Early Entry Research Articles

Pig IFITMs are restriction factors for Japanese encephalitis virus

Objective: Japanese encephalitis virus (JEV) is responsible for one of the most serious epidemics of encephalitis in the world. JEV uses pigs as its main hosts and spreads among vertebrates and humans mediated by Culex mosquitoes. The prevention and control of JEV spread in pigs is one of the most effective measures to protect global public health. Interferon-inducible transmembrane proteins (IFITMs) are small membrane-spanning proteins that were identified as innate antiviral factors against multiple pathogenic viruses, especially enveloped viral pathogens. This study aims to verify whether pig interferon-inducible transmembrane proteins (pIFITMs) inhibit JEV and investigate the related molecular mechanisms of anti-JEV. Methods: Transient expression and RNA interference technology were used to overexpress and silence IFITMs gene. Three different cell lines, PK15, HEK293 and Huh7, were transfected with recombinant pIFITMs-expressing plasmids. The lentiviral vectors harboring RNAi sequences targeting pIFITMs were introduced into PK15 cells. Quantitative real-time PCR was used to determine the antiviral activities of pIFITMs through measuring and analyzing the virus copy number of JEV (SA14-14-2 strain) in the supernant of pIFITMs overexpression or silencing cells 48 hours post transfection. The expression of the related proteins was examined by western blot. The fusion vectors inserted with pig IFITM1-EGFP were constructed and introduced into three different cell lines respectively. Then Laser Co-focus light microscopy was used to observe the subcellular localization. The key active amino acids of pIFITM1 were analyzed by investigating the anti-JEV effect of the cysteine mutants produced with PCR site directed mutagenesistechnology. Results: In three different cell lines, PK15, HEK293 and Huh7, all of three pig IFITM proteins, pIFITM1, pIFITM2, and pIFITM3 could inhibit the replication of JEV whether through transient gene over-expression methods or RNA interference silencing. And that, among three pig IFITMs, pIFITM1 showed the strongest anti-JEV effect. The anti-JEV activity of pIFITM1 manifested at the early entry stage. In PK15, BHK21, and HEK293 cells, before virus infection, pIFITM1 was located in the plasma membrane area, and after infection, transferred to the membranous structures outside the nucleus. The S-palmitoylated cysteines at position 50, 51 and 84 of pIFITM1 had significant effect on virus replication. Conclusions: Pig interferon-inducible transmembrane proteins are restriction factors for JEV infection and have potentials in the prevention of virus spread. Our results provide some new sights into understanding the antiviral activity of pig IFITMs.

Influence of boundary conditions in DEM models of sublevel caving on dilution and recovery

Discrete element modelling is common approach for analysis of dynamical processes that include granular materials. Method is commonly applied for the modeling of the gravity flow of the broken rock in caving operations. Herein, comparison of two different approaches in such modelling was presented. Two models, ring wide and symmetrically divided, were used for comparison. Ore dilution and recovery were monitored as main comparison parameters, but also the processing time of such models and spatial shape of extraction zones. Ring wide model show greater ore dilution and lower recovery, while half wide models show the opposite results. Cumulative dilution differs for 10%, while ore recoveries differ in approximately 15% for same extraction levels. Also, dilution entrance points are different, where ring wide model shows earlier dilution entrance at about 15% of extraction, while dilution enters at 25% in half wide models. Processing speed is increased in half wide model due to the reduction in number of elements, but overall processing time is nearly the same due to the frequent hang ups inside the half wide model. Shape and size of the extraction zones are very different. Half wide model shows much higher extraction zone in contrary with ring wide, while depth of extraction zone is greater in ring wide models which corresponds with higher and earlier entry of dilution.

