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Validation of IDO-1 and B7-H4 assays.

Modulation of PD-L1, IDO-1 and B7-H4 levels by cytokines in lung adenocarcinoma cells.

Purpose: Blockade of the PD-1/PD-L1 axis emerged as a promising new therapeutic option for cancer that has resulted in lasting responses in metastatic renal, lung carcinomas, and melanomas. Tumor PD-L1 protein expression may predict response to drugs targeting this pathway. Measurement of PD-L1 protein is limited by the lack of standardized immunohistochemical methods and variable performance of antibodies. Our goal was to correlate PD-L1 mRNA expression with clinical variables in primary breast carcinomas.Experimental Design: The fluorescent RNAscope paired-primer assay was used to quantify in situ PD-L1 mRNA levels in 636 stage I–III breast carcinomas on two sets of tissue microarrays [YTMA128 (n = 238) and YTMA201 (n = 398)]. Tumor-infiltrating lymphocytes (TIL) were assessed by hematoxylin/eosin stain and quantitative fluorescence.Results: On YTMA128 and YTMA201, 55.7% and 59.5% of cases showed PD-L1 mRNA expression, respectively. Higher PD-L1 mRNA expression was significantly associated with increased TILs (P = 0.04) but not with other clinical variables. Elevated TILs (scores 2 and 3+) occurred in 16.5% on YTMA128 and 14.8% on YTMA201 and was associated with estrogen receptor–negative status (P = 0.01 on YTMA128 and 0.0001 on YTMA201). PD-L1 mRNA expression was associated with longer recurrence-free survival (log-rank P = 0.01), which remained significant in multivariate analysis including age, tumor size, histologic grade, nodal metastasis, hormone receptor, HER2 status, and the extent of TILs (HR, 0.268; CI, 0.099–0.721; P = 0.009).Conclusions: PD-L1 mRNA expression is identified in nearly 60% of breast tumors and it is associated with increased TILs and improved recurrence-free survival. These observations support the evaluation of PD-1/PD-L1–targeted therapies in breast cancer. Clin Cancer Res; 20(10); 2773–82. ©2014 AACR.

PDF file - 1784K, Figure S5. Validation of the prognostic value of PD-L1 mRNA expression in publicly available breast cancer datasets.

Simultaneous detection of PD-L1, IDO-1 and B7-H4 in lung cancer.

PDF file - 10619K, Figure S4. Characterization of TILs subtypes in breast carcinomas with low and high PD-L1 mRNA.

Lung cancer cohorts description.

Association between PD-L1, IDO-1 and B7-H4 signal and survival in lung cancer.

PDF file - 9837K, Figure S1. Validation of in situ PD-L1 mRNA detection using quantitative fluorescence in cell lines and human tissues.

PDF file - 978K, Figure S2, Reproducibility of in situ mRNA measurements.

XLSX file - 13K, Clinico-pathological characteristics of YTMA128 and 201.

Luminescence thermometry represents a technique of choice for measurements in small objects and imaging of temperature distribution. However, most state-of-the-art luminescent probes are limited in spectral characteristics, brightness, photostability, and sensitivity. Molecular thermometers of the new generation utilizing air and moisture-stable zirconium(IV) pyridinedipyrrolide complexes can address all these limitations. The dyes emit pure thermally activated delayed fluorescence without any prompt fluorescence and show a unique combination of attractive features: a) visible light excitation and emission in the orange/red region, b) high luminescence brightness (quantum yields approximate to 0.5 in toluene and 0.8-1.0 in polystyrene matrix), c) excellent photostability, d) suitability for two-photon excitation and e) mono-exponential decay on the order of tens to hundreds of microseconds with strongly temperature-dependent lifetimes (between -2.5 and -2.9% K-1 in polystyrene at 25 degrees C). Immobilization in gas-blocking polymers yields sensing materials for self-referenced decay time read-out that are manufactured in two common formats: planar optodes and water-dispersible nanoparticles. Positively charged nanoparticles are demonstrated to be suitable for nanothermometry in live cells and multicellular spheroids. Negatively charged nanoparticles represent advanced analytical tools for imaging temperature gradients in samples of small volumes such as microfluidic devices.

Este documento, se enfoca en analizar los obstáculos que impiden al teletrabajo consolidarse como una modalidad de empleo, que posibilite reducir la tasa de desocupación de las personas en condición de discapacidad. Este objetivo es desarrollado y estimado a partir de la aplicación de un estudio descriptivo en el cual se aplicaron 50 encuestas a empresas de la ciudad Bogotá, donde se indagó frente a aspectos como: medios para ofertas vacantes, políticas de inclusión laboral, conocimiento de las leyes de fomento, experiencia y percepción sobre el teletrabajo. Los resultados muestran que el teletrabajo tiene una participación minoritaria en las empresas, los empresarios lo asocian con baja productividad, los modelos de contratación siguen siendo excluyentes y existe un desconocimiento de los beneficios otorgados por el gobierno para la contratación de personas con algún tipo de inhabilidad. De esta manera, se recomienda a las empresas iniciar procesos de transformación digital donde se aprovechen las oportunidades del teletrabajo e implementar modelos de contratación incluyentes y utilizar los beneficios otorgados por el gobierno en esta materia. Los resultados, son analizados a partir de una revisión bibliográfica en diferentes bases de datos, especialmente de artículos donde se estudia la relación entre el teletrabajo y los procesos de inclusión laboral en el periodo comprendido entre el 2014 al 2019.

