Search

Notch signaling is important for tumor angiogenesis induced by vascular endothelial growth factor A. Blockade of the Notch ligand Dll4 inhibits tumor growth in a paradoxical way. Dll4 inhibition increases endothelial cell sprouting, but vessels show reduced perfusion. The reason for this lack of perfusion is not currently understood. Here we report that inhibition of Notch signaling in endothelial cell using an inducible binary transgenic system limits VEGFA-driven tumor growth and causes endothelial dysfunction. Neither excessive endothelial cell sprouting nor defects of pericyte abundance accompanied the inhibition of tumor growth and functional vasculature. However, biochemical and functional analysis revealed that endothelial nitric oxide production is decreased by Notch inhibition. Treatment with the soluble guanylate cyclase activator BAY41-2272, a vasorelaxing agent that acts downstream of endothelial nitric oxide synthase (eNOS) by directly activating its soluble guanylyl cyclase receptor, rescued blood vessel function and tumor growth. We show that reduction in nitric oxide signaling is an early alteration induced by Notch inhibition and suggest that lack of functional vessels observed with Notch inhibition is secondary to inhibition of nitric oxide signaling. Coculture and tumor growth assays reveal that Notch-mediated nitric oxide production in endothelial cell requires VEGFA signaling. Together, our data support that eNOS inhibition is responsible for the tumor growth and vascular function defects induced by endothelial Notch inhibition. This study uncovers a novel mechanism of nitric oxide production in endothelial cells in tumors, with implications for understanding the peculiar character of tumor blood vessels. Cancer Res; 74(9); 2402–11. ©2014 AACR.

PDF file - 3357K, Figure S1. DnMAML-mediated EC-specific Notch blockade inhibits larger vessel function. Figure S2. Flow cytometry analysis of PDGFRβ in LLC-VEGFA tumors of control and double Tg. Figure S3. NO production in the microvasculature of LLC-VEGFA tumors in response to EC-specific Notch inhibition. Figure S4 Effect of the NO-independent soluble guanylyl cyclase agonist BAY41-2272 on tumor growth in EC-specific Notch-inhibited mice. Figure S3. NO production in the microvasculature of LLC-VEGFA tumors in response to EC-specific Notch inhibition. Figure S5. dnMAML-mediated EC Notch blockade inhibits growth, functional blood vessels and eNOS activation of B16F10-VEGFA tumors. Figure S6. Expression analysis of vasodilatory genes Nts and Adm in response to EC-specific Notch inhibition in tumors. Table S1. RT-PCR analysis of Ace2, Edn2, Agt and Ednra in bulk and sorted endothelial cells (EC) from LLC-VEGFA tumors grown in control and double Tg VE-cadherin-tTA x TetOS-dnMAML-GFP.

Item does not contain fulltext BACKGROUND: VACTERL association is defined by the presence of 3 or more anomalies in any of the following systems: vertebral, anorectal, cardiac, trachea-esophageal, renal, or limb. This study hypothesized that the presence of VACTERL association would correlate with an increased risk of gynecologic anomalies in patients with anorectal malformation (ARM). METHODS: This study is a cross-sectional, retrospective analysis from the prospectively collected, multicenter registry of the Pediatric Colorectal and Pelvic Learning Consortium (PCPLC). The 834 female patients with ARM who were enrolled in the registry by January 1, 2020 were included in this study. The relationship of VACTERL association with presence of a gynecologic anomaly was evaluated with Fisher's exact test. The relationship of each VACTERL system with presence of a gynecologic anomaly was assessed in patients with cloaca, rectovestibular fistulas and rectoperineal fistulas. P-values reported were based on a 2-sided alternative and considered significant when less than 0.05. RESULTS: 834 patients with ARM underwent VACTERL screening and gynecologic evaluation with the three most common subtypes being cloaca (n = 215, 25.8%), rectovestibular fistula (n = 191, 22.9%) and rectoperineal fistula (n = 194, 23.3%). A total of 223 (26.7%) patients with ARM had gynecologic anomalies. VACTERL association was seen in 380 (45.6%) of patients with ARM. Gynecologic anomalies were present in 149 (39.1%) vs. 74 (16.3%) of subjects with vs. without VACTERL association (p < 0.001). VACTERL association did not significantly increase the risk of gynecologic anomaly in patients with cloaca and VACTERL (n = 88, 61.5%) vs. cloaca without VACTERL (n = 39, 54.2% p = 0.308). VACTERL association increased the risk of gynecologic anomalies in patients with rectoperineal fistulas (n = 7, 14.9% vs n = 9, 6.1% p = 0.014) and rectovestibular fistulas (n = 19, 31.1% vs. n = 13, 10.0% p

Fecal continence is a concern for many patients with idiopathic constipation and can significantly impact quality of life. It is unknown whether racial, ethnic, and socioeconomic disparities are seen in fecal continence within the idiopathic constipation population. We aimed to evaluate fecal continence and associated demographic characteristics in children with idiopathic constipation referred for surgical evaluation.A multicenter retrospective study of children with idiopathic constipation was performed at sites participating in the Pediatric Colorectal and Pelvic Learning Consortium (PCPLC). All patients3y of age with idiopathic constipation diagnosis were included. The primary outcome was fecal continence, categorized as complete (no accidents ever), daytime (no accidents during the day), partial (occasional incontinence day/night), and none (incontinent). We evaluated for associations between fecal continence and race, sex, age, insurance status, and other patient-level factors, employing Kruskal-Wallis and trend tests.458 patients with idiopathic constipation from 12 sites were included. The median age of diagnosis was 4.1 years. Only 25% of patients referred for surgical evaluation were completely continent. Age at the visit was significantly associated with fecal continence level (p = 0.002). In addition, patients with public and mixed public and private insurance had lower levels of continence (p0.001). Patients with developmental delay were also more likely to have lower continence levels (p = 0.009) while diagnoses such as anxiety, ADD/ADHD, autism, depression, obsessive-compulsive disorder were not associated. Approximately 30% of patients had an ACE operation (antegrade continence enema) at a median age of 9.2 years at operation. Black patients were significantly less likely to undergo ACE operation (p = 0.016) when compared to white patients.We observed data that suggest differences in fecal incontinence rates based on payor status. Further investigation is needed to characterize these potential areas of disparate care.Level III.

Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging (“inflammaging”) has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a–null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a−/− myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a−/− HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a−/− mice. miR-146a−/− HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

In developing an effective monitoring program for the wastewater surveillance of SARS-CoV-2 ribonucleic acid (RNA), the importance of sampling methodology is paramount. Passive sampling has been shown to be an effective tool to detect SARS-CoV-2 RNA in wastewater. However, the adsorption characteristics of SARS-CoV-2 RNA on passive sampling material are not well-understood, which further obscures the relationship between wastewater surveillance and community infection. In this work, adsorption kinetics and equilibrium characteristics were evaluated using batch-adsorption experiments for heat-inactivated SARS-CoV-2 (HI-SCV-2) adsorption to electronegative filters. Equilibrium isotherms were assessed or a range of total suspended solids (TSS) concentrations (118, 265, and 497 mg L–1) in wastewater, and a modeled qmax of 7 × 103 GU cm–2 was found. Surrogate adsorption kinetics followed a pseudo-first-order model in wastewater with maximum concentrations achieved within 24 h. In both field and isotherm experiments, equilibrium behavior and viral recovery were found to be associated with wastewater and eluate TSS. On the basis of the results of this study, we recommend a standard deployment duration of 24–48 h and the inclusion of eluate TSS measurement to assess the likelihood of solids inhibition during analysis., Field and bench-scale studies showed that a 24−48 h deployment was ideal for a passive sampling of SARS-CoV-2 in wastewater and that analysis should include eluatimg TSS measurement to understand the role of inhibition in high TSS samples.

Despite several years of targeted interventions, First Nations drinking water systems in Canada remain under-resourced and require substantial improvements in both infrastructure and management to provide communities with safe drinking water. The purpose of this study was to co-develop a risk assessment process integral to the water safety planning methodology to determine if proactive risk assessment provides a beneficial management tool for First Nations water systems. We co-developed a risk assessment web-application with First Nations stakeholders to identify hazards and assess risk in six Atlantic region First Nations communities. Using this application, we were able to successfully identify high-risk hazards in each community, both risks specific to individual systems, and risks common at a regional level. Through semi-structured interviews we identified the following benefits of a risk assessment web application: increased communication, data ownership and centralized data management. However, challenges remain, including current fragmented governance realities, and liability concerns associated with adopting a new risk management strategy. Successful adoption of proactive risk management strategies in First Nations communities will depend on strong co-development of risk assessment tools, transparent communication between stakeholders and clearly defined data ownership and management practices.

Purpose While fecal incontinence is a primary concern for many children with anorectal malformations (ARM), urinary incontinence is also prevalent in this population. Racial, ethnic, and socioeconomic disparities in urinary continence have been observed in other conditions, but have not been previously evaluated in ARM. We aimed to evaluate urinary continence and associated demographic and socioeconomic characteristics in individuals with ARM. Methods We performed a multicenter retrospective study of ARM patients evaluated at sites participating in the Pediatric Colorectal and Pelvic Learning Consortium (PCPLC). We included all patients with ARM 3 years and older. The primary outcome was urinary continence which was categorized as complete (no accidents), daytime (accidents at night), partial (rare or occasional accidents), and none (frequent accidents or no continence). We evaluated for associations between urinary continence and race, sex, age, insurance status, and adoption status, employing Kruskal-Wallis and trend tests. Secondary outcomes included bladder management strategies such as clean intermittent catheterization and continence surgery. P-value Results A total of 525 patients with ARM were included. Overall, 48% reported complete urinary continence, and continence was associated with greater age. For school-aged children (age ≥ 5 years), 58% reported complete continence, while 30% reported none. Public insurance and adoption status were associated with decreased likelihood of incontinence. Conclusions We observed a novel finding of disparities in urinary continence for children with ARM related to insurance and adoption status. Further investigation regarding the etiologies of these inequities is needed in order to affect clinical outcomes.

Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common structural genomic variant in myelodysplastic syndromes (MDS)1. Lenalidomide (LEN) is the treatment of choice for patients with del(5q) MDS, but half of the responding patients become resistant2 within 2 years. TP53 mutations are detected in ~20% of LEN-resistant patients3. Here we show that patients who become resistant to LEN harbour recurrent variants of TP53 or RUNX1. LEN upregulated RUNX1 protein and function in a CRBN- and TP53-dependent manner in del(5q) cells, and mutation or downregulation of RUNX1 rendered cells resistant to LEN. LEN induced megakaryocytic differentiation of del(5q) cells followed by cell death that was dependent on calpain activation and CSNK1A1 degradation4,5. We also identified GATA2 as a LEN-responsive gene that is required for LEN-induced megakaryocyte differentiation. Megakaryocytic gene-promoter analyses suggested that LEN-induced degradation of IKZF1 enables a RUNX1-GATA2 complex to drive megakaryocytic differentiation. Overexpression of GATA2 restored LEN sensitivity in the context of RUNX1 or TP53 mutations by enhancing LEN-induced megakaryocytic differentiation. Screening for mutations that block LEN-induced megakaryocytic differentiation should identify patients who are resistant to LEN.

