Journal articles on the topic '2D RFA model'

ABSTRACT We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3′ primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase α-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase δ and ɛ, we attempted to reconstitute AAV DNA replication by substituting either purified polymerase δ or polymerase ɛ for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.

To investigate the hemodynamic and hemorheological features related to circulatory diseases, in vitro experiments are carried out using a micro-particle image velocimetry (μ-PIV) technique. Numerical simulations using a commercial computational fluid dynamics (CFD) code are also performed to compare with the experimental results. Five different non-Newtonian blood models and a Newtonian water model are employed to investigate the blood flow characteristics through a stenotic right coronary artery (RCA). The in vitro model is made of two-dimensional (2D) polydimethylsiloxane (PDMS) channel based on the clinical angiogram of the RCA with stenotic lesion. The hemodynamic and hemorheologic behaviors in the control volume near the stenotic lesion are evaluated by velocity profiles. The predicted and measured velocity profiles at the center of the channel have a reasonable agreement.

In this paper, a 2D mesoscale finite element (FE) numerical model of mortar, considering the influence of the ITZ, was proposed to evaluate the corrosion of mortar in sodium sulfate. On the mesoscale, the corroded mortar was regarded as a three-phase composite material composed of sand, cement paste, and an interface transition zone (ITZ). Firstly, the volume fractions and mechanical parameters (elastic modulus, Poisson’s ratio, and strength) of the mesoscale phases were obtained. Then, the cement paste and the ITZ were combined to form an equivalent matrix by homogenization methods, and the calibrated constitutive relations of the equivalent matrix were established. Subsequently, a two-dimensional (2D) random circular aggregate (RCA) model and a 2D random polygonal aggregate (RPA) model of corroded mortar were established using the random aggregate model. The failure process of corroded mortar specimens under uniaxial compression was simulated by the mesoscale FE numerical model. Comparing the simulation results with the measured stress–strain curves of the uniaxial compression test, it was found that the simulation results of the 2D RP model were closer to the experimental results than those of the 2D RC model. Meanwhile, the numerical simulation results were in good agreement with the experimental results, and the error values of peak stress between the simulation results and the measured results were within 7%, which showed that the 2D mesoscale FE model could accurately predict the results of a uniaxial compression test of a mortar specimen under sulfate attack.

Abstract Background: The type 1/2 BRAF inhibitors (e.g., vemurafenib) have been used for patients with BRAFV600E-mutant tumors. But the application of the inhibitors was restricted to a few types of tumors (e.g., melanoma). Common adverse effects such as pruritus and skin papilloma have limited the usage. It could be attributed to the paradoxical activation of the MAPK pathway. The paradoxical activation also leads to tumor promotion in BRAF wild-type melanoma and skin toxicity in 30%-70% patients with KRAS mutations. Therefore, development of type II pan-RAF inhibitors which avoid MAPK paradoxical activation is necessary. Methods: The selective pan-RAF inhibitor QLH11906 was tested on A/B/C RAF kinases and a representative kinase panel. Both 2D and 3D proliferation studies were conducted for cell lines with BRAF and various KRAS mutations, followed by both immunoprecipitation and ERK phosphorylation assays to elucidate the mode of action. Combination studies with MEK inhibitors were implemented in both KRAS-mutant cell lines and BRAF/KRAS-mutant patient derived organoids (PDO). Single-agent and combination studies were also carried out in vivo on both BRAF/KRAS-mutant cell line-derived xenograft (CDX) and KRAS-mutant patient-derived xenograft (PDX) models. Results: QLH11906 inhibited wild-type A/B/C RAF and BRAFV600E with the IC50 of 4.9, 1.9, 0.5, and 1.1 nM, respectively, and showed no inhibition at 1 μM in a selectivity panel of 28 kinases except for DDR1 and DDR2. Under 2D culture condition, QLH11906 inhibited proliferation of various BRAF/KRAS-mutant cell lines (IC50 300-1700 nM) and suppressed ERK phosphorylation (IC50 20-600 nM). In 3D cultures, QLH11906 showed higher activity in proliferation suppression in both HCT116 (KRASG13D, IC50 24 nM 3D vs 1186 nM 2D) and Calu-6 (KRASQ61K, IC50 85 nM 3D vs 1411 nM 2D). In immunoprecipitation studies with HCT116 and Calu-6 cells, both BRAF homodimers and BRAF/CRAF heterodimers could be detected under the treatment of QLH11906. QLH11906 showed synergistic effect when combined with MEK inhibitors trametinib or cobimetinib in KRAS-mutant cell lines, including A549 (KRASG12S), Calu-6, HCT116, NCIH2122 (KRASG12C) and Panc1005 (KRASG12D), and 77%-99% tumor suppression was reached at the expected clinical Ctrough concentrations. Synergistic effects with trametinib were observed in PDO models including pancreatic cancers (KRASG12D and KRASG12V), colon cancers (KRASG12D and KRASG13D) and lung cancers (KRASG12V). QLH11906 also promoted tumor shrinkage in vivo as a single agent in Calu-6, HCT116, and Colo-205 (BRAFV600E) CDX models or combined with trametinib in PDX models (KRASG12C lung cancers and KRASG12C colon cancers) Conclusion: QLH11906 is a highly selective pan-RAF inhibitor with promising antitumor activity both in vitro and in vivo. It is hopeful to be a therapeutic agent for BRAF and KRAS-mutant tumor patients. Citation Format: Zhe Cheng, Fei Chen, Xile Liu, Cong Gao, Linchao Jia, Yuanzhi Lao. Preclinical characterization of QLH11906, a novel pan-RAF inhibitor [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 5460.

e15084 Background: Elevated RAS-RAF-MEK-ERK (MAPK pathway) signaling is observed in over half of all solid human tumors, and mutations in RAS or RAF account for a large fraction. Given MEK’s unique position in the MAPK cascade, it remains an attractive target in cancer. However, FDA-registered MEK inhibitors are susceptible to pathway reactivation events that limit their use to RAF mutant disease and cause on-target toxicities stemming from chronic target engagement. IMM-1-104 is a novel, allosteric dual-MEK inhibitor designed for better applicability to RAS mutant tumors by preventing MEK reactivation. Endowed with a short plasma half-life, IMM-1-104 promotes deep cyclic inhibition with a near-zero drug trough, affording normal cells a chance to recover between doses. Methods: We characterized IMM-1-104’s pharmacologic activity across 52 tumor cell lines that spanned 11 distinct tumor types in a humanized, ECM-based 3D tumor growth assay (3D-TGA). The 3D-TGA has better predicted in vivo tumor responses versus 2D culture and more accurately reflects human tumor biology. Tumor models were categorized based on in vivo drug PK limits as sensitive to IMM-1-104 (EC50 < 1uM), intermediate (1uM≤EC50≤10uM and ≥25% inhibition at 10uM) or resistant otherwise. Models were evaluated by whole exome sequencing, along with RNA sequencing in the 3D context, to profile determinants of sensitivity and resistance and to prioritize patient populations most likely to respond to IMM-1-104. Results: Models sensitive to IMM-1-104 were enriched for MAPK driver mutations, consistent with pathway addiction. We reasoned that activation of parallel compensatory pathways that can reduce reliance on MAPK signaling may increase the likelihood of resistance to IMM-1-104. Pathways and genes suspected of contributing to resistance helped refine signatures based on 3D-TGA outcome data. Models with a MAPK driver mutation and compensatory mutations such as PIK3CA or PTEN deletion were more likely to show intermediate response than those with a greater addiction to MAPK drivers. Models lacking a clear MAPK driver mutation but harboring other putative resistance alterations were more likely to be resistant in the 3D-TGA. Conclusions: To better understand the relevance of tumor model responses in the 3D-TGA relative to RAS mutant patient populations, we computationally compared tumor model data to patient somatic alterations, identified in the public resource GENIE, which has cataloged the molecular profiles of over 100,000 cancer patients. Based on model-to-patient molecular mapping, we identified biomarker-defined subsets of sensitive KRAS mutant lung and colorectal models. The most broadly sensitive patient-aligned models in the 3D assay were KRAS mutant pancreatic cancer and NRAS mutant melanoma patients, supporting the inclusion of such patients in planned clinical studies of IMM-1-104.