Discrete-Event Simulation of the Establishment of a Bare Beachhead for Long-Term Joint Logistics over the Shore (JLOTS) Operations

The United States military uses Joint Logistics Over-the-Shore (JLOTS) operations to move soldiers, vehicles, and equipment across the globe for military and humanitarian missions. These logistics operations can only be accomplished through cooperation between commanders in all services. The U.S. Army Engineer Research and Development Center is developing a tool to analyze a set of early entry alternatives to optimize mission effectives and efficiencies in order to facilitate assured mobility and freedom of movement. This program is currently being developed under the name Planning Logistics Analysis Network System (PLANS). PLANS comprehensively covers air, land, and sea transportation infrastructure, regions of avoidance, and more. This research addresses a gap in strategic and operational planning by modeling the establishment of JLOTS operations on bare beach environments. The West Point developed discrete event simulation will determine the amount of time it takes to prepare a beach to sustain JLOTS operations under varying environmental and operational conditions.

Misuse of antenatal care and its association with adverse outcomes of pregnancy in a Southern rural area of Vietnam

ABSTRACTResearchers in Vietnam reported a high percentage of pregnant women attending ANC at least once, but an insufficient utilization of ANC services remains. The evidence demonstrating how the utilization of these services affect pregnancy outcome is not documented in Vietnam. We investigated the association between the misuse of ANC services and pregnancy outcome, and assessed other determinants associated with ANC services utilization. We conducted a prospective community-based study in Trang Bom district, Dong Nai, during 12 consecutive months. Data were collected using a structured questionnaire and medical records. Women were followed up to delivery. Misuse of ANC services, related factors and its association with adverse events were assessed using logistic regression. Out of 3301 pregnant women, 91% initiated an ANC visit within first trimester, 95% attended at least three ANC visits, but a low percentage of pregnant women underwent blood and urine tests at least once (20% and 39%, respectively). Factors significantly associated with a higher risk of adverse outcomes were lack of blood test and urine test, parity ≥ 3, ANC visits < 3, history of an adverse outcome, and having a clinical condition. Blood and urine tests were less frequently used by young women, women from ethnic minorities, women using only private facilities for ANC attendance, and women with a low number of ANC visits. Despite a high percentage of early entry into ANC and of at least three ANC visits, misuse of ANC services still exists and contributes to adverse outcomes. There is a need to increase the awareness of women on the benefits of ANC services by educating young women as well as women with several children. Health workers should be encouraged to propose suitable ANC services to pregnant women.

Vital Signs: Human Immunodeficiency Virus Testing and Diagnosis Delays - United States.

BackgroundPersons unaware of their human immunodeficiency virus (HIV) infection account for approximately 40% of ongoing transmissions in the United States. Persons are unaware of their infection because of delayed HIV diagnoses that represent substantial missed opportunities to improve health outcomes and prevent HIV transmission.MethodsData from CDC’s National HIV Surveillance System were used to estimate, among persons with HIV infection diagnosed in 2015, the median interval (and range) from infection to diagnosis (diagnosis delay), based on the first CD4 test after HIV diagnosis and a CD4 depletion model indicating disease progression and, among persons living with HIV in 2015, the percentage with undiagnosed infection. Data from CDC’s National HIV Behavioral Surveillance were analyzed to determine the percentage of persons at increased risk for HIV infection who had tested in the past 12 months and who had missed opportunities for testing.ResultsAn estimated 15% of persons living with HIV in 2015 were unaware of their infection. Among the 39,720 persons with HIV infection diagnosed in 2015, the estimated median diagnosis delay was 3.0 years (interquartile range = 0.7–7.8 years); diagnosis delay varied by race/ethnicity (from 2.2 years among whites to 4.2 years among Asians) and transmission category (from 2.0 years among females who inject drugs to 4.9 years among heterosexual males). Among persons interviewed through National HIV Behavioral Surveillance, 71% of men who have sex with men, 58% of persons who inject drugs, and 41% of heterosexual persons at increased risk for HIV infection reported testing in the past 12 months. In each risk group, at least two thirds of persons who did not have an HIV test had seen a health care provider in the past year.ConclusionsDelayed HIV diagnoses continue to be substantial for some population groups and prevent early entry to care to improve health outcomes and reduce HIV transmission to others.Implications for Public Health PracticeHealth care providers and others providing HIV testing can reduce HIV-related adverse health outcomes and risk for HIV transmission by implementing routine and targeted HIV testing to decrease diagnosis delays.