First Nations communities in Canada have a documented history of sub-standard water quality. While efforts have been made to address drinking water quality, little has been done to address longstanding challenges in wastewater systems. This study developed a hazard identification checklist using a sanitation safety plan (SSP) framework to characterize potential hazards in 29 First Nations wastewater systems in Atlantic Canada. System types included in this study included centralized, decentralized, and municipal transfer agreements (MTAs). Using past system assessment reports, potential hazardous events were evaluated along the sanitation chain to assess risk within systems. Overall, 69% of hazardous events had an unknown level of risk while 7% were high-risk. This research found that decentralized systems and MTAs have poorly characterized risk due to a lack of documentation and communication. The presence of significant knowledge deficits and high-risk hazards in centralized systems cause risk propagation and accumulation along the sanitation chain, resulting in potential effluent quality concerns. This desktop study demonstrates that an SSP approach offers an alternative assessment process to the regulatory approach currently being used by proposing an enhanced systemic understanding of risk that can inform management practices and integrate the plurality of stakeholders involved in these systems.

The role of roasting in cold brew coffee chemistry is poorly understood. The brewing temperature influences extraction processes and may have varying effects across the roast spectrum. To understand the relationship between brew temperature and roast temperature, hot and cold brew coffees were prepared from Arabica Columbian coffee beans roasted to light, medium, and dark levels. Chemical and physical parameters were measured to investigate the relationships among degree of roast, water temperature, and key characteristics of resulting coffees. Cold brew coffees showed differential extraction marked by decreased acidity, lower concentration of browned compounds, and fewer TDS indicating that cold water brewing extracts some compounds less effectively than hot water brewing. Compounds in coffee did exhibit sensitivity to degree of roast, with darker roasts resulting in decreased concentrations for both hot and cold brew coffees. Total antioxidant capacity (TAC) was only sensitive to degree of roast in cold brew coffees, while hot brew coffees had a constant TAC for all three roast levels. This indicates that the solid bean matrix and its chemical constituents interact with cold water differently than with hot water. Surface wetting, pore dynamics, and solubility all contribute to the extraction potential during brewing and are all functions of water temperature.

Activation of inflammatory pathways is associated with bone marrow failure syndromes, but how specific molecules impact on the marrow microenvironment is not well elucidated. We report a novel role for the miR-145 target, Toll/Interleukin-1 receptor domain containing adaptor protein (TIRAP), in driving bone marrow failure. We show that TIRAP is overexpressed in various types of myelodysplastic syndromes (MDS), and suppresses all three major hematopoietic lineages.. Constitutive expression of TIRAP in hematopoietic stem/progenitor cells (HSPC) promotes upregulation ofIfnγ, leading to bone marrow failure. Myelopoiesis is suppressed through Ifnγ-Ifnγr-mediated release of the alarmin, Hmgb1, which disrupts the marrow endothelial niche. Deletion ofIfnγor Ifnγr blocks Hmgb1 release and is sufficient to reverse the endothelial defect and prevent myelosuppression. In contrast, megakaryocyte and erythroid production is repressed independently of the Ifnγ receptor. Contrary to current dogma, TIRAP-activated Ifnγ-driven marrow suppression is independent of T cell function or pyroptosis. In the absence of Ifnγ, TIRAP drives myeloproliferation, implicating Ifnγ in suppressing the transformation of bone marrow failure syndromes to myeloid malignancy. These findings reveal novel, non-canonical roles of TIRAP, Hmgb1 and Ifnγ function in the marrow microenvironment,and provide insight into the pathophysiology of preleukemic syndromes.Graphical Abstract: Model of proposed mechanism of TIRAP-induced BMFConstitutive TIRAP expression in marrow cells releases Ifnγ, which directly impacts on megakaryocyte and erythroid production, but indirectly suppresses myelopoiesis through the release of the alarmin, Hmgb1, which disrupts the marrow endothelial compartment.

Background: Current guidelines indicate reduced time to reperfusion with thrombectomy is strongly associated with better outcomes. Validated screening tools for large vessel occlusions are available for the prehospital setting. Objective: To reduce door to access times for patients arriving by EMS to an urban 487 bed quaternary hospital. Methods: Starting in January of 2016, EMS crews screened all stroke patients with the Cincinnati - Stroke Triage Assessment Tool (C-STAT) and communicated results to the ED prior to arrival (PTA). In 2017, a team consisting of Neurology, ED, Cath Lab, Neuro Interventional leadership and stroke coordinator agreed to a 6-month pilot project of activating the Cath Lab when EMS radioed a patient was C-STAT positive. Any ED staff who might take EMS radio report were educated on the new process. Instructions were added to the existing Stroke Patient Checklist. Cath Lab and EMS staff were notified of the pilot project. During the pilot period, feedback was provided to EMS and ED leadership if an issue was identified for a specific case and trends were discussed. Results: During the pilot period, median door to access time for C-STAT positive patients who had Cath Lab activated PTA was 66 minutes compared to the 104 minutes the previous year (37% reduction). Unexpectedly, the median door to needle time for these patients also improved from 45 minutes the previous year to 28 minutes (38% reduction). The Cath Lab was activated PTA 16 times for patients EMS reported as C-STAT positive; Only 5 went on to have a thrombectomy. Overcalls were due to lack of C-STAT specificity (n=6) or C-STAT not scored correctly (n=5). In practice as well as when cases associated with assessment errors are removed, C-STAT demonstrated a low specificity for LVO (37.5% and 40% respectively). Lastly, internal process issues lead to 5 additional PTA Cath Lab activations for patients EMS did not report as being C-STAT positive. Conclusions: It was felt additional education would not be enough to make this initiative sustainable given the low specificity. In conclusion, despite marked improvement in door to access and door to needle times, the overactivation of Cath Lab lead to the initiative being cancelled.