e15084 Background: Elevated RAS-RAF-MEK-ERK (MAPK pathway) signaling is observed in over half of all solid human tumors, and mutations in RAS or RAF account for a large fraction. Given MEK’s unique position in the MAPK cascade, it remains an attractive target in cancer. However, FDA-registered MEK inhibitors are susceptible to pathway reactivation events that limit their use to RAF mutant disease and cause on-target toxicities stemming from chronic target engagement. IMM-1-104 is a novel, allosteric dual-MEK inhibitor designed for better applicability to RAS mutant tumors by preventing MEK reactivation. Endowed with a short plasma half-life, IMM-1-104 promotes deep cyclic inhibition with a near-zero drug trough, affording normal cells a chance to recover between doses. Methods: We characterized IMM-1-104’s pharmacologic activity across 52 tumor cell lines that spanned 11 distinct tumor types in a humanized, ECM-based 3D tumor growth assay (3D-TGA). The 3D-TGA has better predicted in vivo tumor responses versus 2D culture and more accurately reflects human tumor biology. Tumor models were categorized based on in vivo drug PK limits as sensitive to IMM-1-104 (EC50 < 1uM), intermediate (1uM≤EC50≤10uM and ≥25% inhibition at 10uM) or resistant otherwise. Models were evaluated by whole exome sequencing, along with RNA sequencing in the 3D context, to profile determinants of sensitivity and resistance and to prioritize patient populations most likely to respond to IMM-1-104. Results: Models sensitive to IMM-1-104 were enriched for MAPK driver mutations, consistent with pathway addiction. We reasoned that activation of parallel compensatory pathways that can reduce reliance on MAPK signaling may increase the likelihood of resistance to IMM-1-104. Pathways and genes suspected of contributing to resistance helped refine signatures based on 3D-TGA outcome data. Models with a MAPK driver mutation and compensatory mutations such as PIK3CA or PTEN deletion were more likely to show intermediate response than those with a greater addiction to MAPK drivers. Models lacking a clear MAPK driver mutation but harboring other putative resistance alterations were more likely to be resistant in the 3D-TGA. Conclusions: To better understand the relevance of tumor model responses in the 3D-TGA relative to RAS mutant patient populations, we computationally compared tumor model data to patient somatic alterations, identified in the public resource GENIE, which has cataloged the molecular profiles of over 100,000 cancer patients. Based on model-to-patient molecular mapping, we identified biomarker-defined subsets of sensitive KRAS mutant lung and colorectal models. The most broadly sensitive patient-aligned models in the 3D assay were KRAS mutant pancreatic cancer and NRAS mutant melanoma patients, supporting the inclusion of such patients in planned clinical studies of IMM-1-104.

Abstract Background: In the US in 2021, invasive melanoma will account for an estimated 106,000 new cases and &gt; 7,000 deaths. Somatic mutations that activate the MAPK signaling pathway are a leading cause of melanoma with 50% harboring oncogenic BRAF alterations and another 20% with activating NRAS mutations. Of note, NRAS mutant melanoma has been shown to be dependent upon RAF signaling via CRAF dimers for downstream activation of MEK/ERK. While targeted therapies are approved for V600 (Class I, monomer-driven) BRAF mutant melanoma, no approved targeted therapy exists for patients with melanoma driven by Class II or Class III dimer-dependent BRAF alterations or NRAS mutations. KIN-2787 is a novel, orally available, potent, and selective pan-RAF inhibitor designed to be effective in RAF-dependent cancers, including all classes of BRAF alterations, by targeting mutant BRAF monomers and RAF dimers, regardless of isoform. Methods: KIN-2787 activity was assessed by suppression of downstream MAPK pathway signaling and subsequent cell growth inhibition in a panel of human melanoma cell lines. In vivo KIN-2787 efficacy was evaluated in BRAF and NRAS mutant melanoma cell- and patient-derived xenograft models. Results: KIN-2787 cellular activity was measured by inhibition of ERK phosphorylation across a panel of melanoma cell lines, including those harboring Class I BRAF alterations, Class II and III BRAF alterations, NRAS mutations, KRAS mutations, and wild type RAF/RAS. In contrast to vemurafenib, an approved BRAF inhibitor with activity limited to Class I BRAF alterations, KIN-2787 was active across all classes of BRAF mutant melanoma cells (EC50 values &lt; 100 nM). NRAS and KRAS mutant cell lines were moderately responsive to KIN-2787 inhibition. Melanoma cells expressing wild type RAS/RAF were the least sensitive to MAPK pathway inhibition by KIN-2787. KIN-2787 also inhibited cell proliferation in BRAF and NRAS mutant melanoma in 2D and 3D cell cultures. Daily KIN-2787 treatment resulted in significant tumor growth inhibition in human melanoma xenograft models bearing Class I, II and III BRAF alterations as well as NRAS mutations and was associated with MAPK pathway suppression. Additionally, KIN-2787 was efficacious in a pre-/post-treatment melanoma PDX pair in which the original tumor was Class I BRAF V600E but acquired a Class II BRAF kinase domain duplication upon progression on dabrafenib + trametinib. Details from the above findings will be presented at the meeting. Conclusions: KIN-2787 is a next-generation, pan-RAF inhibitor with in vitro and in vivo activity against human melanoma driven by BRAF and/or NRAS mutations. Data supports KIN-2787 use in acquired BRAF dimer-dependent resistance to BRAF+MEK inhibitor therapy. A Phase I dose escalation and expansion clinical trial evaluating the safety and efficacy of KIN-2787 is ongoing (NCT04913285). Citation Format: Nichol L. G. Miller, Tim S. Wang, Paul Severson, Ping Jiang, Michelle Perez, Noel Timple, Toufike Kanouni, Aleksandra Franovic, Eric S. Martin, Eric Murphy. Antitumor activity of KIN-2787, a next-generation pan-RAF inhibitor, in preclinical models of human RAF/RAS mutant melanoma [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 2674.

A avaliação da previsão de chuva a curtíssimo prazo com até 3 horas de antecedência na área de cobertura do Radar Meteorológico de São Paulo (RSP) para diferentes tipos de sistemas precipitantes, principalmente os associados às enchentes e deslizamentos na Região Metropolitana de São Paulo (RMSP), foi realizada por meio de um modelo advectivo a partir do campo de vento 2D médio e da velocidade dos campos das taxas de precipitação estimados com o radar e um Esquema Numérico de Terceira-ordem Corrente Acima (ENTOCA), que utiliza um vetor de deslocamento constante. O desempenho obtido para precipitação acumulada foi avaliado com os seguintes parâmetros estatísticos: Índice de Sucesso Crítico (ISC), Probabilidade de Detecção (POD), Razão de Falsos Alarmes (RFA), Erro Quadrático Médio (EQM) e Coeficiente de Correlação (CCOR). O ISC para limiar de acumulo de 0,2 mm, em períodos de acumulação de 60 minutos, apresentou os seguintes valores: 77% para eventos de Frente fria, 67,5% para Linhas de Instabilidade, 58% para Bandas Dispersas, 56,4% para Convecção Isolada e 47% para Brisa Marítima. A previsibilidade obtida foi maior para sistemas estratiformes do que para sistemas convectivos. A partir de períodos de 90 (120) minutos de advecção o CSI diminui exponencialmente para sistemas convectivos (estratiformes).

The RAS/RAF and PI3K/Akt pathways play a key regulatory role in cancer and are often hit by oncogenic mutations. Despite molecular targeting, the long-term success of monotherapy is often hampered by de novo or acquired resistance. In the case of concurrent mutations in both pathways, horizontal combination could be a reasonable approach. In our study, we investigated the MEK inhibitor selumetinib and PI3K/mTOR dual inhibitor BEZ235 alone and in combination in BRAF-only mutant and BRAF + PI3K/PTEN double mutant cancer cells using short- and long-term 2D viability assays, spheroid assays, and immunoblots. In the 2D assays, selumetinib was more effective on BRAF-only mutant lines when compared to BRAF + PI3K/PTEN double mutants. Furthermore, combination therapy had an additive effect in most of the lines while synergism was observed in two of the double mutants. Importantly, in the SW1417 BRAF + PI3K double mutant cells, synergism was also confirmed in the spheroid and in the in vivo model. Mechanistically, p-Akt level decreased only in the SW1417 cell line after combination treatment. In conclusion, the presence of concurrent mutations alone did not predict a stronger response to combination treatment. Therefore, additional investigations are warranted to identify predictive factors that can select patients who can benefit from the horizontal combinational inhibition of these two pathways.

Abstract Systemic inflammatory response syndrome is a costly burden for health care systems both nationally and globally. SIRS results from the systemic release of exorbitant quantities of proinflammatory cytokines, which leads to severe complications including acute lung injury, multiple organ dysfunction syndrome, and even death. RKIP is a regulator of MAPK and NF-κB signaling cascades which are both critical for production of the cytokines responsible for SIRS initiation. Using a T cell dependent mouse model of SIRS, we have shown that RKIP is necessary for the synthesis of pathologic quantities of IFNγ from antigen specific CD8+ T cells, but not CD4+ T cells, which is due to an intrinsic signaling defect downstream of the TCR in RKIP-/- T cells. Importantly, RKIP deficient mice were able to mount a productive response to vaccination, suggesting that RKIP may be critical for cytokine production during serial TCR triggering in SIRS, but less so during primary T cell responses (data submitted for publication). Additionally, we have found that RKIP may also play a role in antigen presenting cell function. This is evidenced by a rapid, but transient, inability to detect RKIP by western blot after stimulation of splenocytes with TLR ligands. Although an exact molecular mechanism underlying RKIP’s effects is presently unclear, we are currently utilizing cutting-edge PF 2D proteomic mapping to further elucidate its role within the signaling machinery of immune cells.

