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In the present work, the immobilized footprint of β-glucosidase (BGL) on silica-coated iron oxide was explored to produce reusable catalysts with flexible active sites for high activity and heightened storage stability. Synthesized iron oxide particles were coated with silica and functionalized with various densities of (3-aminopropyl)triethoxysilane (APTES) to obtain particles with amine densities ranging from 0 to 3 × 10

Understanding how parents, and other primary caregivers, perceive and experience early childhood programs and services is essential for identifying family-centered facilitators and barriers to service utilization. Therefore, this paper aims to explore parent knowledge of and experiences with community efforts of an early childhood system in Illinois: the All Our Kids Early Childhood Networks (AOK Networks). Our research team conducted focus group interviews with 20 parents across four Illinois counties. A semi-structured interview guide was used to examine parent perceptions of an early childhood system’s community efforts in promoting the health and well-being of children aged from birth to five. Thematic network analysis was used to analyze all focus group data. Parents indicated three salient themes, including: (1) comprehensive information sharing practices, (2) diverse service engagement, and (3) barriers to service access. Overall, parents reported general satisfaction with the quality of available services and provided feedback regarding identified areas of need to increase the accessibility and utilization of local services. Engaging parents as partners is essential to the effective implementation of family-centered early childhood services. Families are the experts of their lived experiences, and incorporating their voices in program development and evaluation efforts works to increase positive child and family outcomes.

Background:Despite being central to atrial fibrillation (AF) and ventricular fibrillation (VF) mechanisms and therapy, the factors governing AF and VF termination are poorly understood. It has been noted that ratio of system size (L) and the two-point spatial correlation length (ξ2) are associated with time until termination in transient spatiotemporally chaotic systems, but the relationship between these characteristics and termination has not been systematically studied in human AF and VF.Objective:We aimed assess whether the time to cardiac fibrillation termination can be predicted using a novel estimator, the spatial autocorrelation dimension (Di), defined as the ratio ofLand ξ2, in human AF and VF.Methods:Diwas computed and compared in a multi-centre, multi-system study with data for sustained versus spontaneously terminating human AF/VF. VF data was collected during coronary-bypass surgery; and AF data during clinically indicated AF ablation. We analyzed: i) VF mapped using a 256-electrode epicardial sock (n=12pts); ii) AF mapped using a 64-electrode constellation basket-catheter (n=15pts); iii) AF mapped using a 16-electrode HD-grid catheter (n=42pts). To investigate temporal fibrillation persistence, the response of AF-episodes to flecainide (n=7pts) was also studied.Results:Spontaneously terminating fibrillation demonstrated a lowerDi(PDiwas also seen in paroxysmal compared to persistent AF (P=0.002). Post-flecainide,Didecreased over time (PDiwas also associated with longer-lasting episodes of AF/VF (R2>0.90, PConclusion:Dipredicts the temporal persistence of cardiac fibrillation. This finding provides potentially important insights into a possible common pathway to termination and therapeutic approaches.

Supplemental data regarding cecum morphology, body weights, adhesion molecule expression in spleen and thymus, spleen size, tumor and stroma cell viability, and IFN-g and VEGF serum levels after induction of dysbiosis.

SF1: Tumor growth assays for lung and HN tumors. SF2: The complete set of MCR-derived pure component spectra and tables listing their peak assignments. SF3: Representative MCR score maps for lung and HN tumor datasets. SF4: Histologic images for HN tumor dataset. TS1-4: Results of binary leave-one-mouse-out SVM analyses.

Delay in the assessment of tumor response to radiotherapy continues to pose a major challenge to quality of life for patients with nonresponsive tumors. Here, we exploited label-free Raman spectroscopic mapping to elucidate radiation-induced biomolecular changes in tumors and uncovered latent microenvironmental differences between treatment-resistant and -sensitive tumors. We used isogenic radiation-resistant and -sensitive A549 human lung cancer cells and human head and neck squamous cell carcinoma (HNSCC) cell lines (UM-SCC-47 and UM-SCC-22B, respectively) to grow tumor xenografts in athymic nude mice and demonstrated the molecular specificity and quantitative nature of Raman spectroscopic tissue assessments. Raman spectra obtained from untreated and treated tumors were subjected to chemometric analysis using multivariate curve resolution-alternating least squares (MCR-ALS) and support vector machine (SVM) to quantify biomolecular differences in the tumor microenvironment. The Raman measurements revealed significant and reliable differences in lipid and collagen content postradiation in the tumor microenvironment, with consistently greater changes observed in the radiation-sensitive tumors. In addition to accurately evaluating tumor response to therapy, the combination of Raman spectral markers potentially offers a route to predicting response in untreated tumors prior to commencing treatment. Combined with its noninvasive nature, our findings provide a rationale for in vivo studies using Raman spectroscopy, with the ultimate goal of clinical translation for patient stratification and guiding adaptation of radiotherapy during the course of treatment.Significance:These findings highlight the sensitivity of label-free Raman spectroscopy to changes induced by radiotherapy and indicate the potential to predict radiation resistance prior to commencing therapy.