Introduction: The impact of red blood cell (RBC) storage on alloimmunization rates after transfusion remains controversial, with clinical experience demonstrating conflicting results. Indeed, some reports suggest increased rates of alloimmunization associated with longer storage of RBC units, while others suggest no association between alloimmunization rates and length of storage. We hypothesized that this discrepancy may reflect the differential impact of storage on alloimmunization related to each unique antigen and the immune response elicited. Here we define the effect of red cell storage on two distinct antigens, including the Hel-Ova-Duffy (HOD) and KEL antigens, using a preclinical murine model of RBC storage and transfusion. Methods: RBCs singly expressing HOD (HOD RBC) or KEL (KEL RBC) antigens, in addition to those dually expressing both HOD and KEL (HOD x KEL RBC) antigens, were collected from respective transgenic donor mice into CPDA. Units of HOD, KEL or HOD x KEL RBC were generated and used immediately (fresh RBC), or stored for 8 (D8) or 14 (D14) days at 4C before transfusion into wildtype recipients. Recipient cytokine profiles were assessed 2 hours post-transfusion using a multiplex inflammation panel. Donor RBC survival at 24 hours post-transfusion was assessed by flow cytometry. Serum was collected from recipients on days 5 and 14 post-transfusion and analyzed for IgM or IgG development by flow-cytometric crossmatch using HOD or KEL RBC targets. Results: Post-transfusion RBC survival was decreased with increased storage, but was not significantly different between donor cell types (HOD, KEL or HOD x KEL). Recipient cytokine profiles demonstrated a distinct inflammatory profile associated with storage. Development of IgM antibodies against HOD in mice transfused fresh versus D8 or D14 HOD or HOD x KEL RBCs were not significantly different; however, significantly increased (p Conclusions: Although the impact of RBC storage on various measures of transfusion efficacy have been determined, the impact of storage on alloimmunization remains incompletely understood. Using a murine model of transfusion, we demonstrate that storage differentially impacts immunization to red cell antigens, increasing the response to certain antigens, like HOD, while decreasing the response to others, like KEL. Although it is unclear whether these findings apply to other RBC antigens and what relevance this holds for human patients, our study provides insight into the impact of storage on red cell alloimmunization and may enlighten future storage protocols. Disclosures Stowell: Argenx: Speakers Bureau; Grifols: Speakers Bureau; Alexion: Consultancy.

The myelodysplastic syndrome (MDS) are a group of hematological malignancies with the propensity to develop into either acute myeloid leukemia (AML) or bone marrow failure (BMF). Dysregulation of immune and inflammatory responses has been implicated in MDS and other BMF disorders. There is significant evidence for IFNγ playing a key role in MDS and BMF syndromes. However, there are conflicting theories regarding the mechanism by which IFNγ promotes BMF. There is also very little information on the triggers that underlie upregulation of IFNγ in these BMF syndromes. Interstitial deletion of chromosome 5q is the most common cytogenetic abnormality observed in MDS, accounting for approximately 10% of all cases. Our lab has previously shown that miR-145, which is located on the minimally deleted region of chromosome 5q, targets Toll/Interleukin-1 receptor domain containing adaptor protein (TIRAP) - an innate immune adaptor protein. However, the role of TIRAP in marrow failure has not been well elucidated. In this study, we identify a novel role for TIRAP in dysregulating normal hematopoiesis through activation of Ifnγ. Using bone marrow transplants in wild-type mice, we showed that constitutive expression of TIRAP in wild-type hematopoietic stem and progenitor cells (HSPC) caused peripheral blood cytopenia, suppressed the bone marrow endothelial niche and significantly reduced overall survival of the mice (median survival 9 weeks post-transplant) compared to controls (p < 0.0001). RNA-seq analysis of TIRAP expressing HSPC identified several proinflammatory cytokines to be significantly overrepresented. Geneset enrichment analysis (GSEA) identified the Ifnγ response as the single most significantly enriched pathway of the Hallmark genesets. To test the functional role of Ifnγ in TIRAP-mediated BMF, wild-type recipient mice were transplanted with TIRAP- HSPC from Ifnγ-/- donor mice. Mice that received TIRAP-transduced Ifnγ-/- HSPC were rescued from BMF, as evidenced by normalized blood cell counts and improved median survival (median survival 48.6 weeks) (Ifnγ-/- TIRAP vs. wild-type TIRAP: p = 0.0004). Interestingly, in our model of TIRAP induced BMF, myeloid rather than the conventional T and NK cells were the cells most responsible for the increased production of Ifnγ. Further, when we transplanted TIRAP expressing wild-type HSPC into NSG recipient mice, which are deficient in functional B, T and NK cells, the NSG mice developed BMF with pancytopenia in a similar time-frame as wild-type mice. This suggested that T and NK cells are not central for the development of TIRAP induced BMF. Delving deeper into the mechanism by which the TIRAP-Ifnγ axis causes BMF, we saw that while Ifnγ played a direct role in suppressing erythropoiesis and megakaryopoiesis, it played an indirect, Ifnγ receptor independent role on myelopoiesis. TIRAP-induced activation of Ifnγ released the alarmin, Hmgb1, which suppressed the marrow endothelial niche, which in turn promoted myeloid suppression. Overexpression of TIRAP in Ifnγ -/- background blocked Hmgb1 release. Further, blocking Hmgb1 in presence of TIRAP expression was sufficient to reverse the marrow endothelial defect and restore myelopoiesis in vivo. Our findings highlight a novel, non-canonical effect of aberrant TIRAP expression via the Ifnγ-Hmgb1 axis on the endothelial cell component of the marrow microenvironment and hematopoiesis. Further understanding of this pathway would open up avenues for developing new therapies for BMF. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common structural genomic variant in myelodysplastic syndromes (MDS)