Lung cancer (LC) remains as the leading cause of death by cancer worldwide. In Chile, LC is the second cause of cancer-related deaths. Recently, studies in melanoma and non-small cell lung cancer (NSCLC) patients have highlighted the role of the gut microbiota as an important host factor in mediating the responses/resistance to immunotherapy, suggesting that bacteria present in the gut may modulate the immune response in these tumors. However, the role of extra-intestinal microbiota; bacteria living outside of the gut, in cancer pathogenesis and the response to anti-cancer therapies remains largely undetermined. Here we characterize the composition of intratumoral microbiota of NSCLC and seek to establish a functional relationship between it and the composition of the immune microenvironment and the clinicopathological characteristics of NSCLC patients. From the first 157 FFPE NSCLC samples, we extracted DNA from all collected samples, obtaining adequate material in quality and quantity to later be submitted for analysis to 16S sequencing. Using 16S rRNA gene sequencing, we assessed the general landscape of the NSCLC tumor microbiome, revealing the presence of large number of bacterial communities in the NSCLC tumor samples on the different histological subtypes analyzed. We have detected differences in alpha diversity in the histological subtypes studied. Even more interesting, we have detected significant differences in lung adenocarcinoma depending on the degree of histological differentiation, observing a decrease in proteobacteria and an enrichment of Bacteroidales, as cell differentiation is lost. Additionally, we identified taxonomic differences between differentiated and undifferentiated tumors. Differentiated tumors show enrichment in Akkermansia muciniphila and while undifferentiated tumors show enrichment in Actinobacter Corynebacterium. Suggesting that these taxa could contribute to maintaining a differentiated state or inducing cell dedifferentiation, respectively. Our results reveal the presence of a large number of bacterial communities in lung cancer samples in the different histological subtypes analyzed. Suggesting that these communities could play a key role in tumor differentiation and progression. Citation Format: Erick M. Riquelme, Ivania Valdes, Carlos Aravena, Ilse Valencia, Barbara Mino, Daniel Carvajal, Alberto Martin. Revealing the role of lung cancer microbiota in the tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 651.

BackgroundA strong association between the proportion of Native American ancestry and the risk of gallbladder cancer (GBC) has been reported in observational studies. Chileans show the highest incidence of GBC worldwide, and the Mapuche are the largest Native American people in Chile. We set out to investigate the causal association between Native American Mapuche ancestry and GBC risk, and the possible mediating effects of gallstone disease and body mass index (BMI) on this association.MethodsMarkers of Mapuche ancestry were selected based on the informativeness for assignment measure and then used as instrumental variables in two-sample mendelian randomization (MR) analyses and complementary sensitivity analyses.ResultWe found evidence of a causal effect of Mapuche ancestry on GBC risk (inverse variance-weighted (IVW) risk increase of 0.8% for every 1% increase in Mapuche ancestry proportion, 95% CI 0.4% to 1.2%, p = 6.6×10-5). Mapuche ancestry was also causally linked to gallstone disease (IVW risk increase of 3.6% per 1% increase in Mapuche proportion, 95% CI 3.1% to 4.0%, p = 1.0×10-59), suggesting a mediating effect of gallstones in the relationship between Mapuche ancestry and GBC. In contrast, the proportion of Mapuche ancestry showed a negative causal effect on BMI (IVW estimate -0.006 kg/m2 per 1% increase in Mapuche proportion, 95% CI -0.009 to -0.003, p = 4.4×10-5).ConclusionsThe results presented here may have significant implications for GBC prevention and are important for future admixture mapping studies. Given that the association between Mapuche ancestry and GBC risk previously noted in observational studies appears to be causal, primary and secondary prevention strategies that take into account the individual proportion of Mapuche ancestry could be particularly efficient.