Sampling programs for suspended sediment were carried out in the Usumacinta River and its tributary Grijalva River in Mexico during the years 2016 and 2017. Suspended sediment samples collected during these sampling programs were analyzed in the laboratory using a Rotating Annular Flume (RAF) fitted with a Particle Tracking Velocimetry (PTV) to obtain the 2D images of the suspended sediment particles as they were undergoing floc reconstruction, and subsequently using a glass settling column fitted with inline digital holography set up to obtain 3D holograms of the fully flocculated sediment particles. From these high-resolution hologram images, the fractal dimension of the flocculated sediment particles was obtained using the classical box-counting method and an improved Triangular box-counting method. The estimated fractal dimension of flocculated sediment, which is a measure of floc compactness and structure that control the settling velocity of flocculated sediment was used to validate two empirical models to estimate the fractal dimension in terms of the floc sizes of suspended sediments of these two rivers. It is shown in this study that the floc characteristic can be analyzed in laboratory experiments after floc reconstruction with the use of an RAF and it offers a viable alternative to the costly in-situ sampling that is often carried out in ocean research. The digital holography method employed in this research offers an efficient methodology to obtain the floc fractal dimension. Regarding the innovative aspects and new contribution to science, we can say that we have developed a laboratory protocol to test river waters to establish floc properties such as fractal dimensions of flocs in this research which will help to test river waters on a routine basis with manageable costs. We can also say that we have developed models to predict the relationship between floc fractal dimension and floc size, which did not exist before.

Background: The network interactions link human disease proteins to regulatory cellular pathways leading to better understanding of protein functions and cellular processes. Revealing the network of signaling pathways in cancer through protein-protein interactions at molecular level enhances our understanding of Hepatocellular Carcinoma (HCC). Objective: A rodent model for study of HCC was developed to identify differentially expressed proteins at very early stage of cancer initiation and throughout its progression. Methodology: HCC was induced by administrating N-Nitrosodiethylamine (DEN) and 2-aminoacetylfluorine (2-AAF) to male Wistar rats. Proteomic approaches such as 2D-Electrophoresis, PD-Quest, MALDI-TOF-MS and Western blot analyses have been used to identify, characterize and validate the differentially expressed proteins in HCC-bearing animals vis-a-vis controls. Results: The step-wise analysis of morphological and histological parameters revealed HCC induction and tumorigenesis at 1 and 4 months after carcinogen treatment, respectively. We report a novel protein network of 735 different proteins out of which eight proteins are characterized by MALDI-TOF-MS analysis soon after HCC was chemically induced in rats. We have analyzed four different novel routes representing the association of experimentally identified proteins with HCC progression. Conclusion: The study suggests that A-Raf, transthyretin and epidermal growth factor receptor play major role in HCC progression by regulating MAPK signaling pathway and lipid metabolism leading to continuous proliferation, neoplastic transformation and tumorigenesis.

Abstract Background: The identification of treatment strategies targeting PIK3CA mutant colorectal cancer (CRC) are of great clinical interest. Previous work from our lab has identified MTORC1/2 and HDAC1/2 inhibition with copanlisib (cop; PI3K/MTOR) and romidepsin (romi; HDAC1/2) as a potential therapeutic strategy. We hypothesized that changes in c-MYC protein levels and c-MYC target gene (CTG) alterations might be a potential mechanism of action for this combination. Methods: Known CTGs were identified and their expression levels were examined in PIK3CA mutant vs WT CRCs using the cBioPortal Colorectal Adenocarcinoma (TCGA, PanCancer Atlas) dataset. Murine derived cancer organoids (MDCO) were generated from adenocarcinomas of Apc and Pik3ca mutant transgenic mice (F1 (FVB x B6) Apcfl/+ Pik3caH1047R/+). MDCO results were corroborated using the human 2D isogenic cell lines SW48 and SW48PIK3CA-H1047R (SW48PK) and RAS/RAF WT patient derived cancer organoids (PDCO) generated from CRC patient samples under approved IRB protocols. Immunoblots (IB) were used to assess c-MYC levels after treatment with cop, romi, and the combination across all models. RNA sequencing was conducted on PDCOs and SW48PK cells and changes in 16 CTGs were examined. An aggregate score was created for each treatment group where a statistically significantly altered CTG with log fold change ≥1.5 added one point and ≤-1.5 subtracted one point. All others were scored 0. Results: c-MYC target genes were assessed for differential expression in PIK3CA mutant CRC vs PIK3CA WT CRC with only 1/16 genes decreased in PIK3CA mutant CRC (GADD45A: log ratio -0.21, q=0.01). Next, MDCOs treated with the combination showed a decrease in total c-MYC levels in the combination therapy. These results were corroborated in two human 2D CRC cell lines, SW48 and SW48PK and a panel of PDCOs. Interestingly, c-MYC levels decreased in both romi alone and the combination in both the SW48 and SW48PK cell lines after 24 hours of treatment. However, the extent to which c-MYC levels were decreased was not as substantial in the panel of PDCOs. Across all in vitro models a decrease in PI3K signaling, as illustrated by decreased pRPS6 and p4EBP1 in response to cop treatment and an increase in H3K27 acetylation in response to romi, was observed in the single agent and combination therapies. RNA sequencing demonstrated that cop, romi, and combo had a CTG score of 0, -4, and -7 respectively in the PIK3CA mutant PDCO. Similar results were seen in an additional RAS/RAF WT PDCO (0, 1, and -4, respectively) and SW48PK cells (0, -3, and -7, respectively). Conclusion: A potential mechanism by which cop and romi treatment promote tumor response is through a decrease in c-MYC protein levels and expression of downstream c-MYC target genes. This regimen deserves further mechanistic investigations in vivo. Citation Format: Rebecca A. DeStefanis, Autumn M. Olson, Alyssa K. DeZeeuw, Susan N. Payne, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. MTORC1/2 and HDAC1/2 inhibition promote tumor response through inhibition of MYC [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 1128.

Abstract Resistance to targeted therapies remains an obstacle in curing cancers. A reason for this is testing of therapeutic agents in 2D culture conditions that do not recapitulate growth conditions of tumors in vivo. A critical component for defining the cell survival in vivo is the extracellular matrix (ECM). The ECM not only modulates cell-matrix and cell-cell interactions, but also determines drug accessibility to the tumor tissue. Many labs thus evaluate drug perturbations in 3D cell culture platforms using Matrigel and collagen scaffolds. A caveat with these models is the simplistic use of only basement membrane proteins or individual ECM proteins that, again, do not efficiently mimic the tumor microenvironment (TME). Although in vivo mouse models can be a better alternative, the time to conduct experiments, the associated cost, and inability to recapitulate patient TME remain a crucial challenge. Thus, we aim to regenerate an in vitro melanoma TME by engineering a melanoma extracellular matrix. We aim to do this by first identifying the patient melanoma ECM. On conducting proteomics for ECM proteins on patient tissues we identified collagen VI to be the most dominant ECM protein. However, prior work from other labs reports collagen I as the most abundant protein. This information was used to construct a platform through which we can recapitulate the melanoma ECM. We did this by generating cell-derived matrices (CDM) using human foreskin fibroblasts. On comparing the CDM from HFF1 to patient ECM, we could detect ~80 of ~120 core ECM proteins. Upon performing proteomic studies on CDMs, we again identified collagen VI as the dominant ECM protein. We also tested CDM deposition with cancer-associated fibroblasts, yielding similar results. This brought us to the question of whether collagen VI plays a role in drug resistance in melanoma. To address the role of cell-derived matrices and collagen VI, we created collagen VI KO fibroblasts through CRISPR/Cas9, which were then used to generate these matrices and will be used to evaluate its effect on drug treatments. While building this in vitro melanoma TME, we understood the need for a 3D assay to have a robust readout on effect of drugs in 3D compared to 2D. Although many viability and proliferation assays are used by various labs, these assays do not look at the effect of treatments at a single-cell resolution and neither distinguishes if the drug is cytostatic or cytotoxic. Towards that goal, we established a 3D assay by culturing melanoma cells in collagen and tested for Raf-Mek combination therapies. We made comparisons of cells either cultured in 3D as single cells or spheroids. Imaging of these platforms was performed on epifluorescence and light-sheet microscopes. We tested the platform for both viability and proliferation and the images were analyzed through individual Z planes of the 3D image. While we observed differences in viability and proliferation in 2D vs. 3D upon drug treatments, there was no difference in 3D single cells vs. spheroids. Citation Format: Vasanth Siruvallur Murali, Justin Cillay, Erik Welf, Gaudenz Danuser, Murat Can Cobanoglu. Engineering a 3D melanoma microenvironment and identifying novel therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A27.