Background: The Oregon and South West Washington Cascade Region Stroke Coordinators (CRSC) formed in 2006. By 2015 active membership represented 15 hospitals by 5 health care systems, all certified as either comprehensive or primary stroke centers. Clinical practice guidelines recommend quality improvement processes involving Emergency Medical Services (EMS). April 2017, the CRSC initiated discussions to develop a standardized EMS feedback tool. Purpose: Develop and implement a standardized EMS feedback tool across multiple health care systems to provide feedback on acute ischemic stroke (AIS) treatment cases, while improving stroke coordinator and EMS satisfaction. Methods: April 2017 through April 2018 a core group led efforts, utilizing Lean principles, to develop a standardized EMS feedback tool for AIS treatment cases. The first step was reviewing the variability in existing tools. A survey was created for EMS to identify meaningful elements. From those results a standardized feedback tool was drafted and finalized based on CRSC input. Throughout development, the core-group applied Kurt Lewin’s change model by creating an environment ready for change, actively supporting the CRSC through the change to a new tool, and lastly reinforcing the new tool to sustain the desired change of a standardized EMS feedback tool. A survey 10 weeks post implementation captured stroke coordinators satisfaction. Results: The Portland Standardized Stroke feedback Tool for EMS (PSST EMS) was implemented in April 2018. All sites adopted the tool to provide EMS feedback on AIS treatment cases. Post implementation survey among stroke coordinators had 90% response rate. Seventy-five percent valued a standardized EMS feedback tool even if it meant more work. Ninety percent were satisfied and 10% were neutral with PSST EMS. Anecdotally EMS voiced a high degree of satisfaction with a standardized tool. Conclusion: Applying Kurt Lewin’s change model resulted in a standardized EMS feedback tool for AIS treatment cases in the Cascade region. It is feasible for stroke program coordinator nurses, across multiple health care systems, to collaborate and standardize EMS feedback.

BMS-986094, the prodrug of a guanosine nucleotide analogue (2'-C-methylguanosine), was withdrawn from clinical trials due to serious safety issues. Nonclinical investigative studies were conducted as a follow up to evaluate the potential for BMS-986094-related mitochondrial-toxicity. In vitro, BMS-986094 was applied to human hepatoma cells (HepG2 and Huh-7) or cardiomyocytes (hiPSCM) up to 19 days to assess mitochondrial DNA content and specific gene expression. There were no mitochondrial DNA changes at concentrations ≤10 µM. Transcriptional effects, such as reductions in Huh-7 MT-ND1 and MT-ND5 mRNA content and hiPSCM MT-ND1, MT-COXII, and POLRMT protein expression levels, occurred only at cytotoxic concentrations (≥10 µM) suggesting these transcriptional effects were a consequence of the observed toxicity. Additionally, BMS-986094 has a selective weak affinity for inhibition of RNA polymerases as opposed to DNA polymerases. In vivo, BMS-986094 was given orally to cynomolgus monkeys for 3 weeks or 1 month at doses of 15 or 30 mg/kg/day. Samples of heart and kidney were collected for assessment of mitochondrial respiration, mitochondrial DNA content, and levels of high energy substrates. Although pronounced cardiac and renal toxicities were observed in some monkeys at 30 mg/kg/day treated for 3-4 weeks, there were no changes in mitochondrial DNA content or ATP/GTP levels. Collectively, these data suggest that BMS-986094 is not a direct mitochondrial toxicant.

Red blood cell (RBC) alloimmunization represents a significant immunological challenge for some patients. While a variety of immune constituents likely contribute to the initiation and orchestration of alloantibodies to RBC antigens, identification of key immune factors that initiate alloantibody formation may aid in the development of a therapeutic modality to minimize or prevent this process. To define the immune factors that may be important in driving alloimmunization to an RBC antigen, we determined the specific immune compartment and distinct cells that may initially engage transfused RBCs and facilitate subsequent alloimmunization. Our findings demonstrate that the splenic compartment is essential for formation of anti-KEL antibodies following KEL RBC transfusion. Within the spleen, transfused KEL RBCs are found within the marginal sinus, where they appear to specifically co-localize with marginal zone (MZ) B cells. Consistent with this, removal of MZ B cells completely prevented alloantibody formation following KEL RBC transfusion. While MZ B cells can mediate a variety of key downstream immune pathways, depletion of follicular B cells or CD4 T cells failed to similarly impact the anti-KEL antibody response, suggesting that MZ B cells may play a key role in the development of anti-KEL IgM and IgG following KEL RBC transfusion. These findings highlight a key contributor to KEL RBC-induced antibody formation, wherein MZ B cells facilitate antibody formation following RBC transfusion.

Formalin fixation is the standard method for the preservation of tissue for diagnostic purposes, including pathologic review and molecular assays. However, this method is known to cause artifacts that can affect the accuracy of molecular genetic test results. We assessed the applicability of alternative fixatives to determine whether these perform significantly better on next-generation sequencing assays, and whether adequate morphology is retained for primary diagnosis, in a prospective study using a clinical-grade, laboratory-developed targeted resequencing assay. Several parameters relating to sequencing quality and variant calling were examined and quantified in tumor and normal colon epithelial tissues. We identified an alternative fixative that suppresses many formalin-related artifacts while retaining adequate morphology for pathologic review.

Kulic et al. show that RBPJ, a transcriptional repressor of Notch, is frequently deleted in human cancers and can function as a tumor suppressor. Loss of RBPJ acts to derepress target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumor growth., Aberrant Notch activity is oncogenic in several malignancies, but it is unclear how expression or function of downstream elements in the Notch pathway affects tumor growth. Transcriptional regulation by Notch is dependent on interaction with the DNA-binding transcriptional repressor, RBPJ, and consequent derepression or activation of associated gene promoters. We show here that RBPJ is frequently depleted in human tumors. Depletion of RBPJ in human cancer cell lines xenografted into immunodeficient mice resulted in activation of canonical Notch target genes, and accelerated tumor growth secondary to reduced cell death. Global analysis of activated regions of the genome, as defined by differential acetylation of histone H4 (H4ac), revealed that the cell death pathway was significantly dysregulated in RBPJ-depleted tumors. Analysis of transcription factor binding data identified several transcriptional activators that bind promoters with differential H4ac in RBPJ-depleted cells. Functional studies demonstrated that NF-κB and MYC were essential for survival of RBPJ-depleted cells. Thus, loss of RBPJ derepresses target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumorigenesis.

