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Background: It would be ideal for a non-hyperaemic index to predict fractional flow reserve (FFR) more accurately, given FFR's extensive validation in a multitude of clinical settings. Aims: The aim of this study was to derive a novel non-hyperaemic algorithm based on deep learning and to validate it in an internal validation cohort against FFR. Methods: The ARTIST study is a post hoc analysis of three previously published studies. In a derivation cohort (random 80% sample of the total cohort) a deep neural network was trained (deep learning) with paired examples of resting coronary pressure curves and their FFR values. The resulting algorithm was validated against unseen resting pressure curves from a random 20% sample of the total cohort. The primary endpoint was diagnostic accuracy of the deep learning-derived algorithms against binary FFR ≤0.8. To reduce the variance in the precision, we used a fivefold cross-validation procedure. Results: A total of 1,666 patients with 1,718 coronary lesions and 2,928 coronary pressure tracings were included. The diagnostic accuracy of our convolutional neural network (CNN) and recurrent neural networks (RNN) against binary FFR ≤0.80 was 79.6±1.9% and 77.6±2.3%, respectively. There was no statistically significant difference between the accuracy of our neural networks to predict binary FFR and the most accurate non-hyperaemic pressure ratio (NHPR). Conclusions: Compared to standard derivation of resting pressure ratios, we did not find a significant improvement in FFR prediction when resting data are analysed using artificial intelligence approaches. Our findings strongly suggest that a larger class of hidden information within resting pressure traces is not the main cause of the known disagreement between resting indices and FFR. Therefore, if clinicians want to use FFR for clinical decision making, hyperaemia induction should remain the standard practice.

Background Acute myeloid leukemia (AML) with the t(8;21)(q22;q22) chromosomal translocation is among the most common subtypes of AML and produces the AML1-ETO (RUNX1-ETO, RUNX1-RUNX1T1) oncogenic fusion gene. AML1-ETO functions as an aberrant transcription factor which plays a key role in blocking normal hematopoiesis. Thus, the expression of AML1-ETO is critical to t(8;21) AML leukemogenesis and maintenance. Post-transcriptional regulation of gene expression is often mediated through interactions between trans-factors and cis-elements within transcript 3′-untranslated regions (UTR). AML1-ETO uses the 3′UTR of the ETO gene, which is not normally expressed in hematopoietic cells. Therefore, the mechanisms regulating AML1-ETO expression via the 3’UTR are attractive therapeutic targets. Methods We used RNA-sequencing of t(8;21) patients and cell lines to examine the 3′UTR isoforms used by AML1-ETO transcripts. Using luciferase assay approaches, we test the relative contribution of 3′UTR cis elements to AML1-ETO expression. We further use let-7b microRNA mimics and anti-let-7b sponges for functional studies of t(8;21) AML cell lines. Results In this study, we examine the regulation of AML1-ETO via the 3’UTR. We demonstrate that AML1-ETO transcripts primarily use a 3.7 kb isoform of the ETO 3′UTR in both t(8;21) patients and cell lines. We identify a negative regulatory element within the AML1-ETO 3′UTR. We further demonstrate that the let-7b microRNA directly represses AML1-ETO through this site. Finally, we find that let-7b inhibits the proliferation of t(8;21) AML cell lines, rescues expression of AML1-ETO target genes, and promotes differentiation. Conclusions AML1-ETO is post-transcriptionally regulated by let-7b, which contributes to the leukemic phenotype of t(8;21) AML and may be important for t(8;21) leukemogenesis and maintenance.

Objectives We sought to develop an automatic method for correcting common errors in phasic pressure tracings for physiology‐guided interventions on coronary and valvular stenosis. Background Effective coronary and valvular interventions rely on accurate hemodynamic assessment. Phasic (subcycle) indexes remain intrinsic to valvular stenosis and are emerging for coronary stenosis. Errors, corrections, and clinical implications of fluid‐filled catheter phasic pressure assessments have not been assessed in the current era of ubiquitous, high‐fidelity pressure wire sensors. Methods We recruited patients undergoing invasive coronary physiology assessment. Phasic aortic pressure signals were recorded simultaneously using a fluid‐filled guide catheter and 0.014″ pressure wire before and after standard calibration as well as after pullback. We included additional subjects undergoing hemodynamic assessment before and after transcatheter aortic valve implantation. Using the pressure wire as reference standard, we developed an automatic algorithm to match phasic pressures. Results Removing pressure offset and temporal shift produced the largest improvements in root mean square (RMS) error between catheter and pressure wire signals. However, further optimization

