highly dispersed nickel atomic clusters (Ni-clusters) in the coating matrix undergoing pyrolysis. A thermally reactive polymer precursor is developed to allow the above two measures to act more effectively. The various pyrolysis atmospheres employed include inert Ar, a reducing H2/N2 gas mixture, and weak oxidizing CO2. In the absence of the Ni-clusters, the H2/N2 atmosphere restrains the extent of graphitization through a chain transfer effect of H2 that ceases the free radical chain propagation, whereas CO2, owing to its high critical temperature (Tc) nature, shows the capability to reduce nanograin sizes. As for the catalytic roles of the embedded Ni-clusters, they vary with the pyrolysis atmosphere applied: offering coke nuclei for the growth of carbonaceous grains in Ar, enhancing gasification of carbon in CO2, and repressing the extent of aromatization via hydrogenation in H2/N2. The carbonaceous membranes (CnMs) obtained under the above pyrolysis conditions are distinguished by the distribution density and structure of NCs evolved, which locate primarily in the boundaries of nanograins. The NF of an aqueous solution of methylene blue (MB, 10 ppmw) is utilized to assess these CnMs to show impacts of the NCs on the separation performance.

Porous SiO2 Hollow Spheres as a Solar Reflective Pigment for Coatings.

This study starts with the synthesis of silica hollow spheres (HSs) by utilizing in situ synthesized polystyrene (PS) microspheres as the template for the deposition of a silica (SiO2) shell, followed by a slow gasification step in air to remove the PS core. The size of HS and the thickness of the porous SiO2 shell are tuned by varying the synthesis conditions of the PS latex and those of the sol-gel deposition, respectively. Various HS powder samples are characterized by ultraviolet-visible-near infrared (UV-vis-NIR) spectroscopy to determine their diffuse reflectance. Furthermore, they are used as the filler in an acrylic polymer matrix for the measurement of solar reflectivity on a solar spectrum reflectometer. It turns out that both cavity size and the structure of the SiO2 shell are influential in the reflection of NIR and UV-vis light, respectively. In addition, this study examines the effect on solar reflectivity of a selected metal oxide with a SiO2 HS. In conclusion, the cavity size of the HS has a strong impact on the reflectivity to NIR light whereas the shell itself affects the reflection of UV-blue light.

Proteome Analysis of Drosophila Mutants Identifies a Regulatory Role for 14-3-3ε in Metabolic Pathways.

The evolutionary conserved family of 14-3-3 proteins appears to have a role in integrating numerous intracellular pathways, including signal transduction, intracellular trafficking, and metabolism. However, little is known about how this interactive network might be affected by the direct abrogation of 14-3-3 function. The loss of Drosophila 14-3-3ε resulted in reduced survival of mutants during larval-to-adult transition, which is known to depend on an energy supply coming from the histolysis of fat body tissue. Here we report a differential proteomic analysis of larval fat body tissue at the onset of larval-to-adult transition, with the loss of 14-3-3ε resulting in the altered abundance of 16 proteins. These included proteins linked to protein biosynthesis, glycolysis, tricarboxylic acid cycle, and lipid metabolic pathways. The ecdysone receptor (EcR), which is responsible for initiating the larval-to-adult transition, colocalized with 14-3-3ε in wild-type fat body tissues. The altered protein abundance in 14-3-3ε mutant fat body tissue was associated with transcriptional deregulation of alcohol dehydrogenase, fat body protein 1, and lamin genes, which are known targets of the EcR. This study indicates that 14-3-3ε has a critical role in cellular metabolism involving either molecular crosstalk with the EcR or direct interaction with metabolic proteins.

The role of osteocyte apoptosis in cancer chemotherapy-induced bone loss.

Intensive cancer chemotherapy leads to significant bone loss, the underlying mechanism of which remains unclear. The objective of this study was to elucidate mechanisms for effect of the commonly used anti-metabolite methotrexate (MTX) on osteocytes and on general bone homeostasis. The current study in juvenile rats showed that MTX chemotherapy caused a 4.3-fold increase in the number of apoptotic osteocytes in tibial metaphysis, which was accompanied by a 1.8-fold increase in the number of tartrate-resistant acid phosphatase-positive bone resorbing osteoclasts, and a 35% loss of trabecular bone. This was associated with an increase in transcription of the osteoclastogenic cytokines IL-6 (10-fold) and IL-11 (2-fold). Moreover, the metaphyseal bone of MTX-treated animals exhibited a 37.6% increase in the total number of osteocytes, along with 4.9-fold higher expression of the DMP-1 transcript. In cultured osteocyte-like MLO-Y4 cells, MTX treatment significantly increased caspase-3-mediated apoptosis, which was accompanied by the formation of plasma membrane-born apoptotic bodies and an increase in IL-6 (24-fold) and IL-11 (29-fold) mRNA expression. Conditioned media derived from MTX-treated MLO-Y4 cells was twice as strong as untreated media in its capacity to induce osteoclast formation in primary bone marrow osteoclast precursors. Thus, our in vivo and in vitro data suggested that MTX-induced apoptosis of osteocytes caused higher recruitment of DMP-1 positive osteocytes and increased osteoclast formation, which could contribute towards the loss of bone homeostasis in vivo.

Drosophila 14-3-3ε has a crucial role in anti-microbial peptide secretion and innate immunity.

The secretion of anti-microbial peptides is recognised as an essential step in innate immunity, but there is limited knowledge of the molecular mechanism controlling the release of these effectors from immune response cells. Here, we report that Drosophila 14-3-3ε mutants exhibit reduced survival when infected with either Gram-positive or Gram-negative bacteria, indicating a functional role for 14-3-3ε in innate immunity. In 14-3-3ε mutants, there was a reduced release of the anti-microbial peptide Drosomycin into the haemolymph, which correlated with an accumulation of Drosomycin-containing vesicles near the plasma membrane of cells isolated from immune response tissues. Drosomycin appeared to be delivered towards the plasma membrane in Rab4- and Rab11-positive vesicles and smaller Rab11-positive vesicles. RNAi silencing of Rab11 and Rab4 significantly blocked the anterograde delivery of Drosomycin from the perinuclear region to the plasma membrane. However, in 14-3-3ε mutants there was an accumulation of small Rab11-positive vesicles near the plasma membrane. This vesicular phenotype was similar to that observed in response to the depletion of the vesicular Syntaxin protein Syx1a. In wild-type Drosophila immune tissue, 14-3-3ε was detected adjacent to Rab11, and partially overlapping with Syx1a, on vesicles near the plasma membrane. We conclude that 14-3-3ε is required for Rab11-positive vesicle function, which in turn enables antimicrobial peptide secretion during an innate immune response.

PolyPEGA with predetermined molecular weights from enzyme-mediated radical polymerization in water.

The preparation of acrylic polymers with predetermined molecular weights using metalloenzymes as catalysts, ascorbic acid as reducing agent and alkyl halides as initiators is reported. The mechanism of polymerization resembles an ARGET ATRP process.

Organic polymer composites as robust, non-covalent supports of metal salts.

A novel organic polymer composite for the encapsulation of metal catalysts and applications to synthesis are described here.

Controlled polymerization and self-assembly of a supramolecular star polymer with a guanosine quadruplex core.

A novel supramolecular star polymer is reported, which can be prepared either from atom-transfer radical polymerization (ATRP) of a self-assembled initiator or from self-assembly of a guanosine-capped linear polymer.