BACKGROUND While there is some research on adult recovery from substance use, particularly regarding its definition and correlates, there appears to be little work on defining recovery for adolescents. Definitional research on adolescent recovery, including adolescents’ perceptions of recovery, will create opportunities to enhance adolescent recovery-oriented systems of care. METHODS Data for this study were collected through Qualtrics, an online survey platform. The survey gathered different behavioral and social indicators, including whether adolescents resolved a problem with substances or considered themselves to be in recovery. Youth also responded to a list of statements, indicating whether the item was consistent with their definition of problem resolution or recovery. The final analytic sample included 63 (16.3%) adolescents in recovery and 323 who were not. We analyzed descriptive statistics for both groups and assessed whether perceptions on problem resolution or recovery definitions differed by recovery status using chi-square tests. RESULTS Among all statements presented to youth in reference to problem resolution, the most commonly endorsed was “stop completely” (59.8%), followed by “not hanging out with friends who use a lot” (47.7%) and “getting counseling for alcohol/drug use” (46.1%). Youth in recovery were significantly less likely than those not in recovery to endorse “stop completely” as being associated with problem resolution (p

Antibiotic-induced microbial imbalance, or dysbiosis, has systemic and long-lasting effects on the host and response to cancer therapies. However, the effects on tumor endothelial cells are largely unknown. Therefore, the goal of the current study was to generate matched B16-F10 melanoma associated endothelial cell lines isolated from mice with and without antibiotic-induced dysbiosis. After validating endothelial cell markers on a genomic and proteomic level, functional angiogenesis assays (i.e., migration and tube formation) also confirmed their vasculature origin. Subsequently, we found that tumor endothelial cells derived from dysbiotic mice (TEC-Dys) were more sensitive to ionizing radiotherapy in the range of clinically-relevant hypofractionated doses, as compared to tumor endothelial cells derived from orthobiotic mice (TEC-Ortho). In order to identify tumor vasculature-associated drug targets during dysbiosis, we used tandem mass tag mass spectroscopy and focused on the statistically significant cellular membrane proteins overexpressed in TEC-Dys. By these criteria c-Met was the most differentially expressed protein, which was validated histologically by comparing tumors with or without dysbiosis. Moreover, in vitro, c-Met inhibitors Foretinib, Crizotinib and Cabozantinib were significantly more effective against TEC-Dys than TEC-Ortho. In vivo, Foretinib inhibited tumor growth to a greater extent during dysbiosis as compared to orthobiotic conditions. Thus, we surmise that tumor response in dysbiotic patients may be greatly improved by targeting dysbiosis-induced pathways, such as c-Met, distinct from the many targets suppressed due to dysbiosis.

Nanoscale phenomena are under increasingly intense investigation both in academia and industry. The unique physical and chemical properties stemming from their high surface area and confined space ...

Purpose Ongoing angiogenesis renders the tumor endothelium unresponsive to inflammatory cytokines and interferes with adhesion of leukocytes, resulting in escape from immunity. This process is referred to as tumor endothelial cell anergy. We aimed to investigate whether anti-angiogenic agents can overcome endothelial cell anergy and provide pro-inflammatory conditions. Experimental design Tissues of renal cell carcinoma (RCC) patients treated with VEGF pathway-targeted drugs and control tissues were subject to RNAseq and immunohistochemical profiling of the leukocyte infiltrate. Analysis of adhesion molecule regulation in cultured endothelial cells, in a preclinical model and in human tissues was performed and correlated to leukocyte infiltration. Results It is shown that treatment of RCC patients with the drugs sunitinib or bevacizumab overcomes tumor endothelial cell anergy. This treatment resulted in an augmented inflammatory state of the tumor, characterized by enhanced infiltration of all major leukocyte subsets, including T cells, regulatory T cells, macrophages of both M1- and M2-like phenotypes and activated dendritic cells. In vitro, exposure of angiogenic endothelial cells to anti-angiogenic drugs normalized ICAM-1 expression. In addition, a panel of tyrosine kinase inhibitors was shown to increase transendothelial migration of both non-adherent and monocytic leukocytes. In primary tumors of RCC patients, ICAM-1 expression was found to be significantly increased in both the sunitinib and bevacizumab-treated groups. Genomic analysis confirmed the correlation between increased immune cell infiltration and ICAM-1 expression upon VEGF-targeted treatment. Conclusion The results support the emerging concept that anti-angiogenic therapy can boost immunity and show how immunotherapy approaches can benefit from combination with anti-angiogenic compounds.

Background\udThe TEG 6s is an automated cartridge-based device with limited description of use in obstetric haemorrhage. The aim of this analysis was to describe the utility of TEG 6s in identifying abnormal laboratory results of coagulation and platelet count, and inform an interventional treatment algorithm for postpartum haemorrhage.\udMethods\udA prospective observational cohort study of 521 women with moderate to severe obstetric haemorrhage (>1000 mL blood loss), including 372 women with at least one TEG 6s test. A non-pregnant control group was used for reference. TEG 6s test parameters Citrated Functional Fibrinogen (CFF), Citrated Kaolin TEG (CK) and Citrated Rapid TEG (CRT) were compared with paired laboratory tests of fibrinogen, PT/aPTT and platelet count, obtained during haemorrhage.\udResults\udAmong 456 TEG 6s tests, 389 were matched with laboratory coagulation results. The receiver operator characteristic area-under-the-curve (95% CI) for CFF amplitude by 10 min to detect Clauss fibrinogen ≤2 g/L was 0.95 (0.91 to 0.99) (P