Heterozygous deletions within chromosome 20q, or del(20q), are frequent cytogenetic abnormalities detected in hematologic malignancies. To date, identification of genes in the del(20q) common deleted region that contribute to disease development have remained elusive. Through assessment of patient gene expression, we have identified STK4 (encoding Hippo kinase MST1) as a 20q gene that is downregulated below haploinsufficient amounts in myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN). Hematopoietic-specific gene inactivation in mice revealed Hippo kinase loss to induce splenomegaly, thrombocytopenia, megakaryocytic dysplasia, and a propensity for chronic granulocytosis; phenotypes that closely resemble those observed in patients harboring del(20q). In a JAK2-V617F model, heterozygous Hippo kinase inactivation led to accelerated development of lethal myelofibrosis, recapitulating adverse MPN disease progression and revealing a novel genetic interaction between these 2 molecular events. Quantitative serum protein profiling showed that myelofibrotic transformation in mice was associated with cooperative effects of JAK2-V617F and Hippo kinase inactivation on innate immune-associated proinflammatory cytokine production, including IL-1β and IL-6. Mechanistically, MST1 interacted with IRAK1, and shRNA-mediated knockdown was sufficient to increase IRAK1-dependent innate immune activation of NF-κB in human myeloid cells. Consistent with this, treatment with a small molecule IRAK1/4 inhibitor rescued the aberrantly elevated IL-1β production in the JAK2-V617F MPN model. This study identified Hippo kinase MST1 (STK4) as having a central role in the biology of del(20q)-associated hematologic malignancies and revealed a novel molecular basis of adverse MPN progression that may be therapeutically exploitable via IRAK1 inhibition.

Recent genome analysis of human prostate cancers demonstrated that both AR gene amplification and TP53 mutation are among the most frequently observed alterations in advanced prostate cancer. However, the biological role of these dual genetic alterations in prostate tumorigenesis is largely unknown. In addition, there are no biologically relevant models that can be used to assess the molecular mechanisms for these genetic abnormalities. Here, we report a novel mouse model, in which elevated transgenic AR expression and Trp53 deletion occur simultaneously in mouse prostatic epithelium to mimic human prostate cancer cells. These compound mice developed an earlier onset of high-grade prostatic intraepithelial neoplasia and accelerated prostate tumors in comparison with mice harboring only the AR transgene. Histological analysis showed prostatic sarcomatoid and basaloid carcinomas with massive squamous differentiation in the above compound mice. RNA-sequencing analyses identified a robust enrichment of the signature genes for human prostatic basal cell carcinomas in the above prostate tumors. Master regulator analysis revealed SOX2 as a transcriptional regulator in prostatic basal cell tumors. Elevated expression of SOX2 and its downstream target genes were detected in prostatic tumors of the compound mice. Chromatin immunoprecipitation analyses implicate a coregulatory role of AR and SOX2 in the expression of prostatic basal cell signature genes. Our data demonstrate a critical role of SOX2 in prostate tumorigenesis and provide mechanistic insight into prostate tumor aggressiveness and progression mediated by aberrant AR and p53 signaling pathways.

Cancer vaccines are promising treatments to prevent relapse after chemotherapy in acute myeloid leukemia (AML) patients, particularly for those who cannot tolerate intensive consolidation therapies. Here, we report the development of an AML cell membrane-coated nanoparticle (AMCNP) vaccine platform, in which immune-stimulatory adjuvant-loaded nanoparticles are coated with leukemic cell membrane material. This AMCNP vaccination strategy stimulates leukemia-specific immune responses by co-delivering membrane-associated antigens along with adjuvants to antigen-presenting cells. To demonstrate that this AMCNP vaccine enhances leukemia-specific antigen presentation and T cell responses, we modified a murine AML cell line to express membrane-bound chicken ovalbumin as a model antigen. AMCNPs were efficiently acquired by antigen-presenting cells in vitro and in vivo and stimulated antigen cross-presentation. Vaccination with AMCNPs significantly enhanced antigen-specific T cell expansion and effector function compared with control vaccines. Prophylactic vaccination with AMCNPs enhanced cellular immunity and protected against AML challenge. Moreover, in an AML post-remission vaccination model, AMCNP vaccination significantly enhanced survival in comparison to vaccination with whole leukemia cell lysates. Collectively, AMCNPs retained AML-specific antigens, elicited enhanced antigen-specific immune responses, and provided therapeutic benefit against AML challenge.

In 2011, TRB Geometric Design Committee (AFB10) drafted a research needs statement to complete an updated synthesis of the practice to the 1979 Glare Screen Guidelines, Synthesis of Highway Practice. This study completes the efforts of the 2011 research needs statement on glare screens and their use in road design. The research identifies the uses and types of glare screen in road design, examines national-level glare screen guidance, and provides a summary of the findings from 30 transportation agencies in the United States.

Viral gain‐of‐function mutations frequently evolve during laboratory experiments. Whether the specific mutations that evolve in the lab also evolve in nature and whether they have the same impact on evolution in the real world is unknown. We studied a model virus, bacteriophage λ, that repeatedly evolves to exploit a new host receptor under typical laboratory conditions. Here, we demonstrate that two residues of λ’s J protein are required for the new function. In natural λ variants, these amino acid sites are highly diverse and evolve at high rates. Insertions and deletions at these locations are associated with phylogenetic patterns indicative of ecological diversification. Our results show that viral evolution in the laboratory mirrors that in nature and that laboratory experiments can be coupled with protein sequence analyses to identify the causes of viral evolution in the real world. Furthermore, our results provide evidence for widespread host‐shift evolution in lambdoid viruses.