Types and delivery of emotional support to promote linkage and engagement in HIV care.

PurposeDespite recommendations for early entry into human immunodeficiency virus (HIV) care, many people diagnosed with HIV delay seeking care. Multiple types of social support (ie, cognitive, emotional, and tangible) are often needed for someone to transition into HIV care, but a lack of emotional support at diagnosis may be the reason why some people fail to stay engaged in care. Thus, the purpose of this study was to identify how people living with HIV conceptualized emotional support needs and delivery at diagnosis.MethodWe conducted a secondary analysis of qualitative data from 27 people living with HIV, many of whom delayed entry into HIV care.ResultsParticipants described their experiences seeking care after an HIV diagnosis and identified components of emotional support that aided entry into care – identification, connection, and navigational presence. Many participants stated that these types of support were ideally delivered by peers with HIV.ConclusionIn clinical practice, providers often use an HIV diagnosis as an opportunity to educate patients about HIV prevention and access to services. However, this type of social support may not facilitate engagement in care if emotional support needs are not met.

The E3 ubiquitin ligase APC/CCdh1 degrades MCPH1 after MCPH1-βTrCP2-Cdc25A-mediated mitotic entry to ensure neurogenesis.

Mutations of microcephalin (MCPH1) can cause the neurodevelopmental disorder primary microcephaly type 1. We previously showed that MCPH1 deletion in neural stem cells results in early mitotic entry that distracts cell division mode, leading to exhaustion of the progenitor pool. Here, we show that MCPH1 interacts with and promotes the E3 ligase βTrCP2 to degrade Cdc25A independent of DNA damage. Overexpression of βTrCP2 or the knockdown of Cdc25A remedies the high mitotic index and rescues the premature differentiation of Mcph1-deficient neuroprogenitors invivo MCPH1 itself is degraded by APC/CCdh1, but not APC/CCdc20, in late mitosis and G1 phase. Forced MCPH1 expression causes cell death, underlining the importance of MCPH1 turnover after mitosis. Ectopic expression of Cdh1 leads to premature differentiation of neuroprogenitors, mimicking differentiation defects of Mcph1-knockout neuroprogenitors. The homeostasis of MCPH1 in association with the ubiquitin-proteasome system ensures mitotic entry independent of cell cycle checkpoint. This study provides a mechanistic understanding of how MCPH1 controls neural stem cell fate and brain development.

Autobiography of Mark Twain, Volume 3

Autobiography of Mark Twain, Volume 3

The influence of a cachexia clinic on palliative care integration in oncology.

124 Background: Appetite and weight loss are common in patients with advanced cancer and specialized cachexia clinics have been established to address these symptoms. Given the association between anorexia/cachexia and other adverse symptoms, these patients may also benefit from specialty level palliative care (PC). However, referral to outpatient specialty level PC is often delayed or does not occur. We sought to examine the prevalence of other factors associated with appetite and weight loss in patients with advanced cancer and the impact of a specialized cachexia clinic on identification and treatment of other PC needs. Methods: The records of patients referred by their Oncologist to the cachexia clinic of a cancer center from August 2016 to June 2017 were reviewed retrospectively. Subjects who had been referred to PC by their Oncologist were excluded. Patients had been assessed for symptom burden using the Edmonton Symptom Assessment Scale (ESAS-r). Patients identified with PC needs had been referred to the PC clinic for follow-up within 30 days after cachexia clinic consultation. Results: Thirty subjects were evaluated in the cachexia clinic (average age 68 years; 63% female). The predominant diagnosis was lung cancer (70%). An average of 6 symptoms per patient were in the moderate to severe range on ESAS, excluding appetite. Depression, fatigue and pain were most common. The average cachexia clinic total ESAS score was 51.61. Only 17% of patients had completed advance directives. Ninety-three % of patients were referred to PC and 68% were seen. The average number of PC visits was 2.79. Within the PC clinic, advance directive completion increased to 37%, goals of care discussion occurred with 50% and 17% received hospice referrals. At the most recent follow-up in the PC clinic, the average total ESAS score had decreased by 11.44 (22%) and all ESAS item scores were improved on average. Conclusions: The cachexia clinic proved a useful means to identify other PC needs and achieve effective PC referrals. We suggest this is proof of concept that specialty clinics can be a meaningful way to achieve an earlier entry point to comprehensive PC in patients who were not previously referred by their Oncologists.