BackgroundComplement FH (FH) is a key regulator of complement activity whereas thrombin (FIIa) is central to hemostasis with both pro- and anticoagulant functions. Both have separately been shown to have auxiliary activities across the two systems. The purpose of this study was to determine the effect of FH on pro- and anti-coagulant functions and investigate the interaction between FH and thrombin.MethodsTail bleeding time and hemolysis were measured in FH-deficient mice (CFH−/−). Activated partial thromboplastin time (aPTT) was determined in FH-depleted human plasma. FH effect on fibrin clot generation was investigated in turbidity assays and on activated protein C (APC) generation. Binding affinity of thrombin with FH was determined using surface plasmon resonance (SPR).ResultsTail bleeding time in CFH−/− mice was significantly prolonged compared to wild type mice. The aPTT in FH-depleted human plasma was elevated compared to normal plasma and restored by adding back FH to depleted plasma. Accordingly, FH enhanced thrombin-mediated fibrin clot generation by shortening lag time, increasing rate of clot formation and maximum turbidity, and affected clot structure. Despite this, FH also increased the rate of thrombin-mediated protein C (PC) activation, both in the presence and absence of soluble recombinant thrombomodulin (TM). Nanomolar affinity binding of FH with thrombin, but not prothrombin, was confirmed.ConclusionComplement FH binds thrombin with strong affinity and acts as a novel cofactor that enhances both pro- and anticoagulant actions of thrombin. These data highlight an important role for FH in hemostasis.Key pointsAbsence of FH prolongs tail bleeding time in CFH−/− mice and activated partial thromboplastin time (aPTT) is elevated in human FH-depleted plasma.FH acts a cofactor for thrombin by enhancing fibrin generation, altering fibrin clot structure and enhancing TM-thrombin mediated protein C activation

The crosstalk between the microbiota and the host is crucial to maintaining immune homeostasis. A growing body of evidence shows that microbial imbalance affects cancer susceptibility and progression. A common significant influence on the microbiota in the gastrointestinal (GI) tract is the use of antibiotics. Oral antibiotics severely alter the bacteria in the GI tract by destroying beneficial bacteria alongside pathogenic ones — producing a state of microbial imbalance called dysbiosis. The overall use of antibiotics has increased by more than 30% in recent years, and moreover, many cancer patients are prescribed antibiotics during treatment, as infection is frequently a complication. We hypothesized that antibiotic-induced dysbiosis of the gut microbiota would suppress cytokine profiles in the host, thereby causing stromal immune surveillance suppression. We found that antibiotics as well as tumorigenesis caused changes in the bacterial abundance, composition, or diversity in the GI tract. On the host side, we found that antibiotic-induced dysbiosis caused the small intestine and cecum to enlarge and elongate, yet the colon was not affected. Moreover, B16-F10 melanoma and Lewis Lung carcinoma progressed more quickly during dysbiosis. Mechanistically, tumor progression was mediated by suppressed TNF-α levels, both locally and systemically. In contrast, IFN-γ and VEGF did not change significantly during dysbiosis. The suppressed levels of TNF-α, a pleiotropic regulator of intercellular adhesion molecule-1 (ICAM-1), resulted in reduced expression of tumor endothelial cell associated ICAM-1, and a subsequent decrease in the number of activated and effector CD8+ T-cells in the tumor. However, suppression of ICAM-1 or its binding site, the alpha subunit of lymphocyte function-associated antigen-1 (LFA-1), was not seen in established vasculature during dysbiosis as assessed in the spleen or the thymus. While dysbiosis enhanced tumor growth in ICAM-1+/+ and immunocompetent mice, it did not affect tumor growth in ICAM-1-/- or T-cell deficient mice, highlighting the influence and impact of dysbiosis on T-cell trafficking and extravasation. TNF-α supplementation in dysbiotic mice was able to overcome the dysbiosis-induced suppression as evident by increased ICAM-1 expression, leukocyte trafficking and tumor growth inhibition. Overall, these results demonstrate the importance of commensal bacteria in supporting immune surveillance and expand our understanding of the crosstalk between the gut microbiome and the stroma in distal non-GI tract tumors. Moreover, this knowledge has the potential to influence clinical decision-making when treating patients for solid cancers with dysbiosis-inducing antibiotics and may further inform concurrent and (neo-)adjuvant treatments to overcome the adverse effects of the antibiotics. Citation Format: Samir V. Jenkins, Michael S. Robeson, Eric R. Siegel, Charles M. Quick, Robert J. Griffin, Martin J. Cannon, Kieng B. Vang, Ruud P. Dings. T-cell trafficking and extravasation is suppressed in distal tumors during gastrointestinal tract dysbiosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3529.