The extraction kinetics and equilibrium concentrations of caffeine and 3-chlorogenic acid (3-CGA) in cold brew coffee were investigated by brewing four coffee samples (dark roast/medium grind, dark roast/coarse grind, medium roast/medium grind, medium roast/coarse grind) using cold and hot methods. 3-CGA and caffeine were found at higher concentrations in cold brew coffee made with medium roast coffees, rather than dark roast. The grind size did not impact 3-CGA and caffeine concentrations of cold brew samples significantly, indicating that the rate determining step in extraction for these compounds did not depend on surface area. Caffeine concentrations in cold brew coarse grind samples were substantially higher than their hot brew counterparts. 3-CGA concentrations and pH were comparable between cold and hot brews. This work suggests that the difference in acidity of cold brew coffee is likely not due to 3-CGA or caffeine concentrations considering that most acids in coffee are highly soluble and extract quickly. It was determined that caffeine and 3-CGA concentrations reached equilibrium according to first order kinetics between 6 and 7 hours in all cold brew samples instead of 10 to 24 hours outlined in typical cold brew methods.

Objective APELA is a small, secreted peptide that can function as a ligand for the G-protein coupled receptor, Apelin Receptor (APLNR, APJ). APELA plays an essential role in endoderm differentiation and cardiac development during embryogenesis. We investigated whether APELA exerts any functions in cancer progression. Methods The Cancer Genome Atlas (TCGA) RNA sequencing datasets, microarray from an OCCC mouse model, and RNA isolated from fresh frozen and FFPE patient tissue were used to assess APELA expression. APELA knockout ovarian clear cell carcinoma (OCCC) cell lines were generated using CRISPR/Cas9. Results APELA was expressed in various ovarian cancer histotypes and was especially elevated in OCCC. Disruption of APELA expression in OCCC cell lines suppressed cell growth and migration, and altered cell-cycle progression. Moreover, addition of human recombinant APELA peptide to the OCCC cell line OVISE promoted cell growth and migration. Interestingly, OVISE cells do not express APLNR, suggesting that APELA can function through an APLNR-independent pathway. Furthermore, APELA affected cell growth and cell cycle progression in a p53-dependent manner. In addition, APELA knockdown induced p53 expression in cancer cell lines. Conclusions Our findings uncover a potential oncogenic role for APELA in promoting ovarian tumour progression and provide a possible therapeutic strategy in ovarian cancer by targeting APELA.

Summary: The sterile alpha motif (SAM) and SRC homology 3 (SH3) domain containing protein 1 (Sash1) acts as a scaffold in TLR4 signaling. We generated Sash1−/− mice, which die in the perinatal period due to respiratory distress. Constitutive or endothelial-restricted Sash1 loss leads to a delay in maturation of alveolar epithelial cells causing reduced surfactant-associated protein synthesis. We show that Sash1 interacts with β-arrestin 1 downstream of the TLR4 pathway to activate Akt and endothelial nitric oxide synthase (eNOS) in microvascular endothelial cells. Generation of nitric oxide downstream of Sash1 in endothelial cells affects alveolar epithelial cells in a cGMP-dependent manner, inducing maturation of alveolar type 1 and 2 cells. Thus, we identify a critical cell nonautonomous function for Sash1 in embryonic development in which endothelial Sash1 regulates alveolar epithelial cell maturation and promotes pulmonary surfactant production through nitric oxide signaling. Lung immaturity is a major cause of respiratory distress and mortality in preterm infants, and these findings identify the endothelium as a potential target for therapy. : Surfactant deficiency due to lung immaturity is a major cause of respiratory distress in premature newborns. Coulombe et al. show that endothelial SAM and SH3 domain containing protein 1 (Sash1) drives perinatal lung maturation via nitric oxide signaling to alveolar cells. Sash1 interacts with β-arrestin1 to activate Akt-eNOS and induce alveolar epithelial cell maturation and surfactant synthesis. Keywords: TLR4, Sash1, surfactant, endothelium, alveolar type 2 cells, respiratory distress, lung development, β-arrestin, nitric oxide