A Study on Early Career Formation and Entry Process into a Decent Job of Graduates of Colleges in Regions other than Seoul

이 연구는 비서울 소재 대졸자의 초기 경력탐색과 괜찮은 일자리 진입과정 사례를 분석하여 그들의 특별한 경험과정을 내부적 요인, 외부적 요인 등을 총체적으로 탐색하고 성공요인과 과정을 밝혀내는데 있다. 연구방법은 근거이론이며, 연구 참여자는 2013년과 2014년에 졸업한 비서울 소재 대졸자로 괜찮은 일자리에 입직한 15명을 선정하였다. 연구결과는 다음과 같다. 첫째, 비서울 소재 대졸자의 초기 경력탐색 과정은 ‘꿈 태동기’, ‘흥미 잠복기’, ‘현실 변혁기’, ‘진로 몰입기’ 네 단계인 것으로 밝혀졌다. 참여자들은 대학입학 전까지 다양한 꿈을 탐색하고 대학입학 후에는 꿈을 이루기 위한 준비보다 새로운 환경에 적응하는 과정을 거쳤다. 참여자들은 진로 몰입기 시점이 되어서야 변화의 필요성을 인식하고 실천하는 전환 과정을 겪었다. 둘째, 참여자의 괜찮은 일자리에 진입과정은 ‘목표 설정기’, ‘취업 준비기’, ‘일자리 접촉기’ 세단계의 준비 과정을 거쳐 ‘괜찮은 일자리 안착기’에 들어서는 것으로 나타났다. 대부분의 참여자들은 취업을 준비하는 과정에서 도움을 받을 수 있는 다양한 물리적 자원과 심리적 자원을 확보하였다. 마지막으로 참여자들이 괜찮은 일자리에 입직할 수 있었던 주요 요인은 ‘차별화된 스펙 쌓기’, ‘도전과 좌절 그리고 성찰을 통한 재도전’, ‘꿈-흥미와 적성-능력-일이 연계된 괜찮은 일자리 진입성공’이라는 범주로 도출되었다. 이 연구결과를 바탕으로 비서울 소재 대학생들에게 적합한 경력개발 프로그램 설계 및 프로그램 효과성 측정이 후속 연구로 이루어 질 필요가 있음을 시사한다.The purpose of this study is to inquire into the internal/external factors in their special experiential process comprehensively and make it clear their success factors & process by analyzing the early career formation of graduates of colleges located in the regions other than Seoul, and the case of the entry process into a decent job. With the research method based on the grounded theory, this study selected 15 graduates who were graduates of colleges(in 2013, and 2014) located in the regions other than Seoul, and found a decent job as the research participants. The research results are as follows: First, it was found that the early career formation process of the graduates of colleges located in the regions other than Seoul consisted of 4 phases, i.e. ‘the beginning period of dreams’, ‘interest latency’, ‘reality transformational period’ and ‘career commitment period.’ The research results indicated that the research participants had searched diverse dreams before they entered college, and went through the process of adapting themselves to new environment rather than preparation for achieving their dreams. Also, participants didn’t perceive the necessity of a change and undergo the transitional process of practice until the time for career commitment period came. Second, the participants’ entry process into a decent job was found to enter ‘the safe settlement period’ through the three-phased preparatory process, i..e. ‘ goal-setting period’, ‘preparatory period for employment’, and ‘job contact period.’ In addition, it was found that most of the participants managed to secure diverse physical resources and psychological resources which made it possible for them to get help in the process of preparing for their employment. Lastly, the major factors leading them into finding a decent job were deducted as the categories like ‘differentiated specification building’, ‘re-challenge through challenges, frustration and introspection’, and ‘success in the entry into a decent job in connection with dream-interest and aptitude-ability-work.’ With the core category as the center, the study confirmed the relevancy between continuously appearing categories; as a result, it was classified as three sorts of types, i.e. ‘goal-archiving type’, ‘interest commitment type’, and ‘basic faithfulness type.’ On the basis of these research results, this study implies that there is the necessity of career development program design suited for students of colleges located in the regions other than Seoul, and program effectiveness measurement as a follow-up research.

