3D Biomatrix (United States)

Existen numerosos genes que regulan diferentes funciones en el organismo y cuya expresión se ve afectada por factores ambientales, determinando el fenotipo del individuo. Entre estos se encuentran el gen PPARG2, que es un factor de transcripción implicado en el mantenimiento de la homeostasis de los lípidos, regulando las vías anabólicas. Objetivo: Evaluar la asociación entre el polimorfismo Pro12Ala (rs1801282) del gen PPARG2 con la obesidad, resistencia a la insulina y/o dislipidemia en pacientes adultos sometidos a cirugía bariátrica del Hospital Universitario de Caracas. Materiales y Métodos: Se incluyeron 81 individuos con obesidad mórbida, que fueron sometidos a cirugía bariátrica, y 125 individuos normopeso, como grupo control. A todos los individuos se les realizaron mediciones antropométricas y determinaciones bioquímicas. La determinación del polimorfismo Pro12Ala (rs1801282) del gen PPARG2 fue realizada por PCR-RFLP. Resultados y Discusión: En los pacientes, después de la cirugía bariátrica, se observó una disminución estadísticamente significativa de las concentraciones de glucosa, triglicéridos y colesterol. Asimismo, un año después de la cirugía, presentaron una disminución del 43% y 8% de las concentraciones de triglicéridos y colesterol, respectivamente, y un incremento del 23% en las concentraciones de HDL-c. No se observaron asociaciones entre el polimorfismo Pro12Ala del gen PPARG2 con obesidad o pérdida de peso después de un año de la cirugía bariátrica. Conclusión: La cirugía bariátrica tiene una repercusión positiva sobre la hipertrigliceridemia, hipercolesterolemia y el control de la glicemia. El polimorfismo Pro12Ala del gen PPARG2 no está asociado con la obesidad, ni con la pérdida de peso después de la cirugía bariátrica.

This study was designed to determine if feeding β-hydroxy-β-methylbutyrate (HMB) to pregnant mice would improve birth weight uniformity and growth performance of offspring. Dams (Agouti Avy) were assigned to one of four treatments: control (CON; n = 13), low-level HMB (LL; 3.5 mg/g; n = 14), high-level HMB (HL; 35 mg/g; n = 15), and low-level pulse dose fed from gestational days 6 to 10 (PUL; 3.5 mg/g; n = 14). Randomly selected dams (n = 27) were euthanized on gestational day 18 to collect placentae and pup weights. The remaining dams gave birth and lactated for 28 days. Dams only received HMB during gestation. Dietary HMB did not influence the performance of dams. Dietary treatment during gestation did not affect litter size or birth weight of pups. Variation was not different among treatments in terms of birth weight of offspring. Placental weights were not affected by treatments. Overall, growth performance of offspring after weaning was similar among all treatments. Body composition of offspring at 5 and 8 weeks of age was similar regardless of HMB treatment during gestation. In conclusion, dietary HMB supplementation in pregnant mice did not affect birth weight, variations in birth weight, or growth performance of offspring.

e13047 Background: HER2 targeted therapies, such as HER2 monoclonal antibodies, HER2 tyrosine kinase inhibitors, and HER2 antibody drug conjugates, are approved for the treatment of HER2-positive breast, gastric and colorectal cancers. However, inter- and intra-tumoral heterogeneity in HER2 status presented a significant challenge in identifying patients that may benefit from HER2-targeted therapies. Detection of ERBB2 amplification in Circulating Tumor Cells (CTCs) may circumvent tissue heterogeneity issues and effectively predict the response of patients to anti-HER2 agents as a non-invasive alternative approach. Methods: A twenty-four patient pilot study was designed to compare HER2 status of traditional needle biopsies to high sensitivity CTC based liquid biopsies. HER2 status was determined using immunohistochemistry (IHC) and Fluorescence in Situ Hybridization (FISH) for tissue samples and FISH analysis for CTCs. Currently 21 patient samples have been compared in this study. Results: CTCs with HER2 amplified signals were found in patients with stages 2, 3, or 4 breast cancers. Based on the biopsies of the primary tumor or metastatic disease, 6 patients (29%) had HER2-overexpressing as defined by either HER2 IHC 3+ or HER2 FISH amplified. Analysis of CTCs showed 100% concordance (HER2 amplification detected in CTCs) in these patients with HER2-overexpressing breast cancer. Furthermore, an additional 4 (19%) patients had HER2 amplification by CTC analysis but not on needle biopsy. Conclusions: Our finding demonstrates that LiquidScan can be used as a rapid analysis tool to evaluate HER2 amplification in CTCs. This approach can potentially help identify additional cohort of patients (HER2 non-amplified tumor / HER2 amplified CTCs) who may benefit from the new generation of HER2 targeted therapies. Studies are ongoing to include an expanded cohort of patients with early stage disease, as well as HER2-low disease, in order to evaluating dynamics of ERBB2 gene amplification, expression, and pathway activation while on active systemic therapy.