Microtubules are essential to neuron shape and function. Therefore, the stability of the microtubule cytoskeleton must be carefully regulated. Acetylation of tubulin has the potential to directly tune microtubule stability, and proteomic studies have identified several acetylation sites in α-tubulin. This includes the highly conserved residue lysine 394 (K394), which is located at the αβ-tubulin dimer interface. Using a fly model, we show that α-tubulin K394 is acetylated in the nervous system and is an essential residue. We found that an acetylation-blocking mutation in endogenous α-tubulin, K394R, perturbs the synaptic morphogenesis of motoneurons by reducing microtubule stability. Intriguingly, the K394R mutation has opposite effects on the growth of two functionally and morphologically distinct motoneurons, revealing neuron-type-specific responses when microtubule stability is altered. Eliminating the deacetylase HDAC6 increases K394 acetylation, and the over-expression of HDAC6 reduces microtubule stability similar to the K394 mutant. Thus, our findings implicate α-tubulin K394 and its acetylation in the regulation of microtubule stability and suggest that HDAC6 regulates K394 acetylation during synaptic morphogenesis.

IntroductionThe shape of the glucose curve during an oral glucose tolerance test (OGTT) reflects β-cell function in populations without diabetes but has not been as well studied in those with diabetes. A monophasic shape has been associated with higher risk of diabetes, while a biphasic pattern has been associated with lower risk. We sought to determine if phenotypic or metabolic characteristics were associated with glucose response curve shape in adults with type 2 diabetes treated with metformin alone.Research design and methodsThis is a cross-sectional analysis of 3108 metformin-treated adults with type 2 diabetes diagnosed ResultsThe monophasic profile was the most common (67.8% monophasic, 5.5% biphasic, 26.7% continuous rise). The monophasic subgroup was younger, more likely male and white, and had higher body mass index (BMI), while the continuous rise subgroup was more likely female and African American/black. HOMA2-S and fasting glucose did not differ among the subgroups. The biphasic subgroup had the highest early, late, and total insulin and C peptide responses (all pConclusionsBased on the large multiethnic GRADE cohort, sex, race, age, and BMI were found to be important determinants of the shape of the glucose response curve. A pattern of a continuously rising glucose at 2 hours reflected reduced β-cell function and may portend increased glycemic failure rates.Trial registration numberNCT01794143.

The mechanisms governing cardiac fibrillation remain unclear; however, it most likely represents a form of spatiotemporal chaos with conservative system dynamics. Renewal theory has recently been suggested as a statistical formulation with governing equations to quantify the formation and destruction of wavelets and rotors in fibrillatory dynamics. In this perspective Review, we aim to explain the origin of the renewal theory paradigm in spatiotemporal chaos. The ergodic nature of pattern formation in spatiotemporal chaos is demonstrated through the use of three chaotic systems: two classical systems and a simulation of cardiac fibrillation. The logistic map and the baker's transformation are used to demonstrate how the apparently random appearance of patterns in classical chaotic systems has macroscopic parameters that are predictable in a statistical sense. We demonstrate that the renewal theory approach developed for cardiac fibrillation statistically predicts pattern formation in these classical chaotic systems. Renewal theory provides governing equations to describe the apparently random formation and destruction of wavelets and rotors in atrial fibrillation (AF) and ventricular fibrillation (VF). This statistical framework for fibrillatory dynamics provides a holistic understanding of observed rotor and wavelet dynamics and is of conceptual significance in informing the clinical and mechanistic research of the rotor and multiple-wavelet mechanisms of AF and VF.

Background: The combination of biological variation and nanomaterial heterogeneity makes elucidating the mechanisms of interactions between cells and nanoparticles extremely complicated. Accurate nanoparticle quantification can be extremely challenging, and cellular response can change based on the location of the nanoparticle and the cell type under investigation. These complications are only augmented by the additional of external stimuli. These limitations have yielded a wide range of studies that show effects, but often provide little mechanistic insight. Results: Gold nanomaterials were stably immobilized onto glass coverslips treated with mercaptosilane to control both the average number of nanoparticles that interact with cells and their spatial orientation relative to the cell membrane. Surfaces were characterized optically and by electron microscopy to confirm their surface density and uniformity. The thermal response of Au nanocage-coated surfaces to near infrared laser irradiation was measured in cell culture medium and modelled computationally. The modelling showed a vastly higher thermal dose than would be predicted by bulk temperature measurements. Adherent or non-adherent cell lines were cultured directly on the nanocage-coated surface or in the medium, respectively, in culture wells and laser irradiation was applied. Survival of cells growing in suspension correlated with the bulk temperature increase in the culture medium, as measured by viability assay. Conversely, adherent cells exhibited a much greater susceptibility than expected from the bulk temperature measurement, which is ostensibly related to the close interaction with the nanoparticles on their growth substrate and induction of substantially greater thermal dose upon laser exposure. Conclusions: This platform is designed to be a new tool to determine how many particles need to be in contact with a cell to induce desired physical or biological effects. Here we demonstrate the delivery of precise thermal doses following laser irradiation. The anticipated biological effects based on bulk measurements vastly underestimated the effects that were observed, which is ascribed to the proximity of the nanoparticle to the cell and the extraordinary high surface temperature of the particle. This platform could be expanded to a variety of nanoparticles, external stimuli, and cell types to enable more deliberate and optimized application of nanomedicine.