Nongenetic variation drives viral evolution Bacteriophage λ is a virus that infects bacteria by exploiting various membrane proteins in a well-characterized manner. Petrie et al. show how the evolution of variable folding conformations of isogenic proteins, which do not differ in their genetic sequences, contributed to λ's ability to exploit an additional host receptor for infection. Because the protein can take on two shapes, this genotype can have two phenotypes. Natural selection may thus be able to act on this nongenetic heterogeneity to connect phenotypic heterogeneity, evolvability, and protein stability. Science , this issue p. 1542

This paper describes the design of an extended Kalman filter to obtain the precise relative position of two aircraft in a refueling maneuver while operating in GPS-denied environments. The extended...

Background: Potentially some patients have symptoms that arise from their low-gradient aortic valve stenosis (AS). Comprehensive valve physiology with dobutamine stress remains incompletely characterized in this population. Methods: A cohort of 18 subjects with low-gradient AS underwent graded dobutamine infusion with invasive assessment using 0.014” pressure wires. A subset of 4 subjects received thermodilution cardiac output assessment at each stage. Results: Peak dobutamine hemodynamics could not be predicted from clinical or baseline parameters, reflecting statistically the physiologic heterogeneity of the measured pressure loss versus flow curves. While 0 subjects had a baseline aortic/left ventricular pressure ratio during ejection Conclusion: For low-gradient AS, the hemodynamic changes from resting to peak dobutamine conditions cannot be predicted in advance due to pressure loss versus flow curve heterogeneity. A sizable minority of low-gradient AS reaches a severity during dobutamine stress equivalent to patients undergoing TAVI for established benefit. Whether this subset receives similar clinical advantage remains an unproven but natural hypothesis raised by our study.

To prevent type A aortic dissection (AD), patients with aortic diameters ≥ 5.5 cm are recommended to undergo elective aortic repair, yet AD may occur at smaller sizes. This study aimed to identify results of aortic imaging prior to AD. In a retrospective cohort of 200 patients with AD at our

Emerging evidence has shown that the hepatocyte growth factor (HGF) and its receptor, MET proto-oncogene, receptor tyrosine kinase (MET), promote cell proliferation, motility, morphogenesis, and angiogenesis. Whereas up-regulation of MET expression has been observed in aggressive and metastatic prostate cancer, a clear understanding of MET function in prostate tumorigenesis remains elusive. Here, we developed a conditional Met transgenic mouse strain, H11(Met/+):PB-Cre4, to mimic human prostate cancer cells with increased MET expression in the prostatic luminal epithelium. We found that these mice develop prostatic intraepithelial neoplasia after HGF administration. To further assess the biological role of MET in prostate cancer progression, we bred H11(Met/+)/Pten(LoxP/LoxP):PBCre4 compound mice, in which transgenic Met expression and deletion of the tumor suppressor gene Pten occurred simultaneously only in prostatic epithelial cells. These compound mice exhibited accelerated prostate tumor formation and invasion as well as increased metastasis compared with Pten(LoxP/LoxP):PB-Cre4 mice. Moreover, prostatic sarcomatoid carcinomas and lesions resembling the epithelial-to-mesenchymal transition developed in tumor lesions of the compound mice. RNA-Seq and qRT-PCR analyses revealed a robust enrichment of known tumor progression and metastasis-promoting genes in samples isolated from H11(Met/+)/Pten(LoxP/LoxP):PB-Cre4 compound mice compared with those from Pten(LoxP/LoxP):PB-Cre4 littermate controls. HGF-induced cell proliferation and migration also increased in mouse embryonic fibroblasts (MEFs) from animals with both Met transgene expression and Pten deletion compared with Pten-null MEFs. The results from these newly developed mouse models indicate a role for MET in hastening tumorigenesis and metastasis when combined with the loss of tumor suppressors.

Aims: Echocardiography and tomographic imaging have documented dynamic changes in aortic stenosis (AS) geometry and severity during both the cardiac cycle and stress-induced increases in cardiac output. However, corresponding pressure gradient vs. flow relationships have not been described. Methods and results: We recruited 16 routine transcatheter aortic valve implantations (TAVI's) for graded dobutamine infusions both before and after implantation; 0.014″ pressure wires in the aorta and left ventricle (LV) continuously measured the transvalvular pressure gradient (ΔP) while a pulmonary artery catheter regularly assessed cardiac output by thermodilution. Before TAVI, ΔP did not display a consistent relationship with transvalvular flow (Q). Neither linear resistor (median R2 0.16) nor quadratic orifice (median R2