Compounds in garlic have been shown to contain anti-inflammatory, antioxidant, and immune modulatory properties that may be able to mitigate the effects of nursery pig stressors. The objective of the current experiment was to determine if oral gavage of garlic-derived diallyl disulfide (DADS) and diallyl trisulfide (DATS) could mitigate the effects of a 24-h postweaning feed + water deprivation event in nursery pigs. Pigs (6.0 ± 0.05 kg and 21 d old) were allotted to 4 treatments in a randomized complete block design at weaning with 8 replicate pens per treatment that consisted of with or without a 24-h postweaning feed + water deprivation event and with or without an oral gavage containing 3.6 mg DADS + DATS/kg BW. Growth performance and morbidity were recorded throughout the experiment, and on 1, 6, and 21 d after weaning, 1 pig per pen was selected, blood was collected, the pig was euthanized, and a segment of the distal ileum was subsequently excised for morphological and gene and protein expression measurements. Mucosal gene expression was conducted by reverse transcription PCR for immune, antioxidant, and cellular integrity markers. Furthermore, activity of mucosal superoxide dismutase was measured by colorimetric assay. Immediately following the feed + water deprivation event, there was a decrease ( < 0.01) in growth performance and an increase ( = 0.01) in serum cortisol. The feed + water deprivation event tended ( = 0.10) to decrease ileal villus height and supplementation of DADS + DATS by oral gavage increased ( = 0.03) villus height 1 d after weaning. Supplementation of DADS + DATS by oral gavage decreased ( = 0.03) and tended to decrease ( = 0.08) gene expression of on 6 and 21 d after weaning, respectively. Furthermore, at 1 d after weaning, ileal mucosa SOD activity was decreased ( = 0.01) by the feed + water deprivation and increased ( = 0.04) by oral supplementation of DADS + DATS. Expression of the tight junction genes and were reduced ( ≤ 0.05) due to the feed + water deprivation event 1 d after weaning. Results from the current study show that an acute feed + water deprivation event can impact growth performance, intestinal characteristics, and antioxidant status in nursery pigs, which can be partially mitigated by oral supplementation of garlic compounds DADS + DATS.

Abstract Introduction: Liquid biopsies and cell-based therapies are emerging approaches for the treatment of cancer. Accuracy of diagnosis using liquid biopsies and effectiveness of treatment using cell-based therapies will rely upon the preservation cells for both viability and the information contained in them. Experimental procedures: Biomatrica's proprietary chemical library was used to develop formulations that could preserve cells in multiple formats. Cell preservation was assessed by a variety of cell-based and molecular techniques such as microscopy, flow cytometry and real-time PCR. Results: Blood samples spiked with circulating tumor cells (CTCs) were preserved for several days at room temperature to allow for downstream morphological and transcriptomic analyses. CTC recoveries in preserved samples were as much as 4 times those in non-protected samples over 4 days. Changes in RNA expression of CTC-specific genes in preserved samples ranged from 0 to +/- 2 delta Ct values, while in non-protected samples, changes ranged from 0 to +/-5 delta Ct values over 4 days. In some formulations, CTC viabilities were as much as twice the viabilities of CTCs in non-protected samples. Supporting these viability results was a demonstrated culturability of isolated CTCs. Furthermore, cell-free DNA in plasma of preserved blood samples changed by less than 2 Ct values over 4 days, whereas in control samples, plasma DNA changed by as much as 10 Ct values. Hemolysis in preserved blood samples was also lower by as much as 69% compared to non-protected samples. In a separate set of experiments, activated T-cells were stored in the presence of common buffers (RPMI, PBS) or formulations at room temperature. T-cells cultured with formulations displayed greater 40 to 60% greater cell viability after 3 days compared to those stored in common buffers. Conclusions: Biomatrica has developed a library of cell preservatives that can be used to preserve cells in blood samples and in cell culture. The ability to recover cells in blood samples and determine their genetic information would enable monitoring of a patient's cancer status and personalizing drug treatments. In addition, cell preservation may allow further advances in cell-based therapies as cells could be more reliably recovered from patients, modified (e.g. CAR-T, stem cells), and administered to patients. Citation Format: Cecille Browne, Dan Lu, Daniela Roth, Vasco Liberal. Preservation of cells in blood and culture. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 401.

One of the major challenges in DNA microarrays for their widespread application is assay sensitivity. Therefore, to achieve higher sensitivity, we developed a dual signal amplification technology based on the fluorescence beads (FB). It is the first report of the development of a dual signal amplification technology for DNA microarrays by synthesizing and applying the FB–DNA conjugates. The FB–DNA conjugates and Cy5‐T1‐FB‐mixed‐DNA demonstrated ~250–325‐fold increase in sensitivity when compared with the conventional use of Cy5‐labeled oligonucleotides such as Cy5‐T1. Furthermore, the Cy5‐T1‐FB‐mixed‐DNA demonstrated ~5–1.3‐fold increase in the sensitivity when compared with that of the FB–DNA conjugates alone.