The combination of radiotherapy and immunotherapy may generate synergistic anti-tumor host immune responses and promote abscopal effects. Spatial fractionation of a radiation dose has been found to promote unique physiological responses of tumors, which might promote synergy with immunotherapy. To determine whether spatial fractionation may augment immune activity, whole-tumor or spatial fractionation grid radiation treatment (GRID) alone or in combination with antibodies against immune checkpoints PD1 and CTLA-4 were tested in an immunocompetent mouse model using a triple negative breast tumor (4T1). Tumor growth delay, immuno-histochemistry and flow cytometry were used to characterize the effects of each treatment type. Whole-beam radiation with immune checkpoint inhibition significantly restrained tumor growth in the irradiated tumor, but not abscopal tumors, compared to either of these treatments alone. In mice that received spatially fractionated irradiation, evidence of abscopal immune responses were observed in contralateral tumors with markedly enhanced infiltration of both antigen-presenting cells and activated T cells, which were preceded by increased systemic IFNγ production and led to eventual tumor growth delay. These studies suggest that systemic immune activation may be triggered by employing GRID to a primary tumor lesion, promoting anti-tumor immune responses outside the treatment field. Interestingly, PD-L1 was found to be upregulated in abscopal tumors from GRID-treated mice. Combined radio-immunotherapy therapy is becoming a validated and novel approach in the treatment of cancer. With the potential increased benefit of GRID to augment both local and metastatic disease responses, further exploration of GRID treatment as a part of current standards of care is warranted.

Summary: At the cellular level, α-tubulin acetylation alters the structure of microtubules to render them mechanically resistant to compressive forces. How this biochemical property of microtubule acetylation relates to mechanosensation remains unknown, although prior studies have shown that microtubule acetylation influences touch perception. Here, we identify the major Drosophila α-tubulin acetylase (dTAT) and show that it plays key roles in several forms of mechanosensation. dTAT is highly expressed in the larval peripheral nervous system (PNS), but it is largely dispensable for neuronal morphogenesis. Mutation of the acetylase gene or the K40 acetylation site in α-tubulin impairs mechanical sensitivity in sensory neurons and behavioral responses to gentle touch, harsh touch, gravity, and vibration stimuli, but not noxious thermal stimulus. Finally, we show that dTAT is required for mechanically induced activation of NOMPC, a microtubule-associated transient receptor potential channel, and functions to maintain integrity of the microtubule cytoskeleton in response to mechanical stimulation. : Yan et al. identify the major microtubule acetylase in Drosophila and show that the enzyme and microtubule acetylation broadly control mechanosensation, but not other sensory modalities. Acetylation is required for mechanosensation by the TRP channel NOMPC, and possibly other channels, by virtue of its effects on microtubule mechanical stability and/or dynamics. Keywords: Drosophila, mechanosensation, microtubule acetylation, TRP channel, somatosensory neuron

Hypoxia, a hallmark characteristic of glioblastoma (GBM) induces changes in the transcriptome and the proteome of tumor cells. We discovered that hypoxic stress produces significant qualitative and quantitative changes in the protein content of secreted exosomes from GBM cells. Among the proteins found to be selectively elevated in hypoxic exosomes were protein-lysine 6-oxidase (LOX), thrombospondin-1 (TSP1), vascular derived endothelial factor (VEGF) and a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), well studied contributors to tumor progression, metastasis and angiogenesis. Our findings demonstrate that hypoxic exosomes induce differential gene expression in recipient glioma cells. Glioma cells stimulated with hypoxic exosomes showed a marked upregulation of small nucleolar RNA, C/D box 116–21 (SNORD116-21) transcript among others while significantly downregulated the potassium voltage-gated channel subfamily J member 3 (KCNJ3) message. This differential expression of certain genes is governed by the protein cargo being transferred via exosomes. Additionally, compared to normoxic exosomes, hypoxic exosomes increased various angiogenic related parameters vis-à-vis, overall tube length, branching intervals and length of isolated branches studied in tube formation assay with endothelial progenitor cells (EPCs). Thus, the intercellular communication facilitated via exosomes secreted from hypoxic GBM cells induce marked changes in the expression of genes in neighboring normoxic tumor cells and possibly in surrounding stromal cells, many of which are involved in cancer progression and treatment resistance mechanisms., Highlights • In GBM, hypoxic stress induces profound changes in the protein content of secreted exosomes. • Hypoxic exosomal contents induce angiogenesis and significant changes in recipient GBM cell transcriptome. • Hypoxic exosomes play a major role leading to tumor proliferation, tumor growth and cell survival.

Gold nanoparticles with a graft density of 0.09, 0.30 and 0.40 chains/nm2 of poly(N-isopropylacrylamide) were reproducibly synthesized by varying the ratio of disulfide terminated poly(N-isopropylacrylamide) to gold nanoparticle. The polymer coated nanoparticles were stable at room temperature in 50 mM NaCl, yet agglomerated at 37 °C. Previous studies have observed conflicting results as to the reversibility of this agglomeration. Particle agglomeration with three different graft densities was studied in 50 mM NaCl by measurements of their localized surface plasmon resonance and hydrodynamic diameter, and imaging with electron microscopy. Agglomerates with a polymer graft density of 0.30 and 0.40 chains/nm2 could be dispersed with sonication, while particles with a graft density of 0.09 chains/nm2 irreversibly aggregated. The graft density dependence on whether agglomeration or aggregation occurred is due to changes in collapsed polymer steric effects. Localized surface plasmon resonance measurements of agglomerates were discordant with hydrodynamic diameter measurements in determining agglomeration reversibility, which shed light on reasons previous reports yielded different interpretations on the reversibility of this agglomeration. This work demonstrates how polymer graft density affects thermoresponsive nanoparticle stability in salt solutions and the need for use of complementary techniques when determining agglomeration.