Objectives\ud \ud This study classified and quantified the variation in fractional flow reserve (FFR) due to fluctuations in systemic and coronary hemodynamics during intravenous adenosine infusion.\ud \ud Background\ud \ud Although FFR has become a key invasive tool to guide treatment, questions remain regarding its repeatability and stability during intravenous adenosine infusion because of systemic effects that can alter driving pressure and heart rate.\ud \ud Methods\ud \ud We reanalyzed data from the VERIFY (VERification of Instantaneous Wave-Free Ratio and Fractional Flow Reserve for the Assessment of Coronary Artery Stenosis Severity in EverydaY Practice) study, which enrolled consecutive patients who were infused with intravenous adenosine at 140 μg/kg/min and measured FFR twice. Raw phasic pressure tracings from the aorta (Pa) and distal coronary artery (Pd) were transformed into moving averages of Pd/Pa. Visual analysis grouped Pd/Pa curves into patterns of similar response. Quantitative analysis of the Pd/Pa curves identified the “smart minimum” FFR using a novel algorithm, which was compared with human core laboratory analysis.\ud \ud Results\ud \ud A total of 190 complete pairs came from 206 patients after exclusions. Visual analysis revealed 3 Pd/Pa patterns: “classic” (sigmoid) in 57%, “humped” (sigmoid with superimposed bumps of varying height) in 39%, and “unusual” (no pattern) in 4%. The Pd/Pa pattern repeated itself in 67% of patient pairs. Despite variability of Pd/Pa during the hyperemic period, the “smart minimum” FFR demonstrated excellent repeatability (bias −0.001, SD 0.018, paired p = 0.93, r2 = 98.2%, coefficient of variation = 2.5%). Our algorithm produced FFR values not significantly different from human core laboratory analysis (paired p = 0.43 vs. VERIFY; p = 0.34 vs. RESOLVE).\ud \ud Conclusions\ud \ud Intravenous adenosine produced 3 general patterns of Pd/Pa response, with associated variability in aortic and coronary pressure and heart rate during the hyperemic period. Nevertheless, FFR – when chosen appropriately – proved to be a highly reproducible value. Therefore, operators can confidently select the “smart minimum” FFR for patient care. Our results suggest that this selection process can be automated, yet comparable to human core laboratory analysis.

Viral gain-of-function mutations are commonly observed in the laboratory; however, it is unknown whether those mutations also evolve in nature. We identify two key residues in the host recognition protein of bacteriophage λ that are necessary to exploit a new receptor; both residues repeatedly evolved among homologs from environmental samples. Our results provide evidence for widespread host-shift evolution in nature and a proof of concept for integrating experiments with genomic epidemiology.

Potentially some patients have symptoms that arise from their low-gradient aortic valve stenosis (AS) but are not selected for transcatheter aortic valve replacement (TAVR) treatment yet. Comprehensive valve physiology with dobutamine stress remains incompletely characterized in this population.

ZMIZ2, also named ZIMP7, is a protein inhibitor of activated STAT (PIAS)-like protein and a transcriptional coactivator. In this study, we investigated the interaction between ZMIZ2 and β-catenin, a key regulator of the Wnt signaling pathway. We demonstrated that the expression of exogenous ZMIZ2 augments TCF (T cell factor) and β-catenin-mediated transcription. In contrast, shRNA knockdown of ZMIZ2 expression specifically represses the enhancement of TCF/β-catenin-mediated transcription by ZMIZ2. Using Wnt3a-conditioned medium, we demonstrated that ZMIZ2 can enhance Wnt ligand-induced TCF/β-catenin-mediated transcription. We also showed a promotional role of ZMIZ2 in enhancing β-catenin downstream target gene expression in human cells and in Zmiz2 null (Zmiz2(-/-)) mouse embryonic fibroblasts (MEFs). The regulatory role of Zmiz2 in Wnt-induced TCF/β-catenin-mediated transcription can be restored in Zmiz2(-/-) MEFs that were infected with adenoviral expression vectors for Zmiz2. Moreover, enhancement of Zmiz2 on TCF/β-catenin-mediated transcription was further demonstrated in Zmiz2 knockout and Axin2 reporter compound mice. Furthermore, the protein-protein interaction between ZMIZ2 and β-catenin was identified by co-immunoprecipitation and in vitro protein pulldown assays. We also observed recruitment of endogenous ZMIZ2 onto the promoter region of the Axin 2 gene, a β-catenin downstream target promoter, in a Wnt ligand-inducible manner. Finally, a promotional role of ZMIZ2 on cell growth was demonstrated in human cell lines and Zmiz2 knockout MEFs. Our findings demonstrate a novel interaction between ZMIZ2 and β-catenin and elucidate a novel mechanism for PIAS-like proteins in regulating Wnt signaling pathways.

Using an Lzts2 knock-out mouse model, we characterized the biological role of Lzts2 in tumorigenesis. Both heterozygous and homozygous deletion of the Lzts2-targeted allele in mice shows an increased incidence in spontaneous tumor development, although Lzts2 homozygous knock-out mice show significantly higher incidences than heterozygous mice. Treatment of Lzts2-deficient mice with a carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine, increases the susceptibility to N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder carcinoma development. Examination of human prostate cancer tissue specimens shows a reduction of LZTS2 protein expression in prostate cancer cells. Further analyses of mouse embryonic fibroblasts isolated from Lzts2 knock-out embryos show that loss of Lzts2 enhances cell growth. These data provide the first line of evidence demonstrating that deletion of Lzts2 increases susceptibility to spontaneous and carcinogen-induced tumor development.