Cut to the Quick: The Consequences of Youth Violent Victimization for the Timing of Dating Debut and First Union Formation.

Concentrated in adolescence, violent victimization is developmentally disruptive. It undermines physical, mental, and socioemotional well-being and compromises youths' transitions into and progression through key life course tasks. Youth violent victimization (YVV) has been linked to precocious exits from adolescence and premature entries into adulthood. This includes early entry into coresidential romantic unions, which is but one stage of a relationship sequence generally beginning via dating debut. Using data from the National Longitudinal Study of Adolescent to Adult Health (Add Health) and Cox regression, we examine the effects of YVV on the timing of dating debut and progression to first coresidential unions during adolescence and the transition to adulthood. We pay particular attention to how these effects may be structured by age and gender. Overall, we find that victims begin dating sooner and progress more quickly from dating to first unions than do non-victims. However, youths victimized in early adolescence withdraw from dating and union formation, whereas late adolescent victims appear to overinvest in relationships-at least temporarily-displaying accelerated entry into dating and rapid progression to first unions. We conclude by discussing the implication of these age-graded patterns for intervention efforts and youth well-being more broadly.

A5087 - Anti-diabetic Effects of Ileal Transposition Surgery on Obese mice

On the basis of the nutrient passage patterns, bariatric surgery is broadly divided into restrictive, malabsorptive, and/or metabolic procedures. It has been postulated that the early entry of nutrients into the distal small intestine after Roux-en-Y or other intestinal bypass procedures elicits a neuroendocrine response that causes improved glucose homeostasis, which is called “ileal break” mechanism. Ileal transposition (IT) is a hindgut procedure that produce weight loss and improve glycemic control by translocating a segment of the distal ileum proximally to the upper jejunum without altering the length of gastrointestinal tract or gastric restriction. The antidiabetic mechanisms of IT remain poorly understood. Ileal transposition has not been performed in the mouse model because of difficulty in constructing a functional small bowel anastomosis. The aim of this study was to evaluate the optimal surgical technique to construct an IT procedures in the mouse model and to examine the anti-diabetic effects of IT surgery on high-fat diet (HFD) induced obese mice.

Entry-timing, business groups and early-mover advantage within industry merger waves in emerging markets: A study of Indian firms

Merger waves are periods of intense and concentrated merger activity which exhibit a wave-like pattern. Drawing upon the resource-based view, we examine the timing of entry and early-mover advantage within merger waves. Following a robust simulation-based methodology of wave analysis, we identify merger waves in eight industries during the time period 2000–2014. Firms affiliated to business groups were found to be early movers. A higher degree of internationalization is also associated with early movement of a firm. Within business groups, their multi-entity character is positively associated with early entry whereas their board interlock was negatively related with entry-timing. Further, early moving acquirers reap superior post-acquisition performance, thereby suggesting that early-mover advantages exist within merger waves.

Educational Progress of Looked-After Children in England: A Study Using Group Trajectory Analysis.