This abstract was withdrawn by the authors.

Photodynamic therapy (PDT) of tumors relies on the delivery of a photosensitizer to the tumor site followed by external laser activation. The effectiveness of the therapy is determined by the fluence and irradiation of the laser, intratumoral photosensitizer retention, and availability of molecular oxygen. We hypothesized that retention and resulting therapeutic response may be improved with local hyperthermia. Tumors were grown in the rear limb then locally heated for 1 hour at 42.5 °C immediately following i.v. injection of the photosensitizer in an immunocompetent murine model. Laser exposure was applied at 2 h or 24 h after the heating session. Administration of heating caused a significant growth delay when a single laser treatment was applied only 2 hours following injection. However, in animals given photosensitizer only and irradiated 2 h later there was no measurable anti-tumor effect observed. The marked anti-tumor effects obtained with prior local hyperthermia were observed even as the dose was lowered from 10 mg/kg to 1.3 mg/kg. Additionally, histological analysis of our intial studies revealed that the majority of the tumor tissue (~75%) was necrotic after two days when heat was combined with PDT, while PDT alone resulted in only ~25% necrotic tissue. Additionally, a significant, though less notable, increase in the efficacy was observed if the laser treatment was applied 24 hours after hyperthermia and photosensitizer administration. This increase is in part ascribed to the increased retention of the photosensitizer in the tumor tissue and likely lasting effects on tumor blood flow and oxygenation in the heated vs. control groups.

American chestnut was once a foundation species of eastern North American forests, but was rendered functionally extinct in the early 20th century by an exotic fungal blight (Cryphonectria parasitica). Over the past 30 years, The American Chestnut Foundation (TACF) has pursued backcross breeding to generate hybrids that combine the timber-type form of American chestnut with the blight tolerance of Chinese chestnut. The backcross strategy has been implemented based on the hypothesis that blight tolerance is conferred by few major effect alleles. We tested this hypothesis by developing genomic prediction models for five presence/absence blight phenotypes of 1,230 BC3F2selection candidates and average canker severity of their BC3F3progeny. We also genotyped pure Chinese and American chestnut reference panels to estimate the proportion of BC3F2genomes inherited from parent species. We found that genomic prediction from a method that assumes an infinitesimal model of inheritance (HBLUP) has a similar predictive ability to a method that tends to perform well for traits controlled by major genes (Bayes C). Furthermore, the proportion of BC3F2trees’ genomes inherited from American chestnut was negatively correlated with the blight tolerance of BC3F2trees and their progeny. On average, selected BC3F2trees inherited 83% of their genome from American chestnut and have blight-tolerance that is intermediate between F1hybrids and American chestnut. Results suggest polygenic rather than major gene inheritance for blight tolerance. The blight-tolerance of restoration populations will be enhanced by advancing additional sources of blight-tolerance through fewer backcross generations and by potentially by breeding with transgenic blight-tolerant trees.

Delay in the assessment of tumor response to radiotherapy continues to pose a major challenge to quality of life for patients with nonresponsive tumors. Here, we exploited label-free Raman spectroscopic mapping to elucidate radiation-induced biomolecular changes in tumors and uncovered latent microenvironmental differences between treatment-resistant and -sensitive tumors. We used isogenic radiation-resistant and -sensitive A549 human lung cancer cells and human head and neck squamous cell carcinoma (HNSCC) cell lines (UM-SCC-47 and UM-SCC-22B, respectively) to grow tumor xenografts in athymic nude mice and demonstrated the molecular specificity and quantitative nature of Raman spectroscopic tissue assessments. Raman spectra obtained from untreated and treated tumors were subjected to chemometric analysis using multivariate curve resolution-alternating least squares (MCR-ALS) and support vector machine (SVM) to quantify biomolecular differences in the tumor microenvironment. The Raman measurements revealed significant and reliable differences in lipid and collagen content postradiation in the tumor microenvironment, with consistently greater changes observed in the radiation-sensitive tumors. In addition to accurately evaluating tumor response to therapy, the combination of Raman spectral markers potentially offers a route to predicting response in untreated tumors prior to commencing treatment. Combined with its noninvasive nature, our findings provide a rationale for in vivo studies using Raman spectroscopy, with the ultimate goal of clinical translation for patient stratification and guiding adaptation of radiotherapy during the course of treatment. Significance: These findings highlight the sensitivity of label-free Raman spectroscopy to changes induced by radiotherapy and indicate the potential to predict radiation resistance prior to commencing therapy.