Introduction: Primary dysmenorrhea affects many women, being a major cause of absenteeism and reduced productivity at work and at school. Although non-steroidal anti-inflammatory drugs (NSAIDs) are a good treatment option, up to 18% of women show no response or present allergic reactions and adverse events. Curcumin has antispasmodic, antinociceptive, and both specific and nonspecific anti-inflammatory effects, with good tolerability and safety. To date, no previous trial involving curcumin and dysmenorrhea pain has been performed. Therefore, our main goal is to assess the efficacy of curcumin for pain relief among women with primary dysmenorrhea, along with determining curcumin’s adverse effects and tolerability profile.Methods: A phase II, single-center, randomized, triple-blinded, placebo-controlled, parallel-group, superiority trial to evaluate the effect of curcumin (500 mg/12h) in pain reduction in women (18 to 35-year-old) with primary dysmenorrhea. A first cycle will be used for a passive, observational run-in phase. A sample of 108 participants (54 per group) is necessary to detect a 30% difference in pain sensitivity between groups assessed by visual analogue scale (VAS). Secondary outcomes include side effects, Cox Menstrual Symptom Scale (CMSS), and use of rescue drugs for pain relief. Discussion: Clinical evidence has shown analgesic and anti-inflammatory effects of curcumin and in view of dysmenorrhea’s physiopathology being related to those mechanisms targeted by curcumin, we hypothesize its use could represent an innovative and effective therapy to reduce the severity of this disease and its symptoms. Keywords: primary dysmenorrhea, Curcumin, pain relief, Visual Analogue Scale, Cox Menstrual Symptom Scale.

Las enfermedades asociadas a eosinófilos constituyen un grupo de patologías en que el aumento de los eosinófilos presenta un rol fundamental, encontrándose entre ellas la gastroenteritis eosinofílica, entidad poco frecuente. En la literatura existen pocos datos demográficos, con una prevalencia estimada entre 28-30 /100.000 habitantes. Los síntomas dependen principalmente del segmento afectado del tracto gastrointestinal y de la capa de la pared infiltrada por eosinófilos. Se presenta el caso clínico de un paciente masculino de 22 años con cuadro de diarrea de un mes de evolución, asociado a mucosidad y ocasionalmente estrías hemáticas. El estudio de laboratorio inicial mostró leucocitosis con eosinofilia, realizándose estudios de imágenes que demuestran extenso compromiso del tubo digestivo caracterizado por engrosamiento parietal difuso concéntrico y edema submucoso del estómago como también de asas de intestino delgado, asociado a ascitis. La endoscopia digestiva alta y biopsia confirmaron la presencia de focos de infiltración por eosinófilos. El paciente fue tratado con corticoides y dieta con restricción de lactosa y trigo, con evolución favorable, sin recaída tras la suspensión del tratamiento corticoidal.

The importance of extracellular gradients of biomolecules is increasingly appreciated in the processes of tissue development and regeneration, in health and disease. In particular, the dynamics of extracellular calcium concentration is rarely studied. Here, we present a low affinity Ca

A misconception lingers in the minds of some wine consumers that Champagne wines should not age much after the minimum period of 15 months in contact with dead yeasts, known as maturation on lees. It is certainly a myth, as far as the best cuvees are concerned. Dissolved CO2 being responsible for bubble formation in sparkling wines, keeping it as efficiently as possible in the sealed bottles during aging is therefore a challenge of importance for old vintages likely to mature on lees for several decades. Measurements of dissolved CO2 were done on an outstanding vertical collection of successive vintages from a prestige cuvee, showing maturation on lees ranging from several months up to 35 years at a constant temperature of 12 °C. Progressive losses of dissolved CO2 during aging on lees were evidenced and discussed on the basis of a previously developed exponential-decay type model taking into account the main geometrical parameters of both the cork and bottle. The prestige cuvee (with a narrow bottleneck, and sealed with premium natural cork stoppers) was found to hold much more efficiently dissolved CO2 during aging than a batch of standard Champagne and sparkling wines bottles showing 3 mm wider bottlenecks. By fitting our data with the exponential-decay model, the diffusion coefficient of gas-phase CO2 through the cork stoppers used to seal this prestige cuvee was found to range between about 5 × 10 − 11 and 9 × 10 − 11 m 2 s − 1 . Moreover, by extrapolating the data well beyond the measurement range, it was suggested that very long maturation on lees of the order of 76 ± 19 years should be needed to decrease the level of dissolved CO2 in the prestige cuvee below the critical concentration required for bubbling.

The use of implantable cardiac devices in patients with sudden cardiac arrest has contributed to their survival.To determine the survival rate at 30 days and one year after hospital discharge of patients who had a cardiac arrest with subsequent placement of an implantable cardiac device.Twenty-three patients older than 18 years who presented sudden extra-institutional or intra-institutional death with subsequent implantation of an implantable cardiac device and whose survival was recorded at 30 days and one year, were included. A univariate analysis was performed.Eighteen patients had an extra institutional cardiac arrest. All patients were discharged alive. We could not ascertain the health status of one patient at follow-up. Twenty-one patients had a Cerebral Performance Category (CPC) of 1 at discharge. One patient died of a stroke within 30 days and one patient died due to an arrhythmic electrical storm one year later. Twenty patients survived at least one year after hospital discharge.Survival at 30 days and one year, was high in patients with sudden death or cardiac arrest who required intracardiac devices.