The androgen signaling pathway, mediated through the androgen receptor (AR), is critical in prostate tumorigenesis. However, the precise role of AR in prostate cancer development and progression still remains largely unknown. Specifically, it is unclear whether overexpression of AR is sufficient to induce prostate tumor formation in vivo. Here, we inserted the human AR transgene with a LoxP-stop-loxP (LSL) cassette into the mouse ROSA26 locus, permitting "conditionally" activated AR transgene expression through Cre recombinase-mediated removal of the LSL cassette. By crossing this AR floxed strain with Osr1-Cre (odd skipped related) mice, in which the Osr1 promoter activates at embryonic day 11.5 in urogenital sinus epithelium, we generated a conditional transgenic line, R26hAR(loxP):Osr1-Cre+. Expression of transgenic AR was detected in both prostatic luminal and basal epithelial cells and is resistant to castration. Approximately one-half of the transgenic mice displayed mouse prostatic intraepithelial neoplasia (mPIN) lesions. Intriguingly, four mice (10%) developed prostatic adenocarcinomas, with two demonstrating invasive diseases. Positive immunostaining of transgenic AR protein was observed in the majority of atypical and tumor cells in the mPIN and prostatic adenocarcinomas, providing a link between transgenic AR expression and oncogenic transformation. An increase in Ki67-positive cells appeared in all mPIN and prostatic adenocarcinoma lesions of the mice. Thus, we demonstrated for the first time that conditional activation of transgenic AR expression by Osr1 promoter induces prostate tumor formation in mice. This new AR transgenic mouse line mimics the human disease and can be used for study of prostate tumorigenesis and drug development.

Acute Myeloid Leukemia (AML) is the most common acute leukemia in adults and has a five-year survival rate under 50%. Most patients will relapse even after complete remission is achieved through standard chemotherapy. Thus, one barrier in current AML therapy is how to target the minimal residual disease during remission. Recent developments in understanding cancer cell antigen presentation and immunosuppression have revealed the promise of cancer immunotherapy in activating immune responses to target residual disease. Each leukemia patient has a unique spectrum of cell surface antigens, which are mostly uncharacterized. If these antigens can be efficiently presented to the patient's immune system, immune responses to fight the leukemia can be significantly enhanced. We therefore sought to develop and characterize an AML cell membrane-coated nanoparticle (AMCNP) platform with nanoparticles (NPs) carrying the same surface antigens as the source leukemic cells for use as an anti-cancer vaccine. To demonstrate that our AMCNP vaccines enhance leukemia-specific antigen dendritic cell (DC) presentation and T-cell responses, we modified the C1498 murine AML cell line to express membrane-bound chicken ovalbumin (C1498-mOVA) as a model antigen. We confirmed that the C1498-mOVA line presents the OVA MHC class-I "SIINFEKL" antigen through flow-cytometry and LacZ B3Z T-cell activation assays. The C1498-mOVA line remained leukemogenic when injected into C57BL/6 mice, with survival times between 30 and 55 days. We generated both C1498 and C1498-mOVA membrane-coated nanoparticles, that were packaged with CpG oligo-deoxynucleotides (CpG) as an immune-stimulatory adjuvant. The final AMCNPs exhibit a core-shell structure with uniform coating as shown by transmission electron microscopy. The C1498-mOVA AMCNPs retained mOVA antigen. To confirm that the C1498-mOVA AMCNPs can effectively stimulate DC OVA MHC class I cross-presentation, we pulsed primary bone marrow derived DCs with C1498 AMCNPs or C1498-mOVA AMCNPs; only the C1498-mOVA AMCNP pulsed DCs specifically elicited OVA MHC class-I T-cell activation in lacZ B3Z T-cell activation assays. To verify that the AMCNPs can elicit antigen-specific immune responses in vivo, we vaccinated C57BL/6 mice with C1498 AMCNPs, C1498-mOVA AMCNPs, or equivalent amounts of whole cell lysates. When stimulated ex vivo with OVA peptide, immune-cell preparations from the C1498-mOVA AMCNP vaccinated mice showed significantly enhanced production of OVA-specific T-cells and IFN-γ, demonstrating increased immune responses. To assess if the enhanced cellular immunity afforded by the C1498-mOVA AMCNP formula can translate into functional rejection of leukemia cells, we performed a prophylactic study using the C1498-mOVA model. Mice vaccinated with the C1498-mOVA AMCNPs all survived beyond 120 days post C1498-mOVA cell challenge, compared to mock treated control mice which had a median survival of 60 days. Collectively, we developed an AMCNP platform that carries AML surface antigens and can be packaged with immunostimulatory adjuvants. These AMCNPs retained AML specific antigens, elicited enhanced antigen specific immune responses after in vivo vaccination, and improved immunity against AML challenge. Therefore, using AML cell membrane coated NPs to enhance anticancer immunity is a feasible strategy for AML vaccination immunotherapy. Disclosures Kline: iTeos: Research Funding; Merck: Honoraria, Research Funding.