Abstract Background: KRASG12C inhibitors sotorasib and adagrasib have shown impressive results in clinical studies. These inhibitors efficiently block KRAS downstream effectors such as RAF, MEK, and ERK/p-ERK. However, factors such as feedback reactivation or bypass of KRAS dependence are emerging, resulting in the need for rational combination approaches. KRASG12C inhibitors have limited impact on the parallel oncogenic signaling driven by PI3K/AKT, its regulator molecules such as the Rho GTPases, and their effectors. This signaling pathway can collectively promote p-ERK re-activation thereby blunting the efficacy of KRASG12C targeted drugs. The p21-activated kinase (PAK) family are crucial effectors of the Rho family of GTPases and act as regulatory switches that control important cellular processes including motility, proliferation, and survival. PAK4 is the main effector of Rho GTPase, cell division control protein 42 homolog (Cdc42), and has an established role in mutant RAS-dependent tumor metastasis. In this study, we rationally targeted two parallel signaling pathways by combining KRASG12C inhibitors with the PAK4 inhibitor KPT-9274 in KRASG12C mutant PDAC and NSCLC in vitro and in vivo models. Methods: We evaluated the cytotoxicity and molecular profile of KRASG12C inhibitors in combination with PAK4 inhibitor KPT-9274 as well as positive control PF-3758309 in KRASG12C mutant 2D and 3D cellular/spheroid models. The anti-tumor activity of the combinations was evaluated in KRASG12C mutant cell line-derived xenograft. Tumor volumes were assessed throughout the treatment period using an ultrasound scanner. Results: PAK4 inhibitor KPT-9274 synergized with both sotorasib and adagrasib yielding suppressed growth of KRASG12C mutant PDAC and NSCLC cells in 2D and 3D cultures, but not in KRASwt, KRASG12V, and KRASG12D cell lines (NCI RASless MEFs). In addition, the combination reduced the clonogenic potential of KRASG12C mutant cancer cells. PAK4-KRASG12C inhibitor combinations resulted in enhanced and prolonged inhibition of KRAS downstream effector pathways. In xenograft studies, oral administration of KPT-9274 at sub-optimal dose (100 mg/kg QD x 5 x 3 weeks) and sotorasib at one-fourth of MTD (25 mg/kg QD x 5 x 3 weeks) demonstrated remarkable inhibition of KRASG12C mutant MiaPaCa-2 tumor burden (p&lt;0.001 single agents vs combination treatment). Residual tumor analysis showed a reduction in proliferation index (ki67), activation of pro-apoptotic caspases, and inhibition of mutant RAS effector signaling. Studies in additional KRASG12C xenograft and tumor explant models are ongoing. Conclusion: This is the first study showing that KRASG12C inhibitors can synergize with PAK4 inhibitors. This combination can simultaneously target direct KRAS downstream effectors and the parallel PI3K/AKT oncogenic signaling warranting further clinical investigations. Citation Format: Husain Yar Khan, Sahar Bannoura, Hirva Mamdani, Amro Aboukameel, Yousef Mzannar, Yiwei Li, Mohammad Najeeb Al-Hallak, Ibrahim Azar, Steve Kim, Rafic Beydoun, Ramzi Mohammad, Yosef Landesman, Yue Zhang, Erkan Baloglu, William Senapedis, Ammar Sukari, Misako Nagasaka, Anthony Shields, Asfar S. Azmi. Anti-tumor activity of KRASG12C inhibitors is enhanced when combined with Cdc42 effector p21-activated kinase 4 targeting agents [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 5315.

Objective. The global left ventricular systolic impairment with left ventricular dilatation can manifest due to idiopathic dilated cardiomyopathy or ischemic heart disease and can present a similar clinical picture of severe heart failure. The aim of our investigation was to assess a differential diagnostic value of resting 99mTc-MIBI myocardial perfusion defects in evaluation of the etiology of heart failure. Material and methods. The data of 2D echocardiography, coronary angiography, and myocardial gated single photon emission computed tomography with 99mTc-MIBI investigation were evaluated in 43 patients with global left ventricular systolic impairment, characterized by left ventricular end-diastolic diameter of ≥65 mm and ejection fraction of £40%. The idiopathic dilative cardiomyopathy was diagnosed in 26 patients (Group 1) and ischemic heart failure – in 17 patients (Group 2). The area and the degree (severity) of myocardial perfusion defects (AMPD and DMPD) at rest in regions supplied by three coronary arteries were evaluated in all the patients. Results. The area of perfusion defects in the left anterior descending (LAD) and right coronary artery (RCA) regions in dilative cardiomyopathy patients was smaller than in ischemic heart failure patients (1.43±0.9 vs 2.53±0.53, P=0.001, and 2.19±0.6 vs 2.82±0.56, P=0.02). The degree of perfusion defects was also less severe in the same circulation regions (1.39±0.93 vs 2.59±0.6, P=0.01, and 1.6±0.46 vs 2.71±0.15, P=0.001). We have designed a logistic regression model expressed by formula x=2.52AMPDrca+2.47AMPDlad+2.21DMPDrca. Idiopathic dilative cardiomyopathy was predicted when x was £16 and ischemic heart failure when x was >16. The sensitivity in predicting idiopathic dilative cardiomyopathy was 94.44%, and the specificity was 88.24%. Conclusion. The difference in the area and degree of 99mTc-MIBI myocardial perfusion defects at rest in patients with heart failure caused by idiopathic dilative cardiomyopathy or ischemic heart failure is measurable and has a predictive value for differentiation of the etiology of global left ventricular systolic impairment.

e16263 Background: Pancreatic cancer has a dismal survival rate and no good therapeutic options. Of all types of cancer, it is Pancreatic cancer that is most closely associated with activating mutations in the RAS oncoprotein. Approximately 90% of pancreatic adenocarcinomas carry point mutations activating RAS. Ras directed targeted therapy is the obvious approach to enhancing treatment options for the disease. There is now an FDA approved targeted RAS therapeutic specific to the RAS12C mutant form. However, this particular mutation is uncommon in Pancreatic cancer. Therefore, drugs which act more broadly on RAS are required. There are currently no approved pan-RAS inhibitors. We have developed a small molecule pan-RAS inhibitor which binds directly to all forms of RAS. It acts to block the ability of RAS and to bind and signal through its effectors. It is active in vitro and in vivo against multiple models of pancreatic cancer. Methods: In silico screening of a virtual compound library was performed to identify an initial candidate inhibitor. Iterative rounds of Medicinal Chemistry informed by structural modeling and bioassay in 3D vs 2D growth assays were performed to identify an enhanced activity derivative. Target binding was confirmed by Microscale Thermophoresis and NMR. RAS/RAF complex status was measured by co-immunoprecipitation of the endogenous proteins. Ras signaling was assayed by Western analysis of Phospho-ERK and RAL-GTP pull down assays. In vitro activity was measured using 3D soft agar assays. In vivo activity was measured by ip or po administration against pancreatic tumor cell line xenografts and pdx systems. Results: The current lead, designated RAS-F binds all three main isoforms of RAS at low uM kd and modulates the RAS effector domain in NMR studies. It suppresses RAS signaling pathways in transient assays and suppresses pancreatic tumor cell growth in soft agar with IC50s below 500nM. It suppresses xenograft development of human and mouse RAS driven pancreatic tumor cell lines and reduces the growth of a pancreatic cancer pdx. Conclusions: We have developed a pre-clinical pan-RAS inhibitor that is active in vitro and in vivo against pancreatic cancer models. Funding: NIH 5U01HL127518-03, Kentucky Lung Cancer Research Program (GJC), Qualigen Therapeutics LLC.

e23531 Background: Malignant Peripheral Nerve Sheath Tumors (MPNST) are a rare but deadly form of sarcoma originating from the Schwann cell compartment. They are usually driven by loss of function of the NF1 tumor suppressor. This can happen spontaneously but is also the cause of disease in the inheritable syndrome of Neurofibromatosis. Neurofibromatosis is a member of the family of genetic diseases known as “Rasopathies” and is one of the most common inherited neurological disorders. NF1 serves as an important negative regulator or GAP for the RAS oncoprotein. Loss of function of NF1 thus results in the upregulation of non-mutant RAS activity and this appears to be a driving event in the disease. Therapeutic options remain poor. An obvious approach to effective treatment would be targeted therapy directed at non-mutant Ras proteins. There are currently no approved pan-RAS inhibitors. We have developed a small molecule pan-RAS inhibitor which binds directly to all forms of RAS. It acts to block the ability of RAS and to bind and signal through its effectors. It is active in vitro and in vivo against multiple models of MPNST. Methods: In silico screening of a virtual compound library was performed to identify an initial candidate inhibitor. Iterative rounds of Medicinal Chemistry informed by structural modeling and bioassay in 3D vs 2D growth assays were performed to identify an enhanced activity derivative. Target binding was confirmed by Microscale Thermophoresis and NMR. RAS/RAF complex status was measured by co-immunoprecipitation of the endogenous proteins. Ras signaling was assayed by Western analysis of Phospho-ERK and RAL-GTP pull down assays. In vitro activity was measured using 3D soft agar assays. In vivo activity was measured by ip or po administration against MPNST pdx systems. Results: The current lead, designated RAS-F binds all three main isoforms of RAS at low uM kd and modulates the RAS effector domain in NMR studies. It suppresses RAS signaling pathways in transient assays and suppresses MPNST tumor cell growth in soft agar with IC50s ̃ 250nM. It suppresses MPNST pdx growth. Conclusions: We have developed a pre-clinical pan-RAS inhibitor that is active in vitro and in vivo against MPNST cancer models. The agent also has potential uses against non-tumor manifestations of Neurofibromatosis such as Plexiform and Cutaneous neurofibromas. Funding: NIH 5U01HL127518-03, Kentucky Lung Cancer Research Program CDMRP NF180094 (GJC). Qualigen Therapeutics LLC (GJC, JOT).

Hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days. Equal amounts of proteins from urinary bladder or liver extracts of control and arsenic-treated hamsters were labeled with Cy3 and Cy5 dyes, respectively. After differential in gel electrophoresis and analysis by the DeCyder software, several protein spots were found to be down-regulated and several were up regulated. Our experiments indicated that in the bladder tissues of hamsters exposed to arsenite, transgelin was down-regulated and GST-pi was up-regulated. The loss of transgelin expression has been reported to be an important early event in tumor progression and a diagnostic marker for cancer development [29-32]. Down-regulation of transgelin expression may be associated with the carcinogenicity of inorganic arsenic in the urinary bladder. In the liver of arsenite-treated hamsters, ornithine aminotransferase was up-regulated, and senescence marker protein 30 and fatty acid binding protein were down-regulated. The volume ratio changes of these proteins in the bladder and liver of hamsters exposed to arsenite were significantly different than that of control hamsters. Introduction Chronic exposure to inorganic arsenic can cause cancer of the skin, lungs, urinary bladder, kidneys, and liver [1-6]. The molecular mechanisms of the carcinogenicity and toxicity of inorganic arsenic are not well understood [7-9). Humans chronically exposed to inorganic arsenic excrete MMA(V), DMA(V) and the more toxic +3 oxidation state arsenic biotransformants MMA(III) and DMA (III) in their urine [10, 11], which are carcinogen [12]· After injection of mice with sodium arsenate, the highest concentrations of the very toxic MMA(III) and DMA(III) were in the kidneys and urinary bladder tissue, respectively, as shown by experiments of Chowdhury et al [13]. Many mechanisms of arsenic toxicity and carcinogenicity have been suggested [1, 7, 14] including chromosome abnormalities [15], oxidative stress [16, 17], altered growth factors [18], cell proliferation [19], altered DNA repair [20], altered DNA methylation patterns [21], inhibition of several key enzymes [22], gene amplification [23] etc. Some of these mechanisms result in alterations in protein expression. Methods for analyzing multiple proteins have advanced greatly in the last several years. In particularly, mass spectrometry (MS) and tandem MS (MS/MS) are used to analyze peptides following protein isolation using two-dimensional (2-D) gel electrophoresis and proteolytic digestion [24]. In the present study, Differential In Gel Electrophoresis (DIGE) coupled with Mass Spectrometry (MS) has been used to study some of the proteomic changes in the urinary bladder and liver of hamsters exposed to sodium arsenite in their drinking water. Our results indicated that transgelin was down-regulated and GST-pi was up-regulated in the bladder tissues. In the liver tissues ornithine aminotransferase was up-regulated, and senescence marker protein 30, and fatty acid binding protein were down-regulated. Materials and Methods Chemicals Tris, Urea, IPG strips, IPG buffer, CHAPS, Dry Strip Cover Fluid, Bind Silane, lodoacetamide, Cy3 and Cy5 were from GE Healthcare (formally known as Amersham Biosciences, Uppsala, Sweden). Thiourea, glycerol, SDS, DTT, and APS were from Sigma-Aldrich (St. Louis, MO, USA). Glycine was from USB (Cleveland, OH, USA). Acrylamide Bis 40% was from Bio-Rad (Hercules, CA, USA). All other chemicals and biochemicals used were of analytical grade. All solutions were made with Milli-Q water. Animals Male hamsters (Golden Syrian), 4 weeks of age, were purchased from Harlan Sprague Dawley, USA. Upon arrival, hamsters were acclimated in the University of Arizona animal care facility for at least 1 week and maintained in an environmentally controlled animal facility operating on a 12-h dark/12-h light cycle and at 22-24°C. They were provided with Teklad (Indianapolis, IN) 4% Mouse/Rat Diet # 7001 and water, ad libitum, throughout the acclimation and experimentation periods. Sample preparation and labelling Hamsters were exposed to sodium arsenite (173 mg) in drinking water for 6 days and the control hamsters were given tap water. On the 6th day hamsters were decapitated rapidly by guillotine. Urinary bladder tissues and liver were removed, blotted on tissue papers (Kimtech Science, Precision Wipes), and weighed. Hamster urinary bladder or liver tissues were homogenized in lysis buffer (30mMTris, 2M thiourea, 7M urea, and 4% w/w CHAPS adjusted to pH 8.5 with dilute HCI), at 4°C using a glass homogenizer and a Teflon coated steel pestle; transferred to a 5 ml acid-washed polypropylene tube, placed on ice and sonicated 3 times for 15 seconds. The sonicate was centrifuged at 12,000 rpm for 10 minutes at 4°C. Small aliquots of the supernatants were stored at -80°C until use (generally within one week). Protein concentration was determined by the method of Bradford [25] using bovine serum albumin as a standard. Fifty micrograms of lysate protein was labeled with 400 pmol of Cy3 Dye (for control homogenate sample) and Cy5 Dye (for arsenic-treated urinary bladder or liver homogenate sample). The samples containing proteins and dyes were incubated for 30 min on ice in the dark. To stop the labeling reaction, 1uL of 10 mM lysine was added followed by incubation for 10 min on ice in the dark. To each of the appropriate dye-labeled protein samples, an additional 200 ug of urinary bladderor liver unlabeled protein from control hamster sample or arsenic-treated hamster sample was added to the appropriate sample. Differentially labeled samples were combined into a single Microfuge tube (total protein 500 ug); protein was mixed with an equal volume of 2x sample buffer [2M thiourea, 7M urea, pH 3-10 pharmalyte for isoelectric focusing 2% (v/v), DTT 2% (w/v), CHAPS 4% (w/v)]; and was incubated on ice in the dark for 10 min. The combined samples containing 500 ug of total protein were mixed with rehydration buffer [CHAPS 4% (w/v), 8M urea, 13mM DTT, IPG buffer (3-10) 1% (v/v) and trace amount of bromophenol blue]. The 450 ul sample containing rehydration buffer was slowly pipetted into the slot of the ImmobilinedryStripReswelling Tray and any large bubbles were removed. The IPG strip (linear pH 3-10, 24 cm) was placed (gel side down) into the slot, covered with drystrip cover fluid (Fig. 1), and the lid of the Reswelling Tray was closed. The ImmobillineDryStrip was allowed to rehydrate at room temperature for 24 hours. First dimension Isoelectric focusing (IEF) The labeled sample was loaded using the cup loading method on universal strip holder. IEF was then carried out on EttanIPGphor II using multistep protocol (6 hr @ 500 V, 6 hr @ 1000 V, 8 hr @ 8000 V). The focused IPG strip was equilibrated in two steps (reduction and alkylation) by equilibrating the strip for 10 min first in 10 ml of 50mM Tris (pH 8.8), 6M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 0.5% (w/v) DTT, followed by another 10 min in 10 ml of 50mM Tris (pH 8.8), 6M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 4.5% (w/v) iodoacetamide to prepare it for the second dimension electrophoresis. Second dimension SDS-PAGE The equilibrated IPG strip was used for protein separation by 2D-gel electrophoresis (DIGE). The strip was sealed at the top of the acrylamide gel for the second dimension (vertical) (12.5% polyacrylamide gel, 20x25 cm x 1.5 mm) with 0.5% (w/v) agarose in SDS running buffer [25 mMTris, 192 mM Glycine, and 0.1% (w/v) SDS]. Electrophoresis was performed in an Ettan DALT six electrophoresis unit (Amersham Biosciences) at 1.5 watts per gel, until the tracking dye reached the anodic end of the gel. Image analysis and post-staining The gel then was imaged directly between glass plates on the Typhoon 9410 variable mode imager (Sunnyvale, CA, USA) using optimal excitation/emission wavelength for each DIGE fluor: Cy3 (532/580 nm) and Cy5 (633/670 nm). The DIGE images were previewed and checked with Image Quant software (GE Healthcare) where all the two separate gel images could be viewed as a single gel image. DeCyde v.5.02 was used to analyze the DIGE images as described in the Ettan DIGE User Manual (GE Healthcare). The appropriate up-/down regulated spots were filtered based on an average volume ratio of ± over 1.2 fold. After image acquisition, the gel was fixed overnight in a solution containing 40% ethanol and 10% acetic acid. The fixed gel was stained with SyproRuby (BioRad) according to the manufacturer protocol (Bio-Rad Labs., 2000 Alfred Nobel Drive, Hercules, CA 94547). Identification of proteins by MS Protein spot picking and digestion Sypro Ruby stained gels were imaged using an Investigator ProPic and HT Analyzer software, both from Genomic Solutions (Ann Arbor, MI). Protein spots of interest that matched those imaged using the DIGE Cy3/Cy5 labels were picked robotically, digested using trypsin as described previously [24] and saved for mass spectrometry identification. Liquid chromatography (LC)- MS/MS analysis LC-MS/MS analyses were carried out using a 3D quadrupole ion trap massspectrometer (ThermoFinnigan LCQ DECA XP PLUS; ThermoFinnigan, San Jose, CA) equipped with a Michrom Paradigm MS4 HPLC (MichromBiosources, Auburn, CA) and a nanospray source, or with a linear quadrupole ion trap mass spectrometer (ThermoFinnigan LTQ), also equipped with a Michrom MS4 HPLC and a nanospray source. Peptides were eluted from a 15 cm pulled tip capillary column (100 um I.D. x 360 um O.D.; 3-5 um tip opening) packed with 7 cm Vydac C18 (Vydac, Hesperia, CA) material (5 µm, 300 Å pore size), using a gradient of 0-65% solvent B (98% methanol/2% water/0.5% formic acid/0.01% triflouroacetic acid) over a 60 min period at a flow rate of 350 nL/min. The ESI positive mode spray voltage was set at 1.6 kV, and the capillary temperature was set at 200°C. Dependent data scanning was performed by the Xcalibur v 1.3 software on the LCQ DECA XP+ or v 1.4 on the LTQ [27], with a default charge of 2, an isolation width of 1.5 amu, an activation amplitude of 35%, activation time of 50 msec, and a minimal signal of 10,000 ion counts (100 ion counts on the LTQ). Global dependent data settings were as follows: reject mass width of 1.5 amu, dynamic exclusion enabled, exclusion mass width of 1.5 amu, repeat count of 1, repeat duration of a min, and exclusion duration of 5 min. Scan event series were included one full scan with mass range of 350-2000 Da, followed by 3 dependent MS/MS scans of the most intense ion. Database searching Tandem MS spectra of peptides were analyzed with Turbo SEQUEST, version 3.1 (ThermoFinnigan), a program that allows the correlation of experimental tandem MS data with theoretical spectra generated from known protein sequences. All spectra were searched against the latest version of the non redundant protein database from the National Center for Biotechnology Information (NCBI 2006; at that time, the database contained 3,783,042 entries). Statistical analysis The means and standard error were calculated. The Student's t-test was used to analyze the significance of the difference between the control and arsenite exposed hamsters. P values less than 0.05 were considered significant. The reproducibility was confirmed in separate experiments. Results Analysis of proteins expression After DIGE (Fig. 1), the gel was scanned by a Typhoon Scanner and the relative amount of protein from sample 1 (treated hamster) as compared to sample 2 (control hamster) was determined (Figs. 2, 3). A green spot indicates that the amount of protein from sodium arsenite-treated hamster sample was less than that of the control sample. A red spot indicates that the amount of protein from the sodium arsenite-treated hamster sample was greater than that of the control sample. A yellow spot indicates sodium arsenite-treated hamster and control hamster each had the same amount of that protein. Several protein spots were up-regulated (red) or down-regulated (green) in the urinary bladder samples of hamsters exposed to sodium arsenite (173 mg As/L) for 6 days as compared with the urinary bladder of controls (Fig. 2). In the case of liver, several protein spots were also over-expressed (red) or under-expressed (green) for hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days (Fig. 3). The urinary bladder samples were collected from the first and second experiments in which hamsters were exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days and the controls were given tap water. The urinary bladder samples from the 1st and 2nd experiments were run 5 times in DIGE gels on different days. The protein expression is shown in Figure 2 and Table 1. The liver samples from the 1st and 2nd experiments were also run 3 times in DIGE gels on different days. The proteins expression were shown in Figure 3 and Table 2. The volume ratio changed of the protein spots in the urinary bladder and liver of hamsters exposed to arsenite were significantly differences than that of the control hamsters (Table 1 and 2). Protein spots identified by LC-MS/MS Bladder The spots of interest were removed from the gel, digested, and their identities were determined by LC-MS/MS (Fig. 2 and Table 1). The spots 1, 2, & 3 from the gel were analyzed and were repeated for the confirmation of the results (experiments; 173 mg As/L). The proteins for the spots 1, 2, and 3 were identified as transgelin, transgelin, and glutathione S-transferase Pi, respectively (Fig. 2). Liver We also identified some of the proteins in the liver samples of hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days (Fig. 3). The spots 4, 5, & 6 from the gels were analyzed and were repeated for the confirmation of the results. The proteins for the spots 4, 5, and 6 were identified as ornithine aminotransferase, senescence marker protein 30, and fatty acid binding protein, respectively (Fig. 3) Discussion The identification and functional assignment of proteins is helpful for understanding the molecular events involved in disease. Weexposed hamsters to sodium arsenite in drinking water. Controls were given tap water. DIGE coupled with LC-MS/MS was then used to study the proteomic change in arsenite-exposed hamsters. After electrophoresis DeCyder software indicated that several protein spots were down-regulated (green) and several were up-regulated (red). Our overall results as to changes and functions of the proteins we have studied are summarized in Table 3. Bladder In the case of the urinary bladder tissue of hamsters exposed to sodium arsenite (173 mg As/L) in drinking water for 6 days, transgelin was down-regulated and GST-pi was up-regulated. This is the first evidence that transgelin is down-regulated in the bladders of animals exposed to sodium arsenite. Transgelin, which is identical to SM22 or WS3-10, is an actin cross linking/gelling protein found in fibroblasts and smooth muscle [28, 29]. It has been suggested that the loss of transgelin expression may be an important early event in tumor progression and a diagnostic marker for cancer development [30-33]. It may function as a tumor suppressor via inhibition of ARA54 (co-regulator of androgen receptor)-enhanced AR (androgen receptor) function. Loss of transgelin and its suppressor function in prostate cancer might contribute to the progression of prostate cancer [30]. Down-regulation of transgelin occurs in the urinary bladders of rats having bladder outlet obstruction [32]. Ras-dependent and Ras-independent mechanisms can cause the down regulation of transgelin in human breast and colon carcinoma cell lines and patient-derived tumorsamples [33]. Transgelin plays a role in contractility, possibly by affecting the actin content of filaments [34]. In our experiments loss of transgelin expression may be associated or preliminary to bladder cancer due to arsenic exposure. Arsenite is a carcinogen [1]. In our experiments, LC-MS/MS analysis showed that two spots (1 and 2) represent transgelin (Fig. 2 and Table 1). In human colonic neoplasms there is a loss of transgelin expression and the appearance of transgelin isoforms (31). GST-pi protein was up-regulated in the bladders of the hamsters exposed to sodium arsenite. GSTs are a large family of multifunctional enzymes involved in the phase II detoxification of foreign compounds [35]. The most abundant GSTS are the classes alpha, mu, and pi classes [36]. They participate in protection against oxidative stress [37]. GST-omega has arsenic reductase activity [38]. Over-expression of GST-pi has been found in colon cancer tissues [39]. Strong expression of GST-pi also has been found in gastric cancer [40], malignant melanoma [41], lung cancer [42], breast cancer [43] and a range of other human tumors [44]. GST-pi has been up-regulated in transitional cell carcinoma of human urinary bladder [45]. Up-regulation of glutathione – related genes and enzyme activities has been found in cultured human cells by sub lethal concentration of inorganic arsenic [46]. There is evidence that arsenic induces DNA damage via the production of ROS (reactive oxygen species) [47]. GST-pi may be over-expressed in the urinary bladder to protect cells against arsenic-induced oxidative stress. Liver In the livers of hamsters exposed to sodium arsenite, ornithine amino transferase was over-expressed, senescence marker protein 30 was under-expressed, and fatty acid binding protein was under-expressed. Ornithine amino transferase has been found in the mitochondria of many different mammalian tissues, especially liver, kidney, and small intestine [48]. Ornithine amino transferase knockdown inhuman cervical carcinoma and osteosarcoma cells by RNA interference blocks cell division and causes cell death [49]. It has been suggested that ornithine amino transferase has a role in regulating mitotic cell division and it is required for proper spindle assembly in human cancer cells [49]. Senescence marker protein-30 (SMP30) is a unique enzyme that hydrolyzes diisopropylphosphorofluoridate. SMP30, which is expressed mostly in the liver, protects cells against various injuries by stimulating membrane calcium-pump activity [50]. SMP30 acts to protect cells from apoptosis [51]. In addition it protects the liver from toxic agents [52]. The livers of SMP30 knockout mice accumulate phosphatidylethanolamine, cardiolipin, phosphatidyl-choline, phosphatidylserine, and sphingomyelin [53]. Liver fatty acid binding protein (L-FABP) also was down- regulated. Decreased liver fatty acid-binding capacity and altered liver lipid distribution hasbeen reported in mice lacking the L-FABP gene [54]. High levels of saturated, branched-chain fatty acids are deleterious to cells and animals, resulting in lipid accumulation and cytotoxicity. The expression of fatty acid binding proteins (including L-FABP) protected cells against branched-chain saturated fatty acid toxicity [55]. Limitations: we preferred to study the pronounced spots seen in DIGE gels. Other spots were visible but not as pronounced. Because of limited funds, we did not identify these others protein spots. In conclusion, urinary bladders of hamsters exposed to sodium arsenite had a decrease in the expression of transgelin and an increase in the expression of GST-pi protein. Under-expression of transgelin has been found in various cancer systems and may be associated with arsenic carcinogenicity [30-33). Inorganic arsenic exposure has resulted in bladder cancer as has been reported in the past [1]. Over-expression of GST-pi may protect cells against oxidative stress caused by arsenite. In the liver OAT was up regulated and SMP-30 and FABP were down regulated. These proteomic results may be of help to investigators studying arsenic carcinogenicity. The Superfund Basic Research Program NIEHS Grant Number ES 04940 from the National Institute of Environmental Health Sciences supported this work. Additional support for the mass spectrometry analyses was provided by grants from NIWHS ES06694, NCI CA023074 and the BIOS Institute of the University of Arizona. Acknowledgement The Author wants to dedicate this paper to the memory of his former supervisor Dr. H. VaskenAposhian who passed away in September 6, 2019. He was an emeritus professor of the Department of Molecular and Cellular Biology at the University of Arizona. 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AbstractThe roughness property of rocks is significant in engineering studies due to their mechanical and hydraulic performance and the possibility of quantifying flow velocity and predicting the performance of wells and rock mass structures. However, the study of roughness in rocks is usually carried out through 2D linear measurements (through mechanical profilometer equipment), obtaining a coefficient that may not represent the entire rock surface. Thus, based on the hypothesis that it is possible to quantify the roughness coefficient in rock plugs reconstructed three-dimensionally by the computer vision technique, this research aims to an alternative method to determine the roughness coefficient in rock plugs. The point cloud generated from the 3D model of the photogrammetry process was used to measure the distance between each point and a calculated fit plane over the entire rock surface. The roughness was quantified using roughness parameters ($$R_a$$ R a ) calculated in hierarchically organized regions. In this hierarchical division, the greater the quantity of division analyzed, the greater the detail of the roughness. The main results show that obtaining the roughness coefficient over the entire surface of the three-dimensional model has peculiarities that would not be observed in the two-dimensional reading. From the 2D measurements, mean roughness values ($$R_a$$ R a ) of $$0.35\,\upmu \hbox {m}$$ 0.35 μ m and $$0.235\,\upmu \hbox {m}$$ 0.235 μ m were obtained for samples 1 and 2, respectively. By the same method, the results of the $$R_a$$ R a coefficient applied three-dimensionally over the entire rocky surface were at most $$0.165\,\upmu \hbox {m}$$ 0.165 μ m and $$0.166\,\upmu \hbox {m}$$ 0.166 μ m , respectively, showing the difference in values along the surface and the importance of this approach.