e15088 Background: The neurotrophic receptor tyrosine kinase genes NTRK1-3 encode the tropomyosin receptor kinase proteins TRKA, TRKB, and TRKC, respectively. TRK fusions lead to overexpression of the chimeric protein, resulting in constitutively active, ligand-independent downstream signaling. NTRKs act as oncogenes, they can be targeted, and it is estimated that they are present in up to 0.3% of tumors. The incidence of actionable alterations in these genes is currently unknown in Latin-American patients. We investigated the presence of NTRK1-3 mutations/rearrangements in 3 independent patient series from several tertiary hospitals from Chile, Brazil and Peru. Methods: 1795 FFPE tumor samples from multiple institutions divided in 3 series were analyzed using 2 NGS panels: Oncomine Focus Assay (OFA; 52 genes) and Oncomine Comprehensive Assay (OCA; 161 genes. Data on NTRK1-3 SNVs, indels, CNVs and fusions was obtained. Bioinformatic workflows were used to study the biologic significance of these alterations. Results: Using OCA (series 1-2), 31 somatic variants were found in gastric, CRC, gallbladder, lung and pancreatic cases out of 300 patients. From these, 13 were located in the tyrosine kinase domain. Using OFA, no NTRK alteration were detected (series 3, 1495 NSCLC cases). Conclusions: NTRK alterations are rare events in Latin-American cancer patients. In 3 series including 1,795 patients, 31 somatic variants were detected. No NTRK fusions or CNVs were identified. The presence of NTRK mutations in the tyrosine kinase domain warrants further research into their potential to benefit from FDA/EMA-approved targeted therapies. [Table: see text]

Background: According to the 2013 guidelines breast cancers are defined as Her2 positive if there is evidence of protein expression in at least 10% of tumor cells by IHC and/or gene amplification by FISH. Nevertheless, there are still IHC 2+ and FISH equivocal breast cancers resulting in repeat testing. It is also known that not all Her2 positive breast cancers respond to Trastuzumab, while up to 8% of Her2 "negative" classified patients benefit from Her2 targeting regimens. Toward the goal of generating a more accurate test, we report in situ quantification of Her2 protein levels on a prospectively collected cohort of breast cancers and comparison to conventional IHC and FISH evaluation. Materials and Methods: A prospectively designed study was initiated at Yale University, comparing quantitative, in situ measurement of Her2 protein levels with conventional IHC and FISH evaluation. All breast cancer specimens were analyzed by IHC and FISH in our routine clinical laboratory, read and signed out by the breast pathologists. Serial sections were then stained and quantified for Her2 expression levels using the AQUA method of quantitative immunofluorescence (QIF). Data for all assays were obtained on 120 samples over a period of 6 months. Staining was performed using the DAKO Herceptest and the Epitomics EP3 Her2 antibody for IHC, the DAKO rabbit polyclonal antibody for QIF. The 30 highest cases were then retested for QIF and IHC using the Biocare c-erbB-2 [CB11] antibody. Each staining run included an index tissue microarray (TMA) consisting of 80 cases, cell lines and normal tissue for quality control, assay reproducibility and threshold definition of AQUA scores correlated to HER2 over expression and amplification. Results: Out of 120 specimens analyzed for HER2, 13 were diagnosed as IHC 2+/3+, FISH amplified, 1 case had an equivocal score, 14 cases were IHC 2+/non amplified, 2 cases IHC 1+/FISH amplified and 89 specimens IHC 0/1+ non amplified. The continuous AQUA scores for Her2 expression of the samples significantly correlate with traditional clinical Her2 scoring. However, 5 IHC 0/1+, non amplified cases revealed high AQUA scores in the range of HER2 overexpression/amplification. Repeat testing of these by both QIF and IHC showed reproducibility of the results. AQUA scores of one IHC 3+/amplified sample were lower than the threshold of HER2 overexpression/amplification. Conclusions: QIF measurement of HER2 protein levels in a prospectively collected cohort of 120 breast cancer specimens reveals significant association between continuous HER2 protein levels and the ordinal conventional scoring system. However, five discordant cases that were above the threshold for HER2 protein by QIF, were classified as negative by conventional methods. Given the accuracy and reproducibility of the QIF test, it raises the possibility that some of these patients might benefit from HER2 targeted therapy. In summary, while continuous scoring of HER2 protein correlates well with conventional methods, it identifies a subset of patients that are discordant with current methods. Further comparative studies in a patient cohort with response to targeted therapy need to be evaluated. Citation Format: Neumeister VM, Yan SS, McGuire JA, Carvajal DE, Prasad ML, Rimm DL. Quantitative immuno-fluorescent evaluation of Her2 expression levels in a prospectively collected cohort of breast cancer cases: Comparison to conventional IHC scoring and FISH. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-22.