Alternative polyadenylation (APA) can alter the three prime untranslated region (3'UTR) length of mRNAs, crucial for regulating mRNA metabolism and gene expression. Despite the prevalence of APA post-transcriptional regulation in cancers, changes in 3'UTR length by APA and its contribution to leukemia development have not been thoroughly studied. In this study, we demonstrated the significance of APA of leukemic fusion genes in acute myelogenous leukemia (AML) development. T(8;21) is the most common chromosomal abnormality in AML and encodes the AML1-ETO (AE) fusion gene. The AE 3'UTR has a full length of 5.2kb that contains 4 canonical polyadenylation sites (PAS), such that APA can result in several mRNA isoforms with varying 3'UTR lengths. However, RNA-seq and absolute quantification qPCR revealed that AE mainly uses 2 PAS: 5.2kb (long 3'UTR) and 3.7kb (short 3'UTR). Moreover, the AE short 3'UTR is the major isoform in both t(8;21) AML primary patients and cell lines. Based on these findings, we next hypothesize that changes in PAS usage and thus AE 3'UTR length, can modulate fusion gene expression. The single cell based dynamic array revealed that AE+ non-leukemic differentiated cells coexisted with AML blasts in the diagnostic bone marrow and these non-leukemic AE+ cells expressed much lower AE compared to AML blasts (monocytes, 0.19-fold and granulocytes, 0.11-fold, p The selective inhibition of AE short 3'UTR PAS by morpholino antisense oligonucleotide caused an increase of AE long 3'UTR usage, decrease of total AE expression, and an increase in apoptosis. These results indicate that the inhibition of AE short 3'UTR PAS increases the long 3'UTR usage and the PAS usage of leukemic fusion genes could be a potential therapeutic target. To find the key regulator of this 3'UTR change, we comprehensively knocked down all APA machinery members by shRNA in t(8;21) AML cell lines. We discovered that CPSF1 (cleavage and polyadenylation specific factor 1) knockdown increased long 3'UTR usage (37% in total AE) and down-regulated overall AE mRNA (0.47-fold). Importantly, CPSF1 was highly expressed in t(8;21) AML patients' leukemia cells compared to healthy CD34+ cells (2.7-fold, p=0.01). CPSF1 knockdown also promoted apoptosis and Gene Set Enrichment Analysis revealed that CPSF1 knockdown showed the similar gene signature to AE knockdown. These data suggest that CPSF1 promotes AE 3'UTR shortening, resulting in AE stability and a growth advantage of AML cells. Importantly, we also found that AE regulates CPSF1 expression, suggesting a positive feedback loop of AML1-ETO and CPSF1 in t(8;21) AML. Finally, we also checked APA of leukemic fusion gene MLL-AF9, which has 2 canonical PAS (0.1kb short 3'UTR and 0.7kb long 3'UTR). Compared to healthy CD34+ cells, THP-1 cells (MLL-AF9+ AML cell line) predominantly used MLL-AF9 short 3'UTR. Knockdown of CPSF1, which is also up-regulated in MLL-associated AML, showed increase of MLL-AF9 long 3'UTR usage, decrease of MLL-AF9 expression, and an increase of apoptosis in THP-1 cells. Collectively, we conclude that the polyadenylation regulator CPSF1 controls 3'UTR length of leukemic fusion transcripts, and extending the fusion gene 3'UTR by knocking down CPSF1 reduces fusion gene mRNA stability and restricts cell growth in both AE+ and MLL-AF9+ AMLs. These results suggest that 3'UTR shortening of oncogenic fusion gene transcripts contributes to leukemogenesis and controlling CPSF1 and 3'UTR usage may be a useful approach to inhibit leukemia progression. Disclosures Akashi: Pfizer: Research Funding; Taiho Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Eli Lilly Japan: Research Funding; Otsuka Pharmaceutical: Research Funding; Eisai: Research Funding; Astellas Pharma: Research Funding; MSD: Research Funding; sanofi: Research Funding; Novartis pharma: Research Funding; Chugai Pharma: Research Funding; Celgene: Research Funding, Speakers Bureau; Asahi-kasei: Research Funding; Ono Pharmaceutical: Research Funding.

Bladder cancer represents a significant human tumor burden, accounting for about 7.7% and 2.4% of all cancer cases in males and females, respectively. While men have a higher risk of developing bladder cancer, women tend to present at a later stage of disease and with more aggressive tumors. Previous studies have suggested a promotional role of androgen signaling in enhancing bladder cancer development. To directly assess the role of androgens in bladder tumorigenesis, we have developed a novel transgenic mouse strain, R26hARLoxP/+:Upk3aGCE/+, in which the human AR transgene is conditionally expressed in bladder urothelium. Intriguingly, both male and female R26hARLoxP/+:Upk3aGCE/+ mice display a higher incidence of urothelial cell carcinoma (UCC) than the age and sex matched control littermates in response to the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). We detect expression of the human AR transgene in CK5-positive and p63-positive basal cells in bladder urothelium. Further analyses of UCC tissues from R26hARLoxP/+:Upk3aGCE/+ mice showed that the majority of tumor cells are of urothelial basal cell origin. Positive immunostaining of transgenic AR protein was observed in the majority of tumor cells of the transgenic mice, providing a link between transgenic AR expression and oncogenic transformation. We observed an increase in Ki67 positive cells within the UCC lesions of transgenic AR mice. Manipulating endogenous androgen levels by castration and androgen supplementation directly affected bladder tumor development in male and female R26hARLoxP/+:Upk3aGCE/+ mice, respectively. Taken together, our data demonstrate for the first time that conditional activation of transgenic AR expression in bladder urothelium enhances carciongen-induced bladder tumor formation in mice. This new AR transgenic mouse line mimics certain features of human bladder cancer and can be used to study bladder tumorigenesis and for drug development.