American chestnut was once a foundation species of eastern North American forests, but was rendered functionally extinct in the early 20th century by an exotic fungal blight (Cryphonectria parasitica). Over the past 30 years, the American Chestnut Foundation (TACF) has pursued backcross breeding to generate hybrids that combine the timber‐type form of American chestnut with the blight resistance of Chinese chestnut based on a hypothesis of major gene resistance. To accelerate selection within two backcross populations that descended from two Chinese chestnuts, we developed genomic prediction models for five presence/absence blight phenotypes of 1,230 BC3F2 selection candidates and average canker severity of their BC3F3 progeny. We also genotyped pure Chinese and American chestnut reference panels to estimate the proportion of BC3F2 genomes inherited from parent species. We found that genomic prediction from a method that assumes an infinitesimal model of inheritance (HBLUP) has similar accuracy to a method that tends to perform well for traits controlled by major genes (Bayes C). Furthermore, the proportion of BC3F2 trees' genomes inherited from American chestnut was negatively correlated with the blight resistance of these trees and their progeny. On average, selected BC3F2 trees inherited 83% of their genome from American chestnut and have blight resistance that is intermediate between F1 hybrids and American chestnut. Results suggest polygenic inheritance of blight resistance. The blight resistance of restoration populations will be enhanced through recurrent selection, by advancing additional sources of resistance through fewer backcross generations, and by potentially by breeding with transgenic blight‐tolerant trees.

The overall use of antibiotics has increased significantly in recent years. Besides fighting infections, antibiotics also alter the gut microbiota. Commensal bacteria in the gastrointestinal tract are crucial to maintain immune homeostasis, and microbial imbalance or dysbiosis affects disease susceptibility and progression. We hypothesized that antibiotic-induced dysbiosis of the gut microbiota would suppress cytokine profiles in the host, thereby leading to changes in the tumor microenvironment. The induced dysbiosis was characterized by alterations in bacterial abundance, composition, and diversity in our animal models. On the host side, antibiotic-induced dysbiosis caused elongated small intestines and ceca, and B16-F10 melanoma and Lewis lung carcinoma progressed more quickly than in control mice. Mechanistic studies revealed that this progression was mediated by suppressed TNFα levels, both locally and systemically, resulting in reduced expression of tumor endothelial adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1) and a subsequent decrease in the number of activated and effector CD8+ T cells in the tumor. However, suppression of ICAM-1 or its binding site, the alpha subunit of lymphocyte function-associated antigen-1, was not seen in the spleen or thymus during dysbiosis. TNFα supplementation in dysbiotic mice was able to increase ICAM-1 expression and leukocyte trafficking into the tumor. Overall, these results demonstrate the importance of commensal bacteria in supporting anticancer immune surveillance, define an important role of tumor endothelial cells within this process, and suggest adverse consequences of antibiotics on cancer control. Significance: Antibiotic-induced dysbiosis enhances distal tumor progression by altering host cytokine levels, resulting in suppression of tumor endothelial adhesion molecules and activated and effector CD8+ T cells in the tumor.

Background: Overdose deaths from fentanyl and its analogs have increased significantly since 2013. There are limited data regarding the prevalence of fentanyl exposure among emergency department (ED) patients with active opioid use. Methods: We conducted a cross-sectional study at an urban hospital from May 20 to July 30, 2018. A convenience sample of adult ED patients with active opioid use, defined as opioid use within seven days prior to ED visit, were enrolled. Rapid Response® Single Drug Test Strip (BTNX Inc., Markham, Canada) was used to detect fentanyl in urine samples. Information on demographic, substance use history, and knowledge of fentanyl was obtained using a brief survey tool. Our primary outcome was the prevalence of fentanyl exposure; secondary outcomes included patients’ knowledge regarding potency, risk of overdose death from fentanyl and intentional purchase of fentanyl. Results: During our study period, 451 patients reported active substance use. Of these, 208 reported active opioid use and 165 consented for the study. The median age was 49 years [interquartile range: 38, 57] and 77.0% (n = 127) were male; 42 participants (25.5%) presented to ED after an acute overdose event. Heroin was the preferred opioid of use in 90.8% of the participants, primarily via intranasal route (64.6%). Polysubstance use was reported in 98.8%, most commonly with cocaine (57.6%; n = 95). Fentanyl was detected in 104 out of 129 urine samples tested (80.6%). 84.2% (n = 139) identified fentanyl as highly potent and 85.5% (n = 141) recognized highest risk of death in fentanyl overdose. A larger proportion of non-overdose participants intentionally purchased fentanyl (34.1%; n = 42) compared to the overdose group (16.7%, n = 7; p = .04). Conclusions: The majority of ED patient with active opiate use were exposed to fentanyl while one in three participants intentionally purchased fentanyl despite their awareness of its potency and the high-risk of death from overdose.

A major challenge in photothermal treatment is generating sufficient heat to eradicate diseased tissue while sparing normal tissue. Au nanomaterials have shown promise as a means to achieve highly localized photothermal treatment. Toward that end, the synthetic peptide anginex was conjugated to Au nanocages. Anginex binds to galectin-1, which is highly expressed in dividing endothelial cells found primarily in the tumor vasculature. The skin surface temperature during a 10 min laser exposure of subcutaneous murine breast tumors did not exceed 43°C and no normal tissue damage was observed, yet a significant anti-tumor effect was observed when laser was applied 24 h post-injection of targeted nanocages. Untargeted particles showed little effect in immunocompetent, tumor-bearing mice under these conditions. Photoacoustic, photothermal, and ICP-MS mapping of harvested tissue showed distribution of particles near the vasculature throughout the tumor. This uptake pattern within the tumor combined with a minimal overall temperature rise were nonetheless sufficient to induce marked photothermal efficacy and evidence of tumor control. Importantly, this evidence suggests that bulk tumor temperature during treatment does not correlate with treatment outcome, which implies that targeted nanomedicine can be highly effective when closely bound/distributed in and around the tumor endothelium and extensive amounts of direct tumor cell binding may not be a prerequisite of effective photothermal approaches.