Sometimes, a designer needs to share a “creation heritage” to support the generativity of his pairs, in the form of a book. What should be its content? The literature has shown that knowledge in such books might be fixating or defixating, leading to inconclusive results. Using recent advances in design theories we model the features of a heritage oriented towards generativity. Relying on the literary tradition in Cuisine, we validate our model. We show that transferring knowledge implies sharing objects structure, value criteria, desired unknowns, progress principles, and creative reasoning.

Objetivo. Establecer el estado nutricional en pacientes de dos instituciones hospitalarias de Villavicencio. Método. Estudio de corte transversal, prospectivo, mediante una encuesta en 2 instituciones hospitalarias de Villavicencio, Colombia, en febrero de 2015. Se incluyeron pacientes adultos hospitalizados en servicios quirúrgicos. Los datos fueron registrados en Excel y se realizaron análisis estadísticos con SPSS a fin de encontrar asociaciones entre las variables de estudio y los desenlaces. El nivel de significancia se estableció cuando la p

There is considerable interest in finding re- placements for the synthetic fungicides and preservatives that are currently used to control fungal pathogens; how- ever, any alternative compounds must be safe and must prevent the development of microbial resistance. In this regard, essential oils have received special attention. Pro- tium colombianum Cuatrecasas is an endemic tree found in tropical rainforests and possesses aromatic and resinous characteristics. To date, there have been no reports con- cerning the chemistry of this species, which belongs to a genus that represents an interesting source of essential oils that are occasionally used as antimicrobial agents. There- fore, the chemical composition of the essential oils found in P. colombianum collected from two life zones over a five-month period was analyzed using gas-chromatogra- phy/mass-spectrometry. A total of 92 components, com- prising 97.7-99.9 % of the total composition, were identified based on their mass spectra and the retention indices. Precedence markers were identified for each life zone, and seasonality affected the samples from one life zone when rainfall was maximum. The in vitro activity against Fusarium oxysporum isolates from carnation and chrysanthemum, as well as a Fusarium solani isolate from chrysanthemum, was tested by determining the minimum inhibitory concentration. Assays using essential oils and control substances showed that terpenoid and phenolic compounds are mainly responsible for the observed anti- fungal activity. These results suggest that P. colombianum essential oils exhibit cytotoxicity against Fusarium isolates and that the search for new sources of anti-fungal essential oils should include sources that contain different ratios of terpenoid and phenolic compounds.

The laparoscopic cholecystectomy has allowed the detection of an increasing number of incidental gallbladder cancers (IGBC). Although laparoscopy is employed in the management of a variety of abdominal tumors, its use in gallbladder cancer is reduced and controversial. This study analyzes the role of laparoscopy in gallbladder cancer with the focus in IGBC. We evaluated our prospective series of 51 patients with an IGBC who were treated by laparoscopy between 2006 and 2016 at the Clinica Alemana in Santiago, Chile. The series comprised 7 men and 44 women. Age ranged from 43 to 76 years (mean age 60). Regarding wall involvement, 29 patients had a T2 tumor, which was the most common. 8 and 14 patients had T1b and T3 tumors, respectively. Of the patients, 17 underwent only laparoscopic exploration. This was due to the presence tumor dissemination not being observed in the preoperative staging. 10 patients had to be converted to complete the resection, whereas 24 patients were laparoscopically resected. The quality of the resected material was not different between those who were converted and those who were treated by laparoscopy. In the laparoscopic group, the average number of harvested lymph nodes was 7.9, not statistically different from the converted group. The mean of hospital stay in the laparoscopic group (4.3 days) was significantly lower than the converted group. Laparoscopy has been shown to be a safe and feasible method for the management of IGBC. This method not only allows for a complete exploration, identifying a previously unseen residual tumor, but also makes it possible to accomplish the same oncology objectives as the open procedure. Therefore, laparoscopy should be considered a valid alternative in the management of IGBC.

Recent strategies targeting the interaction of the programmed cell death ligand-1 (PD-L1, B7-H1, CD274) with its receptor, PD-1, resulted in promising activity in early phase clinical trials. In this study, we used various antibodies and in situ mRNA hybridization to measure PD-L1 in non-small cell lung cancer (NSCLC) using a quantitative fluorescence (QIF) approach to determine the frequency of expression and prognostic value in two independent populations. A control tissue microarray (TMA) was constructed using PD-L1-transfected cells, normal human placenta and known PD-L1-positive NSCLC cases. Only one of four antibodies against PD-L1 (5H1) validated for specificity on this TMA. In situ PD-L1 mRNA using the RNAscope method was similarly validated. Two cohorts of NSCLC cases in TMAs including 340 cases from hospitals in Greece and 204 cases from Yale University were assessed. Tumors showed PD-L1 protein expression in 36% (Greek) and 25% (Yale) of the cases. PD-L1 expression was significantly associated with tumor-infiltrating lymphocytes in both cohorts. Patients with PD-L1 (both protein and mRNA) expression above the detection threshold showed statistically significant better outcome in both series (log-rank P=0.036 and P=0.027). Multivariate analysis showed that PD-L1 expression was significantly associated with better outcome independent of histology. Measurement of PD-L1 requires specific conditions and some commercial antibodies show lack of specificity. Expression of PD-L1 protein or mRNA is associated with better outcome. Further studies are required to determine the value of this marker in prognosis and prediction of response to treatments targeting this pathway.