Looked-after children in local authority care are among the most disadvantaged, and measures of their well-being, including educational outcomes, are poorer than other children's. The study sample consisted of all children in England born in academic years 1993 to 1994 through 1997 to 1998 who were in local authority care at any point during the academic years 2005 to 2006 through 2012 to 2013 and for whom results of national tests in literacy and numeracy were available at ages 7, 11, and 16 (N = 47 500). Group trajectory analysis of children's educational progress identified 5 trajectory groups: low achievement, late improvement, late decline, predominant, and high achievement. Being looked after earlier was associated with a higher probability of following a high achievement trajectory and a lower probability of following a late decline trajectory. For children first looked after between ages 7 and 16, having a longer total time looked after by age 16 was associated with a higher probability of following a high achievement trajectory. For children with poor outcomes at ages 7 and 11, being looked after by age 16 was associated with an increased chance of educational improvement by age 16. This study provides evidence that early entry into care can reduce the risk of poor educational outcomes. It also establishes group trajectory analysis as an effective method for analyzing the educational progress of looked-after children, with the particular strength that it allows factors associated with a late decline or improvement in educational progress to be identified.

Innovative Models for High-Risk Patients Use Care Coordination and Palliative Supports to Reduce End-of-life Utilization and Spending.

Background and ObjectivesCare coordination and palliative care supports are associated with reduced anxiety, fewer hospital admissions, and improved quality of life for patients and their families. Early palliative care can result in savings in the end-of-life period, but there is limited evidence that larger-scale models can improve both utilization and the cost of care. Three models that received Health Care Innovation Awards from the Centers for Medicare & Medicaid Services aimed to improve quality of care and reduce cost through the use of innovative care coordination models. This study explores the total cost of care and selected utilization outcomes at the end-of-life for these innovative models, each of which enrolled adults with multiple chronic conditions and featured care coordination with advance care planning as a component of palliative care. These included a comprehensive at-home supportive care model for persons predicted to die within a year and two models offering advance care planning in nursing facilities and during care transitions.Research Design and MethodsWe used regression models to assess model impacts on costs and utilization for high-risk Medicare beneficiaries participating in the comprehensive supportive care model (N = 3,339) and the two care transition models (N = 587 and N = 277) who died during the study period (2013–2016), relative to a set of matched comparison patients.ResultsComparing participants in each model who died during the study period to matched comparators, two of the three models were associated with significantly lower costs in the last 90 days of life ($2,122 and $4,606 per person), and the third model showed nonsignificant differences. Two of the three models encouraged early hospice entry in the last 30 days of life. For the comprehensive at-home supportive care model, we observed aggregate savings of nearly $19 million over the study period. One care transition model showed aggregate savings of over $500,000 during the same period. Potential drivers of these cost savings include improved patient safety, timeliness of care, and caregiver support.Discussion and ImplicationsTwo of the three models achieved significant lower Medicare costs than a comparison group and the same two models also sustained their models beyond the Centers for Medicare & Medicaid Services award period. These findings show promise for achieving palliative care goals as part of care coordination innovation.

Respiratory syncytial virus (RSV) entry is inhibited by serine protease inhibitor AEBSF when present during an early stage of infection

BackgroundHost proteases have been shown to play important roles in many viral activities such as entry, uncoating, viral protein production and disease induction. Therefore, these cellular proteases are putative targets for the development of antivirals that inhibit their activity. Host proteases have been described to play essential roles in Ebola, HCV, HIV and influenza, such that specific protease inhibitors are able to reduce infection. RSV utilizes a host protease in its replication cycle but its potential as antiviral target is unknown. Therefore, we evaluated the effect of protease inhibitors on RSV infection.MethodsTo measure the sensitivity of RSV infection to protease inhibitors, cells were infected with RSV and incubated for 18 h in the presence or absence of the inhibitors. Cells were fixed, stained and studied using fluorescence microscopy.ResultsSeveral protease inhibitors, representing different classes of proteases (AEBSF, Pepstatin A, E-64, TPCK, PMSF and aprotinin), were tested for inhibitory effects on an RSV A2 infection of HEp-2 cells. Different treatment durations, ranging from 1 h prior to inoculation and continuing for 18 h during the assay, were evaluated. Of all the inhibitors tested, AEBSF and TPCK significantly decreased RSV infection. To ascertain that the observed effect of AEBSF was not a specific feature related to HEp-2 cells, A549 and BEAS-2B cells were also used. Similar to HEp-2, an almost complete block in the number of RSV infected cells after 18 h of incubation was observed and the effect was dose-dependent. To gain insight into the mechanism of this inhibition, AEBSF treatment was applied during different phases of an infection cycle (pre-, peri- and post-inoculation treatment). The results from these experiments indicate that AEBSF is mainly active during the early entry phase of RSV. The inhibitory effect was also observed with other RSV isolates A1998/3–2 and A2000/3–4, suggesting that this is a general feature of RSV.ConclusionRSV infection can be inhibited by broad serine protease inhibitors, AEBSF and TPCK. We confirmed that AEBSF inhibition is independent of the cell line used or RSV strain. The time point at which treatment with the inhibitor was most potent, was found to coincide with the expected moment of entry of the virion with the host cell.