Background Here we investigated the influence of different stabilization and storage strategies on the quality and composition of the fecal microbial community. Namely, same-day isolated murine DNA was compared to samples stored for 1 month in air at ambient temperature, with or without preservative buffers (i.e. EDTA and lysis buffer), different temperatures (i.e. 4 °C, − 20 °C, and − 80 °C), and hypoxic conditions. Results Only storage in lysis buffer significantly reduced DNA content, yet without integrity loss. Storage in EDTA affected alpha diversity the most, which was also reflected in cluster separation. Distinct changes were also seen in the phyla and bacterial species abundance per storage strategy. Metabolic function analysis showed 22 pathways not significantly affected by storage conditions, whereas the tyrosine metabolism pathway was significantly changed in all strategies except by EDTA. Conclusion Each long-term storage strategy introduced a unique post-collection bias, which is important to take into account when interpreting data. Electronic supplementary material The online version of this article (10.1186/s12866-018-1359-5) contains supplementary material, which is available to authorized users.

Strategies to increase the proportion of neural stem cells that differentiate into neurons are vital for therapy of neurodegenerative disorders. In vitro, the extracellular matrix composition and topography have been found to be important factors in stem cell differentiation. We have developed a novel artificial extracellular matrix (aECM) formed by attaching gold nanocages (AuNCs) to glass coverslips. After culturing rat neural stem cells (rNSCs) on these gold nanocage-coated surfaces (AuNC-aECMs), we observed that 44.6% of rNSCs differentiated into neurons compared to only 27.9% for cells grown on laminin-coated glass coverslips. We applied laser irradiation to the AuNC-aECMs to introduce precise amounts of photothermally induced heat shock in cells. Our results showed that laser-induced thermal stimulation of AuNC-aECMs further enhanced neuronal differentiation (56%) depending on the laser intensity used. Response to these photothermal effects increased the expression of heat shock protein 27, 70, and 90α in rNSCs. Analysis of dendritic complexity showed that this thermal stimulation promoted neuronal maturation by increasing dendrite length as thermal dose was increased. In addition, we found that cells growing on AuNC-aECMs post laser irradiation exhibited action potentials and increased the expression of voltage-gated Na+ channels compared to laminin-coated glass coverslips. These results indicate that the photothermal response induced in cells growing on AuNC-aECMs can be used to produce large quantities of functional neurons, with improved electrochemical properties, that can potentially be transplanted into a damaged central nervous system to provide replacement neurons and restore lost function.

Resistance to conventional antibiotics is a growing public health concern that is quickly outpacing the development of new antibiotics. This has led the Infectious Diseases Society of America (IDSA) to designate Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species as "ESKAPE pathogens" on the basis of the rapidly decreasing availability of useful antibiotics. This emphasizes the urgent need for alternative therapeutic strategies to combat infections caused by these and other bacterial pathogens. In this study, we used Staphylococcus aureus (S. aureus) as a proof-of-principle ESKAPE pathogen to demonstrate that an appropriate antibiotic (daptomycin) can be incorporated into polydopamine-coated gold nanocages (AuNC@PDA) and that daptomycin-loaded AuNC@PDA can be conjugated to antibodies targeting a species-specific surface protein (staphylococcal protein A; Spa) as a means of achieving selective delivery of the nanoconstructs directly to the bacterial cell surface. Targeting specificity was confirmed by demonstrating a lack of binding to mammalian cells, reduced photothermal and antibiotic killing of the Spa-negative species Staphylococcus epidermidis, and reduced killing of S. aureus in the presence of unconjugated anti-Spa antibodies. We demonstrate that laser irradiation at levels within the current safety standard for use in humans can be used to achieve both a lethal photothermal effect and controlled release of the antibiotic, thus resulting in a degree of therapeutic synergy capable of eradicating viable S. aureus cells. The system was validated using planktonic bacterial cultures of both methicillin-sensitive and methicillin-resistant S. aureus strains and subsequently shown to be effective in the context of an established biofilm, thus indicating that this approach could be used to facilitate the effective treatment of intrinsically resistant biofilm infections.

Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function. Fibroblasts expressed 6–9 x 104 TF molecules/cell but had low specific activity for FXa generation. We confirmed that this was associated with minimal externalized PS and PE and characterised for the first time the molecular species of PS/PE demonstrating that these differed from those found in platelets. Mechanical damage of fibroblasts, used to simulate vascular injury, increased externalized PS/PE and led to a 7-fold increase in FXa generation that was inhibited by annexin V and an anti-TF antibody. Platelet-derived extracellular vesicles (EVs), that did not express TF, supported minimal FVIIa-dependent FXa generation but substantially increased fibroblast TF activity. This enhancement in fibroblast TF activity could also be achieved using synthetic liposomes comprising 10% PS without TF. In conclusion, despite high levels of surface TF expression, healthy fibroblasts express low levels of external-facing PS and PE limiting their ability to generate FXa. Addition of platelet-derived TF-negative EVs or artificial liposomes enhanced fibroblast TF activity in a PS dependent manner. These findings contribute information about the mechanisms that control TF function in the fibroblast membrane.