Self-assembly in the presence of external forces is an adaptive, directed organization of molecular components under nonequilibrium conditions. While forces may be generated as a result of spontaneous interactions among components of a system, intervention with external forces can significantly alter the final outcome of self-assembly. Superimposing these intrinsic and extrinsic forces provides greater degrees of freedom to control the structure and function of self-assembling materials. In this work we investigate the role of electric fields during the dynamic self-assembly of a negatively charged polyelectrolyte and a positively charged peptide amphiphile in water leading to the formation of an ordered membrane. In the absence of electric fields, contact between the two solutions of oppositely charged molecules triggers the growth of closed membranes with vertically oriented fibrils that encapsulate the polyelectrolyte solution. This process of self-assembly is intrinsically driven by excess osmotic pressure of counterions, and the electric field is found to modify the kinetics of membrane formation, and also its morphology and properties. Depending on the strength and orientation of the field we observe a significant increase or decrease of up to nearly 100% in membrane thickness, as well as the controlled rotation of nanofiber growth direction by 90 degrees, resulting in a significant increase in mechanical stiffness. These results suggest the possibility of using electric fields to control structure in self-assembly processes involving diffusion of oppositely charged molecules.

We report here on the formation of a bioactive hierarchically structured membrane by self-assembly. The membrane is formed with hyaluronic acid and peptide amphiphiles with binding affinity for heparin, and its hierarchical structure contains both an amorphous zone and a layer of fibrils oriented perpendicular to the membrane plane. The design of bioactivity is based on the potential ability to bind and slowly release heparin-binding growth factors. Human mesenchymal stem cells seeded on these membranes attached and remained viable. Basic fibroblast growth factor (FGF2) and vascular endothelial growth factor (VEGF) were incorporated within the membrane structure prior to self-assembly and released into media over a prolonged period of time (14 days). Using the chicken chorioallantoic membrane (CAM) assay, we also found that these membranes induced a significant and rapid enhancement of angiogenesis relative to controls.

Introduction: T-cells recognizing tumor antigens can express diverse immune inhibitory receptors such as PD-1, LAG-3 and TIM-3. Blockade of PD-1 induces prominent clinical benefit in patients with melanoma, lung, bladder and gastric carcinomas. However, the relative expression and significance of immune inhibitory receptors across different tumors is unknown. Methods: Using multiplexed quantitative immunofluorescence (QIF) we measured the levels of CD3 (rabbit polyclonal, Dako), PD-1 (EH33, CST), LAG-3 (17B4, Abcam) and TIM-3 (D5D5R, CST) in >1,300 human malignancies including 119 triple negative breast carcinomas (TNBC), 225 ovarian carcinomas, 382 melanomas, 259 colorectal tumors, 229 bladder urothelial carcinomas and 130 gastric malignancies. The cohorts included FFPE tumor tissues and were studied using tissue microarrays (TMAs). The targets were measured in all cells of the preparation using fluorescence co-localization with DAPI and specifically in CD3-positive T-lymphocytes. Associations between the markers across tumors and with survival were studied. Results: PD-1, LAG-3, and TIM-3 were expressed predominantly in T-cells and showed a positive association with each other. The highest levels were detected in gastric adenocarcinomas followed by urothelial bladder tumors, ovarian malignancies, melanoma, TNBC and colorectal carcinomas. Gastric and bladder tumors showed comparably high levels of all three markers and were significantly higher than the other tumor types (P Conclusion: PD-1, LAG-3 and TIM-3 are differentially expressed in human solid tumors and show limited prognostic value. Gastric and bladder carcinomas show the highest levels of the targets, while TNBC and colorectal cancer show the lowest. Despite having the highest T-cell infiltration, melanomas show intermediate levels of the immune inhibitory receptors. These data demonstrate the differences in the immune composition of human solid tumors and could be used for optimal design and interpretation of clinical trials. Citation Format: Micaela Morgado, Ila Datar, Jun Wang, Miguel F. Sanmamed, Kristen McEachern, David Jenkins, Lieping Chen, Daniel Carvajal-Hausdorf, David L. Rimm, Roy S. Herbst, Kurt A. Schalper. Simultaneous measurement and significance of PD-1, LAG-3 and TIM-3 expression in human solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1681.