Background: Exposure to red blood cell (RBC) alloantigens during pregnancy or transfusion can lead to the development of alloantibodies and result in transfusion-related complications, including hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. However, the factors that regulate RBC alloimmunization remain incompletely understood. Several studies suggest that alterations in factors that regulate RBC clearance may impact RBC uptake and antigen presentation, directly influencing the likelihood of RBC alloimmunization. To test this, we directly examined the potential role of CD47, a master regulator of RBC removal previously shown to be altered during RBC senescence and cold storage. To accomplish this, we crossed transgenic mice that express the model HOD antigen (a fusion protein consisting of hen egg lysozyme fused to ovalbumin and human Duffy b) with CD47 knock out (KO) mice to generate HOD RBC donors with wild type, heterozygous or homozygous KO levels of CD47 and used these donors to define the impact of CD47 on antibody formation following RBC transfusion. Methods: HOD transgenic mice expressing the HOD antigen exclusively on RBCs were crossed with CD47-/- mice to produce HOD CD47+/- or HOD CD47-/- mice. HOD and CD47 levels were assessed by flow cytometric analysis using anti-HOD and anti-CD47 antibodies. HOD CD47+/+, HOD CD47+/- or HOD CD47-/- RBCs were transfused into C57BL6 recipients, followed by serum collection on days 14 and 28 post transfusion and evaluation of anti-HOD antibodies by flow cytometry crossmatch. To determine the CD4 T cell response to transfusion, TCR transgenics specific to ovalbumin (OTII) were labeled with CFSE, followed by adoptive transfer, transfusion of HOD CD47+/+, HOD CD47+/- or HOD CD47-/- RBCs and evaluation of T cell proliferation, activation and cytokine secretion. Cellular removal of HOD RBCs was determined by flow cytometric examination of CFSE-labeled HOD RBCs. Finally, antigen levels on HOD RBCs was determined by staining cells with anti-HEL antibodies followed by flow cytometric examination. All three groups were subjected to one-way ANOVA analysis with a p value Results: While HOD CD47+/+ RBCs expressed levels of CD47 comparable to WT RBCs, HOD CD47+/- RBCs exhibited significantly reduced CD47 levels (nearly half that observed on WT HOD RBCs) and HOD CD47-/-RBCs failed to express any detectable CD47 (p < 0.0001). Following transfusion, HOD CD47+/- and HOD CD47-/- RBCs produced significantly higher levels of IgG anti-HOD antibodies than HOD CD47+/+ RBCs on days 14 and 28 post-transfusion (p < 0.001). However, while HOD CD47-/- RBCs displayed increased clearance consistent with the possible enhancement of a CD4 T cell response (p < 0.0001), HOD CD47+/- RBCs failed to exhibit any different in clearance when compared to HOD CD47+/+ RBCs overtime. To examine the potential impact of differences in HOD RBC clearance on CD4 T cell activation, OTII T cell proliferation was evaluated. While HOD CD47-/- RBC transfusion resulted in significantly increased 33D1+ dendritic cell uptake, OTII proliferation, activation and cytokine secretion (p < 0.05), no difference in 33D1+ dendritic cell uptake or T cell response was observed following HOD CD47+/+ RBC or HOD CD47+/- RBC transfusion. Instead, HOD CD47+/- RBCs exhibited enhanced antigen removal, while also displaying an increased ability to activate HEL specific B cells when compared to HOD CD47+/+ or HOD CD47-/- RBC transfusion. Conclusions: These results demonstrate that alterations in CD47, which occur during normal RBC senescence and cold storage, directly influence RBC alloimmunization through different mechanisms depending on the extent of CD47 loss. While complete loss of CD47 results in enhanced RBC clearance, antigen presentation and CD4 T cell activation, reductions in CD47 to half WT levels failed to impact RBC clearance or T cell activation, but instead enhanced antigen specific B cell activation. These results demonstrate that even partial loss of CD47 is capable of significantly enhancing alloantibody formation completely independent of its role in regulating RBC clearance. In doing so, these findings provide novel insight into the role of CD47 as a key regulator of RBC alloimmunization. Disclosures Stowell: Grifols: Honoraria.

ImmunoGen’s newest antibody-drug conjugate (ADC) design uses the novel maytansinoid linker-payload, DM21-C that bears a peptidase/protease cleavable linker. These ADCs show good bystander killing of proximal antigen negative cells, suggesting the generation of cell-permeable catabolites. The goal of this study was to identify the catabolites generated upon incubation in antigen-positive cancer cells (both cell pellet and media), in mouse plasma, as well as in in vitro catabolic systems. Upon incubation with a cysteine-conjugated DM21-C ADC, the small molecule fraction was extracted with an organic solvent and analyzed by high resolution mass spectrometry after chromatographic separation. Through comparison to reference standards, mass spectral signal extraction of possible products, and database search of unknown peaks, the potential in vitro catabolite products of DM21-C conjugate were identified. Information on linker-payload stability in these model systems, including plasma stability, in vitro metabolism of the ADC, and catabolic products that were both retained and effluxed from cancer cells was obtained. As a result, we identified DM51 (the thiol- resulting from self-immolation of the cleaved linker-payload) as a major catabolite of the DM21-C ADC. Citation Format: Janet Lau, Paulin Salomon, Kerstin Sinkevicius, Juliet Costoplus, Megan Fuller, Raymond Xu, Stuart Hicks, Ravi Chari, Wayne Widdison, Nicholas Yoder, Thomas Keating. LC-MS based catabolite identification study of an ADC with DM21-C, a novel maytansinoid linker-payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 538.

Recognition of microbial products by TLRs is critical for mediating innate immune responses to invading pathogens. In this study, we identify a novel scaffold protein in TLR4 signaling called SAM and SH3 domain containing protein 1 (SASH1). Sash1 is expressed across all microvascular beds and functions as a scaffold molecule to independently bind TRAF6, TAK1, IκB kinase α, and IκB kinase β. This interaction fosters ubiquitination of TRAF6 and TAK1 and promotes LPS-induced NF-κB, JNK, and p38 activation, culminating in increased production of proinflammatory cytokines and increased LPS-induced endothelial migration. Our findings suggest that SASH1 acts to assemble a signaling complex downstream of TLR4 to activate early endothelial responses to receptor activation.

The application of cosmetic substances such as: Vitamin E, Shea butter or Olive Oil on textile materials have varied. The goal of cosmetic textiles is to transfer substances such as these directly to the skin or scalp, therefore the efficacy of the method is of utmost importance. Consumers want a lasting, effective product. A consumer of cosmetic textiles wants to know that fiber properties are inherent in their garment or textile, and that they last (Nelson, 2002). Shea Butter and Olive Oil are two of the most sought after natural ingredients in the world. But how would consumers know that: 1) these ingredients have really been applied to these products? 2) product use will yield the stated results? The purpose of this study is to investigate the ingredients of the Qfitt Double Mesh Wrap product, to confirm that the natural ingredients cited on the packaging are present in measurable amounts.

Triple modular redundancy (TMR) is the traditional mitigation technique for field-programmable gate arrays (FPGAs) subject to single-event upsets (SEUs) in high-radiation environments. Reduced-precision redundancy (RPR) as an alternative to TMR for communications systems is demonstrated. RPR reduces the number of "catastrophic" SEUs in several simple communications receivers by over 95%, thus increasing the mean time to failure (MTTF) by 25 to 99 times, while consuming less than half of the resources that TMR does in most cases.

Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1 + CD31 dim vascular endothelial (VE)-cadherin − CD45 − precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1 + precursor cell, but not the VE-cadherin + endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1 + precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1 + local precursor to VSMC in a spatiotemporal fashion across all vascular beds.

The immunomodulatory drug lenalidomide (LEN) is the treatment of choice for del(5q) MDS patients. LEN has been shown to trigger the specific degradation of CSNK1A1 and IKZF1 proteins after binding the E3-ligase substrate adaptor CRBN. When brought below a certain expression threshold, CSNK1A1 deficiency activates a p53-dependent apoptotic response. Thus, the unique sensitivity of del(5q) cells to LEN is explained by CSNK1A1 haploinsufficiency in del(5q) MDS patients. Despite its efficacy, 50% of LEN-treated patients eventually relapse within an interval of 2-3 years after treatment. Treatment failure is associated to low platelet counts and occurrence of additional mutations, such as TP53. To identify novel genetic determinants of LEN resistance, we have compared whole genome sequencing data of paired samples from six del(5q) patients who have been treated with LEN and eventually became resistant to the treatment. We identified 2 patients with mutations in TP53. The remaining four presented RUNX1 alterations: two patients had protein coding mutations in RUNX1 and two had a significant reduction in RUNX1, but not TP53, transcript levels. As a model of sensitivity, we studied the response to LEN in two human del(5q) cell lines, MDS-L and KG-1a. RUNX1 protein levels are postranscriptionally upregulated upon exposure to LEN, accompanied by increased levels of RUNX1 activity. Deletion of CRBN expression cancelled these effects. RUNX1 overexpression inhibited clonogenic growth and induced apoptosis. We then generated RUNX1 knock-out (KO) clones derived from MDS-L cells using CRISPR/Cas9 system. RUNX1 KO cells presented increased proliferation, increased colony growth and reduced apoptosis in the presence of LEN compared to wild-type (WT) control clones. These results were validated with different shRNAs against RUNX1. Genetic rescue experiments showed that RUNX1 mutants were unable to restore sensitivity to the drug compared to RUNX1 WT. Finally, modeling RUNX1 loss-of-function (LOF) in CSNK1A-depleted human CD34+ cells abrogated the effects of LEN on colony forming cell assays. Thus, RUNX1 function is required for the elimination of del(5q) cells by LEN. To understand the molecular mechanisms underlying the resistant phenotype, we performed RNA-seq on MDS-L cells treated with LEN for 24h. We observed a significant upregulation of Platelet specific genes (ITGB3, ITGA2B, VWF, THBD, SELP, TREML1, GATA1) coupled to downregulation of Cell Cycle genes (E2F2, E2F1, MCM5, CDKN1A), suggesting that LEN induces differentiation in to the Megakaryocytic (Meg) lineage. We found a significant upregulation of CD41+/CD61+ double positive cells after LEN exposure in vitro and in vivo, associated to the appearance of multinucleated cells. Importantly, the apoptotic response was associated to the emergence of the differentiating population. At the molecular level, CRBN is required for LEN-induced differentiation. Further downstream we identified IKZF1 degradation as key trigger, as IKZF1 overexpression restrained Meg differentiation and a IKZF1 dominant negative isoform enhanced it. In contrast, CSNK1A overexpression did not alter differentiation after LEN, but did reduce apoptotic induction. Moreover, we identified GATA2 targets enriched in LEN-regulated genes and showed that GATA2 overexpression or downregulation using shRNAs significantly increased or reduced LEN induced differentiation respectively. Finally, gene expression analysis after LEN exposure showed that Meg signatures were not enriched in resistant RUNX1 KO cells compared to WT control. Accordingly, RUNX1 KO cells did not undergo differentiation upon LEN exposure. RUNX1 LOF in CSNK1A-depleted primary human CD34+ cells blocked CFU-Mk growth in LEN treated cells. GATA2 overexpression was unable to restore LEN effects in RUNX1 deficient cells, suggesting a cooperative mechanism between both transcription factors. Luciferase assays using the human CD41 promoter showed that RUNX1 mutants reduced promoter transactivation compared to RUNX1 WT. Remarkably, we observed a similar phenotype for LEN-resistant TP53 KO cells. As a conclusion, our results suggest that GATA2, RUNX1 and TP53 cooperate to drive Meg differentiation after LEN-mediated degradation of IKZF1 protein. Loss of function mutations affecting RUNX1 or TP53 alter the activity of GATA2 transcriptional complex, rendering del(5q) cells unresponsive to LEN. Disclosures Platzbecker: Celgene: Research Funding.

This research investigates the storm-water quality at road-salt-storage facilities located at Virginia Department of Transportation (VDOT) winter maintenance locations and investigates the feasibility of a sustainable solution to better manage the salt-contaminated storm-water runoff. Collection ponds are currently used at most salt-storage sites to contain highly saline runoff and prevent its release into the environment. During a synoptic, winter-time sampling, chloride-ion concentrations in these ponds were found to be significantly greater than state and federal regulatory guidelines for surface-water-quality criteria, with individual values exceeding 2000 mg/L. The pond water is currently treated as a waste product by VDOT, resulting in significant costs for disposal. However, this saline pond water can potentially be recycled to produce concentrated brine solutions, which can then be used by VDOT for either prewetting dry salt during application to roadways or for direct brine application. Laboratory and field tests have been performed using a bench-scale brine generation system to quantify the effects of hydraulic retention time, temperature, and influent-water quality on system performance. Results of these studies have found that the storm-water runoff captured in collection ponds requires no pretreatment before entering the brine generation system and can effectively produce brine at the target salt concentration. Results of a cost-benefit analysis indicate that it is possible under multiple scenarios to recover the investment capital of implementing brine generation at all VDOT winter maintenance locations, typically within a 4-year horizon. © 2009 American Institute of Chemical Engineers Environ Prog, 2009