The precise role of Wnt/β-catenin signaling during prostatic development and tumorigenesis is unclear. Axin2 is a direct transcriptional target of β-catenin. Recent studies have shown that Axin2-expressing cells have stem/progenitor cell properties in a variety of mouse tissues. Here, we genetically labeled Axin2-expressing cells at various time points and tracked their cellular behavior at different developmental and mature stages. We found that prostatic Axin2-expressing cells mainly express luminal epithelial cell markers and are able to expand luminal cell lineages during prostatic development and maturation. They can also survive androgen withdrawal and regenerate prostatic luminal epithelial cells following androgen replacement. Deletion of β-catenin or expression of stabilized β-catenin in these Axin2-expressing cells results in abnormal development or oncogenic transformation, respectively. Our study uncovers a critical role of Wnt/β-catenin-responsive cells in prostatic development and regeneration, and that dysregulation of Wnt/β-catenin signaling in these cells contributes to prostatic developmental defects and tumorigenesis. Stem Cells 2015;33:3356–3367

Emerging evidence has demonstrated the critical roles for both androgen and Wnt pathways in prostate tumorigenesis. A recent integrative genomic analysis of human prostate cancers (PCas) has revealed a unique enrichment of androgen and Wnt signaling in early-onset PCas, implying their clinical significance in the disease. Additionally, interaction between the androgen receptor (AR) and β-catenin has long been detected in PCa cells. However, the consequence of this interaction in prostate tumorigenesis is still unknown. Because mutations in adenomatous polyposis coli, β-catenin and other components of the destruction complex are generally rare in PCas, other mechanisms of aberrant Wnt signaling activation have been speculated. To address these critical questions, we developed Ctnnb1(L(ex3)/+)/R26hAR(L/+):PB-Cre4 mice, in which transgenic AR and stabilized β-catenin are co-expressed in prostatic epithelial cells. We observed accelerated tumor development, aggressive tumor invasion and a decreased survival rate in Ctnnb1(L(ex3)/+)/R26hAR(L/+):PB-Cre4 compound mice compared with age-matched Ctnnb1(L(ex3)/+):PB-Cre4 littermate controls, which only have stabilized β-catenin expression in the prostate. Castration of the above transgenic mice resulted in significant tumor regression, implying an essential role of androgen signaling in tumor growth and maintenance. Implantation of the prostatic epithelial cells isolated from the transgenic mice regenerated prostate intraepithelial neoplasias and prostatic adenocarcinoma lesions. Microarray analyses of transcriptional profiles showed more robust enrichment of known tumor- and metastasis-promoting genes: Spp1, Egr1, c-Myc, Sp5, and Sp6 genes, in samples isolated from Ctnnb1(L(ex3)/+)/R26hAR(L/+):PB-Cre4 compound mice than those from Ctnnb1(L(ex3)/+):PB-Cre4 and R26hAR(L/+):PB-Cre4 littermate controls. Together, these data demonstrate a confounding role of androgen signaling in β-catenin-initiated oncogenic transformation in prostate tumorigenesis.

The RUBI project [2,3,4,5,6,7,8,9] started in 2004 with the goal of designing sociable robots for early childhood education. The project relies on an immersive, iterative approach to robot development and scientific progress. From the beginning of the project scientists and engineers immersed themselves at the Early Childhood Education Center at UC San Diego, designed robot prototypes to interact and teach toddlers in close collaboration with teachers, toddlers, and parents. As part of this process we learned many lessons on how to develop hardware and software components, that are safe, reliable, and that can withstand the rigors of daily interactions with toddlers. Here we present our last robot platforms: RUBI-5. The robot components are off-the-shelf and the 3D-CAD files are being made available for free to the research community to ease replication and adaptation by other groups. One of the goals of the new platform is to greatly accelerate the design, data-gathering, and statistical analysis of early education experiments.