Through recurrent disasters, both natural and man-made, the US government has developed a sophisticated emergency and disaster response system, ranging from local to federal government responses. But in large-scale disasters, the number of professional responders and the response times may be inadequate both for the physical magnitude of the disaster area involved and the number of victims. With that experience in hand, the Los Angeles City Fire Department promoted the concept of citizen response and training in 1985, which is now known as the Community Emergency Response Teams (CERT). The CERT program seeks to educate the lay public in disaster preparedness and train volunteers in basic disaster response skills. Training has been made available through the Federal Emergency Management Agency, the Emergency Management Institute, and the National Fire Academy (http://www.citizencorps.gov/cert/). These teams can be used to promote awareness programs in the community and to be readily available in the event of a local incident. Their proximity to the event and knowledge of the area can be a valuable asset both prior to and after the arrival of professional responders. But building such a team from scratch can be a daunting challenge. Known more for their football program, this article describes the system built by the undergraduate student body of the University of Miami Hurricanes.

Drop-shape analysis was used to study the binding of streptavidin to biotin at the interface between water and a pendant chloroform droplet. Polyethylene oxide molecules were synthesized with a hydrophobic tail at one end of the molecule and a hydroxyl or biotin group at the other end. The interfacial tension of the water/chloroform interface was measured before and after addition of these amphiphiles to the chloroform phase and before and after addition of streptavidin to the aqueous phase. The hydroxyl-terminated amphiphiles eliminate nonspecific adsorption of the streptavidin to the interface, while streptavidin binds irreversibly to the biotin-terminated molecules. Molecular interactions within this bound layer were studied by measuring changes in the interfacial pressure as the layer is contracted and expanded by changing the volume of the chloroform droplet. A picture of the interfacial structure was obtained from quantitative comparisons between the experimental results and a molecular theory of protein binding to tethered ligands. These comparisons suggest that protein binding is controlled by the extension of the PEO tethers away from the interface.

Overexpression/amplification of the HER2 gene in advanced gastric cancer is a predictor of response to adjuvant therapy with monoclonal antibodies.To determine the frequency of HER2 gene overexpression and amplification in advanced gastric cancer.One hundred nine advanced gastric cancer biopsy specimens, from 76 men and 33 women aged 67 ± 14 and 62 ± 12 years respectively, were selected. Three histological patterns (diffuse, intestinal and mixed) were recognized. Automated immunohistochemistry was performed with monoclonal c-erbB-2 (NCL-356) Novocastra. Fluorescent in situ hybridization (FISH) for HER2 was performed in positive cases.In 39% of cases, immunohistochemical staining was negative. It was 1+, 2+ and 3+ positive in 15, 36 and 11% of cases, respectively. It was positive in 16% and 3% of intestinal type and mixed carcinomas, respectively. It was negative in all diffuse carcinomas. FISH was performed in 39 (2 +) cases and in 11 (3 +) cases. The gene amplification was positive in two (2 +) and 11 (3 +) cases (11.9%). The overall concordance between immunohistochemical staining and in situ hybridization was 85%.In advanced gastric cancer, HER2 gene overexpression or amplification was observed in 11% and 12% of cases, respectively.

The binding of streptavidin to biotin located at the terminal ends of poly(ethylene oxide) tethered to a planar surface is studied using molecular theory. The theoretical model is applied to mimic experiments (Langmuir 2008, 24, 2472) performed using drop-shape analysis to study receptor-ligand binding at the oil/water interface. Our theoretical predictions show very good agreements with the experimental results. Furthermore, the theory enables us to study the thermodynamic and structural behavior of the PEO-biotin+streptavidin layer. The interfacial structure, shown by the volume fraction profiles of bound proteins and polymers, indicates that the proteins form a thick layer supported by stretched polymers, where the distribution of bound proteins is greater than the thickness of the height of one layer of proteins. When the polymer spacer is composed of PEO (3000), a thick layer with multi-layers of proteins is formed, supported by the stretched polymer chains. It was found that thick multi-layers of proteins are formed when long spacers are present or at very high protein surface coverages on short spacers. This shows that the flexibility of the polymer spacer plays an important role in determining the structure of the bound proteins due to their ability to accommodate highly distorted conformations to optimize binding and protein interactions. Protein domains are predicted when the amount of bound proteins is small due to the existence of streptavidin-streptavidin attractive interactions. As the number of proteins is increased, the competition between attractive interactions and steric repulsions determines the stability and structure of the bound layer. The theory predicts that the competition between these two forces leads to a phase separation at higher protein concentrations. The point where this transition happens depends on both spacer length and protein surface coverage and is an important consideration for practical applications of these and other similar systems. If the goal is to maximize protein binding, it is favorable to be above the layer transition, as multiple layers can accommodate greater bound protein densities. On the other hand, if the goal is to use these bound proteins as a linker group to build more complex structures, such as when avidin or streptavidin serves as a linker between two biotinylated polymers or proteins, the optimum is to be below the layer transition such that all bound linker proteins are available for further binding.