Abstract Background Previous studies show contradictory results of the effect of chronic total occlusions (CTO) revascularization on LVEF. Deformation parameters based on speckle tracking (2D-ST) echo allows the evaluation of the systolic regional function. Purpose To examine the usefulness of 2D-ST to detect changes in regional ventricular mechanics in a consecutive cohort of patients with successful CTO percutaneous revascularization. Methods Prospective study that included 13 consecutive patients (72±8 y, 85% men). Revascularization was justified based on the evidence of viability of the underlying territory. 2D-ST was performed before, one and three months after of the procedure. Based on 16 segments model (n: 208), these were classified in two groups (dependent/non-dependent) according to their relationship with the treated CTO. Strain rate (Sr) and Strain (S) of the three myocardial layers were measured. Results 208 segments analyzed. 62 (30%) perfusion dependent, (mean 5 segments/patient), non-dependent: 146 (70%). During follow-up (median: 3.3 m), there were no significant differences with respect to LVEF (56±6 vs. 59±6, p: 0.2). Although non-dependent segments showed no significant changes of deformation parameters after the procedure, dependent segments showed an increase (Δ) of both the S and Sr, being statistically significant the Δ of the three layers S at 3 months vs. non-dependent. Table 1 PRE POST p Δ Non-Dependent segments (N: 146) S-epicardial −16.6±4.9 −16.0±4.0 0.06 0.6±4.0 S-mesocardiac −17.4±4.8 −16.8±4.2 0.08 0.6±3.9 S-endocardial −18.1±5.1 −17.7±4.9 0.337 0.4±4.5 Sr −1.15±0.4 −1.09±0.4 0.080 0.07±0.47 Dependent segments (N: 62) S-epicardial −13.7±5.8 −16.3±4.5 <0.001 −2.6±5.2* S-mesocardiac −14.7±6.1 −17.0±4.5 <0.05 −2.3±5.2* S-endocardial −15.8±6.7 −17.9±4.9 <0.05 −2.1±5.6* Sr −0.98±0.4 −1.03±0.4 0.353 −0.05±0.42 *p<0.05 ΔNon-dependents vs. ΔDependents. S (%) improvement post RCA CTO treatment Conclusions This study shows that CTO treatment improves regional myocardial function in LV segments dependent on CTO at three months of follow-up, without changes in LVEF. 2D-ST allows to examine the effect of flow restoration, providing new information on the potential short-term benefits of this strategy