Abstract Chemical proteomics is a rapidly evolving technology that allows for drug target identification for small molecule inhibitors. Here we present an approach utilizing a chloroalkane (CA) moiety capture handle, which can be chemically attached to small molecules and used to pull-down protein binding partners using the HaloTag technology. CA-modified compounds are cell permeable and have minimal impact on potency, allowing for phenotypic assays of the derivatized compound to be recapitulated. Here we present a study looking at the targets of two compounds - a Brutons Tyrosine Kinase (BTK) small molecule inhibitor and a Phospho-inositol 3 Kinase (PI3K) small molecule inhibitor. BTK is a non-receptor tyrosine kinase expressed in most hematopoietic cells except T cells, it is essential for B cell receptor (BCR) signaling in B cells and as such is the therapeutic focus in many autoimmune diseases, including rheumatoid arthritis (RA) and lupus. Upon binding to growth factor receptor, PI3K is activated which initiates a signaling cascade involving AKT and mTOR that promotes cell cycle progression and cell proliferation. Mutations of the catalytic subunit of PI3KA are observed in several types of cancers. Inhibition of PI3K is therefore an important therapeutic focus. Taselisib, a selective inhibitor of PIK3CA, and G02599124, an inhibitor of BTK, were derivitized with the CA linker. Phenotypic screens compared to the parental compound showed comparable IC50 values. Both CA-compounds showed good binding to HaloTag and were cell permeable in a U2OS stable cell line. Control and test experiments were performed with BTK-CA compound using Jurkat (T cells) and Ramos (B cells) cell lines. Taselib-CA was studied after 1h and 8h treatment in the HCC1954 cell line. Cells were lysed and the CA compounds, together with the bound targets, were rapidly captured onto magnetic resin coated with HaloTag. Unmodified compound was used to competitively elute interacting proteins. Eluted proteins were processed using SDS-PAGE and in-gel digestion. Peptides were analyzed using nanoscale LC-MS/MS coupled with an Orbitrap Velos Pro mass spectrometer. Using this approach we were able to specifically isolate BTK when compared to control experiments in T cells. In the Taselib-CA experiment, we were able to specifically isolate the PIK3CA catalytic subunit and PIK3R1 and PIK3R2 regulatory subunits. Observation of the wild type and mutant forms of PI3KCA will be discussed. Citation Format: Victoria Pham, James Crawford, Steve Saben, Michael Ford, Richard Jones, Danette Daniels, Thomas Kirkland, Marjeta Urh, Jennie Lill. Chemical Proteomic Analysis of BTK and PI3K Using Chloroalkane-Derivatized Small Molecule Inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B10.

The impact of acute stressors (24-h feed or water deprivation) on growth performance, intestinal characteristics, and serum stress markers in weaned pigs was evaluated. Pigs (6.21 ± 0.29 kg) were allotted in a randomized complete block design to 4 treatments on the basis of BW at the time of weaning. There were 8 mixed-sex pigs in each of 12 pens per treatment. Treatments were arranged as a 2 × 2 factorial and consisted of a feed or water stressor that included a 0- or 24-h deprivation period postweaning, and pigs were subsequently allowed access to feed and water. Growth performance was measured 1, 7, 14, and 28 d postweaning. Serum and intestinal samples were taken 1 and 7 d postweaning. Serum was analyzed for cortisol and corticotrophin-releasing factor, and villus height, crypt depth, and mast cell density were measured in the jejunum and the ileum. Expression of mucin (MUC2), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), claudin 1 (CL-1), occludin (OC), and zonula occludens 1 (ZO-1) genes were measured on d 1 and 7 postweaning in the jejunum and ileum by real-time PCR. There was a decrease (P < 0.05) in ADG with the water stressor 1 d postweaning, although subsequently, there were improvements (P < 0.05) in ADG and feed efficiency. Furthermore, the water stressor reduced ADFI during the last 14 d of the trial and cumulatively (P < 0.05). Seven days postweaning there was an increase (P < 0.05) in jejunal villous height to depth ratio due to the feed stressor and a decrease (P < 0.05) in the ileal villous height to depth ratio due to the water stressor. There was an increase (P < 0.05) in serum cortisol levels due to the water stressor both 1 and 7 d postweaning. Furthermore, there was an increase in serum corticotrophin-releasing factor 1 d but not 7 d postweaning due to the water stressor (P < 0.05). The feed stressor reduced (P < 0.05) TNF-α gene expression, and the water stressor reduced (P < 0.05) OC gene expression in the jejunum 1 d postweaning. In the ileum, there was a reduction in CL-1 and ZO-1 gene expression (P < 0.05) due to the water stressor 7 d postweaning. The results from the current investigation showed that a 24-h feed or water deprivation at the time of weaning has negative impacts on growth performance, intestinal characteristics, and serum stress responses immediately following the stress event and throughout the nursery period.