Aortic injuries were traditionally thought to be the result of severe frontal crashes. Newer data has suggested other crash types such as nearside crashes may also be important in aortic injury. We hypothesized the implementation of recent safety measures would decrease the incidence of aortic injury associated with fatal motor vehicle crashes.The autopsy reports of all traffic fatalities for motor vehicle occupants in a large urban county for the years 1993 to 2004 were examined. The demographics, impact types, safety measures used, and the presence of any aortic injury were recorded. Trends were evaluated for significance by weighted linear regression.The incidence of aortic injury associated with fatal motor vehicle crashes has remained unchanged during the past 12 years (r = 0.057, p = 0.45). There is a trend toward decreased aortic injuries associated with frontal crashes (r = 0.26, p = 0.089) but no change in aortic injuries associated with nearside or farside crashes (r = 0.053, p = 0.47), when the crash resulted in a fatality. This is despite an increase in seat belt use and increased presence of airbags during the same time period.Despite improved safety measures designed to minimize the occurrence of aortic injuries, the incidence of blunt aortic injury in fatal motor vehicle crashes has not decreased during the past decade. Although not statistically significant, there is a trend toward decreased frontal impacts in fatal motor vehicle crashes associated with aortic injuries. The nearside crash mechanism continues to play a prominent role, and efforts at improving vehicle safety should be focused on crash mechanisms as they relate to aortic injury.

11075 Background: Programmed cell death ligand-1 (PDL1) is expressed on various human cancers and is a major mechanism for immune evasion. Preliminary evidence suggests that PDL1 expression on cancer cells predicts response to anti-PD-1 therapy. Here we report our findings using automated quantitative immunofluorescence (QIF) to determine the frequency and prognostic value of PDL1 in two independent NSCLC cohorts. Methods: Five antibodies against PDL1 were screened for sensitivity and specificity using PDL1 transfected cells, normal human placenta and tonsil. Only one monoclonal antibody (clone 5H1) met criteria for specificity. This antibody was used to assess two cohorts of NSCLC cases in TMAs. The cohorts represented 204 cases from Yale University and 340 cases from hospitals in Greece. PDL1 protein was measured using QIF analysis using AQUA method. In addition, in-situ PDL1 mRNA levels were measured in the Greek cohort using the RNAscope paired-primer assay with AQUA method. Results: PDL1 protein expression was detected in 25% and 36% of the studied cohorts, respectively. PDL1 positivity was significantly associated with the presence of tumor-infiltrating lymphocytes (TIL) in both series. Patients with PDL1 protein positivity had better outcome in both series (Log Rank: p=0.036 for Yale cohort, p=0.027 for Greek cohort). Multivariate analysis showed that PDL1 expression was associated with better outcome independent of histology, presence of TILs, and stage in the Greek cohort and marginally significant in the Yale cohort. These findings were further validated by the PDL1 in-situ mRNA measurement in the Greek cohort. PDL1 protein and mRNA levels showed a positive non-linear relationship (R2=0.14, P

Drop shape analysis of pendant drops is a commonly used technique to measure the interfacial tension of liquid–vapor or liquid–liquid interfaces. The technique is increasingly being used to investigate the mechanical response of molecular structures that form at these interfaces as well, utilizing analysis techniques developed by the solid mechanics community. The aim of this highlight is to summarize the formal analysis methods needed to analyze these types of experiments and to make the connection between liquid-like behavior of the interface, and the more general solid-like case where the two principal interfacial tensions are not necessarily equal to one another. Several illustrative examples are used to accomplish this, including protein complexes formed at the oil–water interface, ionically crosslinked block copolymer monolayers at oil–water interfaces, and block copolymer-based thermoplastic elastomers at the air–water interface. In addition to describing the mechanical analysis of these types of experiments, we provide a stability map that defines the experimental window within which useful pendant drop experiments can be conducted for interfacial layers with liquid-like behavior.

The mechanical properties and water permeability of hierarchical self-assembling membranes and sacs formed from oppositely charged high molecular weight hyaluronic acid (HA) and small molecule peptide amphiphiles (PAs) were studied. Techniques to make reproducible 2D planar membranes and 3D spherical sacs from these materials were developed while membrane inflation and osmotic swelling were used to quantify the mechanical properties and water permeability of these structures. It was found that incubation time and concentration of HA used had an effect on the area modulus and water permeability of the membranes. These factors also affected the kinetics of membrane growth as evidenced in SEM micrographs, which showed differences in the structure. Area modulus of membranes changed from about 6 N m−1 for the lower weight percent HA system at the shortest incubation time of 3 minutes, up to 12 N m−1 for the higher weight percent HA system at the longest incubation time of 60 minutes. Water permeability decreased with incubation time, but the lower weight percent HA system showed a lower water permeability when compared to the higher weight percent HA system at the same incubation time. This type of characterization and understanding of the structure–property relationships in self-assembling systems are necessary steps in both using these structures for specific applications and applying this knowledge to design new and better materials in the future.