Abstract INTRODUCTION An early evaluation of patients with non-ST elevation acute coronary syndrome patients (NSTE-ACS) is important to choose the appropriate treatment strategy. In this setting of patients, conventional echocardiographic assessment may reveal normal myocardial kinesis in 25 to 76% of cases. Global and territorial longitudinal strain (GLS and TLS, respectively) may be an early and accurate non-invasive tool for prediction of multivessel CAD in patients with NSTE-ACS. AIM To evaluate the ability of TLS to predict culprit lesions in patients with NSTE-ACS. Material and method We studied 183 patients diagnosed with NSTE-ACS, in our Institution over 2 years of time. Conventional echocardiography and 2D speckle tracking echocardiography (STE) imaging were performed by two experienced echocardiographers, who were blinded to patient characteristics. The TLS was identified as the mean value of the segments'strain as respect to each vessel territory.Coronary angiography was performed in all patients. Significant CAD (luminal stenosis more than 70% in a major epicardial coronary vessel) and culprit lesion were identified and threated by PTCA when appropriate. Results A significant difference between mono- and tri-vessel CAD in the variation of WMSI has been demonstrated. There was a statistically significant difference between both 3-vessels vs 1-vessel disease and 2-vessels vs 1-vessel disease in changing of TLS-LAD, TLS-RCA and TLS-Cx values (p-value &lt;0.001). There was a significant difference between 3-vessels vs 2-vessels disease for TLS-RCA values. There was a statistically significant difference for WMSI-LAD, WMSI-CX and WMSI-RCA values whether the respective artery was involved or not. Variations of TLS were statistically significant both when the territorial tributary artery was involved and also if the artery represented the culprit lesion (p-value TLS-LAD &lt;0.001, TLS-LAD culprit &lt;0.001, TLS-CX&lt;0.001, TLS-cx culprit &lt;0.001, TLS-RCA &lt;0.001, p-value TLS-RCA culprit 0.022). A regression model was performed comparing the variation of WMSI as respect to the variation of WMSI+TLS in the territory of culprit lesions. For WMSI- LAD the OR was 0.94 and for TLS-LAD the OR was 1.19 and the p-value of the addition was 0.001. The OR of WMSI-CX was 1.76 and for TLS-CX the OR was 1.40 and the p- value of the addition was 0.001. The OR of WMSI- RCA was 0.71 and for TLS- RCA the OR was 1.17, the p- value of the addition was 0.019. Conclusion TLS allows an accurate identification of the culprit lesion in patients presenting with NSTE-ACS. TLS can be considered as part of routine echocardiography on top of WMSI in early evaluation for a better clinical assessment in this subset of patients. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Policlinico di Bari

This paper makes a comparative analysis between the Dipole-dipole, Wenner and Schlumberger arrays through electric resistivity tomography (ERT), about the sensitivity and resolution in the spatial characterization of infiltrating pollutants in septic tank and cone of depression in supply well, both contained in unconfined aquifer. Data acquisition consisted of electrical resistivity rea-dings using five parallel lines with 105m long, electrode spacing and lines of 5m. The data from each line were subjected to 2D inversion and then interpolated to generate 3D blocks, which were extracted from a fixed resistivity isosurface (620 W.m), which enabled the modeling of volumes related to the flow structures. The results for the Dipole-dipole array allowed the modeling of the cylindrical structure associated to the supply well, and an isosurface deformation associated to the septic tank, but did not allow the modeling of the plume. The data for the Schlumberger array cannot allow for the cone recognition, but resulted in a drop shape model, associated to the septic tank and similar to a contamination plume. The Wenner array resulted in a model with structu-re in elongated keel format associated to the cone of depression, and another that is similar to the drop shape model, also associated to the septic tank. The comparative analysis shows that the Dipole-dipole array is recommended in works of modeling vertically integrated three-dimensional structures of high resistivity in the saturated zone. The Wenner and Schlumberger arrays are recommended for modeling vertically integrated three-dimensional structures of low resistivity in unsaturated zone, with emphasis on the Schlumberger array.