Members of the leucine zipper putative tumor suppressor (LZTS) family play crucial roles in transcription modulation and cell cycle control. We previously demonstrated that LZTS2 functions as a novel β-catenin-interacting protein and represses β-catenin-mediated transcription on T-cell factor/lymphoid enhancing factor. Here, we investigate the biological role of LZTS2 using newly established Lzts2 KO mice. Homozygosity for loss-of-function of the Lzts2-targeted allele resulted in severe kidney and urinary tract developmental defects, including renal/ureteral duplication, hydroureter, and hydronephrosis, which were visible prenatally. Altered ureteric bud outgrowth was identified in Lzts2 null embryos. Further analysis indicated that β-catenin subcellular localization was altered in fibroblasts isolated from Lzts2 null embryos. In addition, Wnt growth factor-induced β-catenin-mediated transcriptional activity was increased in Lzts2 null fibroblasts, suggesting a direct role for Lzts2 in the Wnt signaling pathway. These data demonstrate a critical role of LZTS2 in renal development and implicate LZTS2 as a critical regulator of β-catenin-mediated nephrogenesis.

This work reports a miniaturized laparoscopic zoom camera that can significantly improve vision for minimally invasive surgery (MIS), also known as laparoscopic surgery. The laparoscopic zoom camera contains bioinspired fluidic lenses that can change curvature and focal length in a manner similar to the crystalline lenses in human eyes. The traditional laparoscope is long, rigid, and made of fixed glass lenses with a fixed field of view. The constricted vision of a laparoscope is often an inconvenience and plays a role in many surgical injuries. To further advance MIS technology, we developed a new type of laparoscopic camera that has a total length of less than 17 mm, greater than 4× optical zoom, and 100 times higher sensitivity than today's laparoscope allowing it to work under illumination as low as 300 lux. All these unique features are enabled by the technology of bioinspired fluidic lenses having a dynamic range over 100 diopters and being convertible between a convex and concave shape.

We developed a new type of optical lens device that can change its curvature like crystalline lens in human eye. The curvature changing capability of the lens allows for a tremendous tuning range in its optical power and subsequently enables miniaturized imaging systems that can perform autofocus, optical zoom, and other advanced functions. In this paper, we study the physical properties of bio-inspired fluidic lenses and demonstrate the optical functionality through miniaturized optical systems constructed with such lenses. We report an auto-focusing optical system that can turn from a camera to a microscope, and demonstrate more than 4X optical zoom with a very short total track length. Finally, we demonstrate the benefits of fluidic lens zoom camera through minimally invasive gallbladder removal surgery.

We developed a miniaturized laparoscopic zoom camera that is 17 mm long, has >4X optical zoom, and works under 300 lux. This camera is suitable for advancing minimally invasive surgery. Demonstration surgery (cholecystectomy) was performed.

Intraocular lens (IOL) is a replacement lens for patients with crystalline lens problems. One key problem for today's IOL is its limited ability to retain the original accommodation capability inherent to human eyes. Unlike conventional optical lenses, a bio-inspired fluidic lens changes the curvature of the lens under an external force, resulting in a lens tuning power about 5 times as large as the current devices. By mimicking this desirable feature, a fluidic intraocular lens can achieve a large accommodation range as well. The device is designed that the lens can fully utilize the ciliary muscle force and the deformation of capsular bag during accommodation. Experimental results on fluidic IOL demonstrated a record high 12 Diopter (D) tuning range with a movement around its equator as small as 0.3 mm, achieved under a modest amount of force of 6 grams or 0.06 Newtons. The tuning range of the bio-inspired fluidic lens is comparable to a young, healthy eye, but the force and the movement required for ciliary muscle in order to achieve the wide accommodation range is comparable to an aged eye.

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancer. Using Osr1 (odd skipped related 1)-Cre mice, we generated a novel conditional Pten knockout mouse strain, Pten(LoxP):Osr1-Cre. Conditional biallelic and monoallelic Pten knockout mice were viable. Deletion of Pten expression was detected in the prostate of Pten(LoxP/LoxP):Osr1-Cre mice as early as 2 weeks of age. Intriguingly, Pten(LoxP/LoxP):Osr1-Cre mice develop high-grade prostatic intraepithelial neoplasms (PINs) with high penetrance as early as one-month of age, and locally invasive prostatic tumors after 12-months of age. Pten(LoxP/+):Osr1-Cre mice show only mild oncogenic changes after 8-weeks of age. Castration of Pten(LoxP/LoxP):Osr1-Cre mice shows no significant regression of prostate tumors, although a shift of androgen receptor (AR) staining from the nuclei to cytoplasm is observed in Pten null tumor cells of castrated mice. Enhanced Akt activity is observed in Pten null tumor cells of castrated Pten(LoxP/LoxP):Osr1-Cre. This study provides a novel mouse model that can be used to investigate a primary role of Pten in initiating oncogenic transformation in the prostate and to examine other genetic and epigenetic changes that are required for tumor progression in the mouse prostate.

The mechanism of the chromic acid oxidation of 2-propanol in trifluoroacetic acid solutions has been studied. The general features of this reaction strongly resemble those observed in aqueous solutions of other strong acids. A "normal" primary kinetic isotope effect is observed, similar thermodynamic parameters are obtained, and the rate behaves in similar way towards changes in acidity.An extensive study of the effect of added salts on this reaction has substantiated that the identity of added anions has a profound effect on the rate of reaction and that any complete reaction mechanism must take this into consideration.