Abstract Background and purpose Previous studies have shown that the role of global longitudinal 2DS in diagnosing and detecting coronary artery disease is modest. However, data on the sensitivity of the regional 2DS assessment is lacking (1–6). The aim of this study was to determine whether in echocardiographic evaluation of myocardial ischemia, regional 2D strain (2DS) assessment is more sensitive and specific in detecting ischemia due to critical stenosis of one or more coronary arteries in comparison with current standard of visual assessment of regional wall motion abnormalities (RWMA). Material and methods This retrospective study included 123 patients admitted through the Emergency Department across 36 months with the diagnosis of NSTE-ACS without previously known CAD or LBBB who underwent coronary angiography within the first 24 hours and had an echocardiogram with RWMA analysis done prior to procedure. 2DS analysis of regional longitudinal peak systolic strain (LPSS) according to the 18-segment model was performed by four clinicians, blinded to patient's clinical data. Results A significant correlation was found between individual parameters of LPSS and significant stenosis of all three major coronary vessels (&gt;70% narrowing on coronary angiography, Table 1). Lower regional LPSS correlated well anatomically with the supplying arteries; for example, patients with LAD stenosis had significantly lower regional LPSS in all apical segments, lateral, anteroseptal and anterior mid segments and anterior basal segment. Similar patterns were seen in the LCx and RCA regions. Three graphs show the sensitivity and specificity of the most sensitive segments for each artery. The diagnostic accuracy of endocardial and epicardial regional LPSS was similar to the average regional LPSS. Conclusion We have shown that regional 2DS could be a good predictor of the presence and localization of coronary stenosis for each of the three main coronary arteries but further studies are needed to confirm this assumption. Funding Acknowledgement Type of funding sources: None.

Summary Core measurements are used for rock classification and improved formation evaluation in both cored and noncored wells. However, the acquisition of such measurements is time-consuming, delaying rock classification efforts for weeks or months after core retrieval. On the other hand, well-log-based rock classification fails to account for rapid spatial variation of rock fabric encountered in heterogeneous and anisotropic formations due to the vertical resolution of conventional well logs. Interpretation of computed tomography (CT) scan data has been identified as an attractive and high-resolution alternative for enhancing rock texture detection, classification, and formation evaluation. Acquisition of CT scan data is accomplished shortly after core retrieval, providing high-resolution data for use in petrophysical workflows in relatively short periods of time. Typically, CT scan data are used as two-dimensional (2D) cross-sectional images, which is not suitable for quantification of three-dimensional (3D) rock fabric variation, which can increase the uncertainty in rock classification using image-based rock-fabric-related features. The methods documented in this paper aim to quantify rock-fabric-related features from whole-core 3D CT scan image stacks and slabbed whole-core photos using image analysis techniques. These quantitative features are integrated with conventional well logs and routine core analysis (RCA) data for fast and accurate detection of petrophysical rock classes. The detected rock classes are then used for improved formation evaluation. To achieve the objectives, we conducted a conventional formation evaluation. Then, we developed a workflow for preprocessing of whole-core 3D CT-scan image stacks and slabbed whole-core photos. Subsequently, we used image analysis techniques and tailor-made algorithms for the extraction of image-based rock-fabric-related features. Then, we used the image-based rock-fabric-related features for image-based rock classification. We used the detected rock classes for the development of class-based rock physics models to improve permeability estimates. Finally, we compared the detected image-based rock classes against other rock classification techniques and against image-based rock classes derived using 2D CT scan images. We applied the proposed workflow to a data set from a siliciclastic sequence with rapid spatial variations in rock fabric and pore structure. We compared the results against expert-derived lithofacies, conventional rock classification techniques, and rock classes derived using 2D CT scan images. The use of whole-core 3D CT scan image-stacks-based rock-fabric-related features accurately captured changes in the rock properties within the evaluated depth interval. Image-based rock classes derived by integration of whole-core 3D CT scan image-stacks-based and slabbed whole-core photos-based rock-fabric-related features agreed with expert-derived lithofacies. Furthermore, the use of the image-based rock classes in the formation evaluation of the evaluated depth intervals improved estimates of petrophysical properties such as permeability compared to conventional formation-based permeability estimates. A unique contribution of the proposed workflow compared to the previously documented rock classification methods is the derivation of quantitative features from whole-core 3D CT scan image stacks, which are conventionally used qualitatively. Furthermore, image-based rock-fabric-related features extracted from whole-core 3D CT scan image stacks can be used as a tool for quick assessment of recovered whole core for tasks such as locating best zones for extraction of core plugs for core analysis and flagging depth intervals showing abnormal well-log responses.

Introduction: High serum levels of lipoprotein (a) (Lp(a)) are associated with adverse outcomes after acute myocardial infarction (AMI). Lp(a) exerts its atherogenic and thrombogenic properties, which suggests greater lipid component of plaque and thrombi at the culprit lesion of AMI with elevated Lp(a). The risk of coronary artery disease is increased with a threshold serum Lp(a) level of 25 mg/dl. Purpose: We sought to evaluate impact of Lp(a) on myocardial viability and left ventricular function after AMI. Methods: The study included 156 patients (66±10.7 yrs, mean±sd, men 122, LAD/LCX/RCA 80/16/60) with first AMI within 24 hours from the onset who underwent emergent PCI. Serum Lp(a) was measured at admission. Serum CPK values were measured at admission and at intervals of 4 hours. Peak CPK value was determined. Within 2 weeks, Tl/BMIPP SPECT was performed to measure perfusion-metabolism mismatch score, an indicator of viable myocardium, from each total defect score of Tl/BMIPP using 17-segment model and semiquantitative visual score (0:normal, to 4:no uptake). Left ventricular ejection fraction (LVEF) was measured by two-dimensional echocardiography (2D trace) within 1 week (baseline LVEF) and at 6 months to 1 year (follow-up LVEF). Results: The study patients were divided into the group with serum Lp(a)≧25mg/dl at admission (n=42, Lp(a) 44.0±19.4mg/dl) and the group with Lp(a)<25 (n=114, Lp(a) 10.8±6.5). There were no significant differences between the two groups with respect to age, gender, peak CPK (2732±2060 IU/L (Lp(a)≧25), 2971±2254 (Lp(a)<25)). The mismatch score was significantly lower in Lp(a)≧25 group than in Lp(a)<25 (4.4±3.3 vs 5.9±3.9, p<0.05, respectively). Δ%LVEF (100*(follow-up LVEF - baseline LVEF)/ baseline LVEF) was significantly lower in Lp(a)≧25 than Lp(a)<25 (-2.0±11.0 vs 7.4±13.0, p<0.05, respectively). Conclusions: Elevated Lp(a) was associated with less myocardial viability and less LVEF recovery after AMI.

Background: Our previous studies show that intracerebroventricular (ICV) delivery of Insulin-like growth factor (IGF)-1 reduces MCAo-induced infarction in middle-aged female rats. Analysis of ischemic tissue at 4h showed that IGF1 treatment significantly reduced the expression of a group of miRNAs. To determine if inhibiting these miRNAs could replicate IGF-1-mediated neuroprotection, we examined the effects of antagomirs to miR-33a (A-mir33a) and miR-92b (A-mir92b) in in vivo and in vitro models of ischemia. Methods: Middle-aged (12 mo) acyclic female rats were subject to intraluminal MCAo for 90 min followed by reperfusion. Animals received a single tail vein injection of A-miR-33a, A-miR-92b, A-miR-33a+A-miR-92b or scrambled oligos (7ug/kg bwt) after 4h of reperfusion. We evaluated infarct volume (TTC staining) at 2d post stroke and behavioral recovery using neurological scores and adhesive removal test (ART). In parallel, human brain endothelial cell cultures were exposed to oxygen-glucose deprivation (OGD) in the presence of A-mir-33a, A-mir-92b or scrambled oligos. The integrity of the cell monolayer was assessed by tomato-lectin staining and cell death was assayed by LDH. Results: Single injection of A-mir92b during the acute stroke phase reduced infarct volume (p<0.04) and improved sensorimotor behavior (p<0.04) while A-mir33a treated animals were not different from scrambled control. However, the combination of A-mir-33a+A-mir-92b resulted in significantly smaller infarct volumes (p<0.04), less motor impairment (neurological score; p<0.02) and sensorimotor deficits (ART; p<0.05). In vitro studies showed that neither of the antagomirs reduced media LDH under OGD. As reported previously, OGD caused cell retraction and A-mir33a notably preserved the cell geometry (continuous lectin staining) to maintain the monolayer integrity and was similar to the normoxic control. Conclusions: IGF-1 regulated microRNAs appear to modulate specific aspects of stroke recovery. IV delivery of a combination of miRNA inhibitors may replicate the neuroprotective actions of IGF-1, thus providing a safer alternate to ICV delivery of IGF-1. Supported by R56 NS074895 and RF1 AG04218906