Capabilities development and deployment activities in born global B-to-B firms for early entry into international markets

This paper sets out to understand how entrepreneurial founders of born global firms acquire, transform and deploy new knowledge resources for early internationalization. Adopting a dynamic capabilities view and using a sample of high-tech B-to-B firms, we report that the new firm's early entry into international markets is executed through three transitionary phases. Founders transform the operational capabilities they endow to the firm, develop dynamic capabilities for use in opportunity exploitation, and deploy these to develop knowledge-intensive products that they take to chosen niche markets. The paper contributes to the B-to-B global marketing literature by uniting it with born global and INV internationalization research, and elucidating the three phases through which founders manage early internationalization. The roles played by entrepreneurial founders and particular capabilities are discussed.

A role for random, humidity-dependent epiphytic growth prior to invasion of wheat by Zymoseptoria tritici

Zymoseptoria tritici causes Septoria leaf blotch of wheat. The prevailing paradigm of the Z. tritici-wheat interaction assumes fungal ingress through stomata within 24–48h, followed by days of symptomless infection. This is extrapolated from studies testing the mode of fungal ingress under optimal infection conditions. Here, we explicitly assess the timing of entry, using GFP-tagged Z. tritici. We show that early entry is comparatively rare, and extended epiphytic growth possible. We test the hypotheses that our data diverge from earlier studies due to: i. random ingress of Z. tritici into the leaf, with some early entry events; ii. previous reliance upon fungal stains, combined with poor attachment of Z. tritici to the leaf, leading to increased likelihood of observing internal versus external growth, compared to using GFP; iii. use of exceptionally high humidity to promote entry in previous studies. We combine computer simulation of leaf-surface growth with thousands of in planta observations to demonstrate that while spores germinate rapidly on the leaf, over 95% of fungi remain epiphytic, growing randomly over the leaf for ten days or more. We show that epiphytic fungi are easily detached from leaves by rinsing and that humidity promotes epiphytic growth, increasing infection rates. Together, these results explain why epiphytic growth has been dismissed and early ingress assumed. The prolonged epiphytic phase should inform studies of pathogenicity and virulence mutants, disease control strategies, and interpretation of the observed low in planta growth, metabolic quiescence and evasion of plant defences by Zymoseptoria during symptomless infection.

Abstract 306: Antitumor effect of Wee1 inhibitor in gastric cancer cells

Abstract Background: A cell cycle checkpoint is a key step to watch any damages and, it is needed to maintain genomic stability. Thus, to jeopardize cell cycle checkpoint is an attractive strategy for cancer treatment. Wee1 is a G2 cell cycle regulator that phosphorylate cdc2 on tyrosine 15 for inhibiting its activity. The activation of Wee1 can delay G2 phase progression and cells can have a chance to repair DNA damages. Therefore, to inhibit wee1 can accelerate DNA damage accumulation in the cell. AZD1775 is a first-in-class wee1 inhibitor. So far, previous studies showed the antitumor effect of AZD1775 as a mono-therapeutic agent or combination treatment with other chemo-agents. However, the activity of wee1 in gastric cancer (GC) cells is not yet fully understood. Thus, we would like to study whether AZD1775 has an antitumor effect against GC or not. Materials and methods: We determined the antitumor effects of AZD1775 on GC cells using a cytotoxicity assay, cell cycle analysis, immunofluorescence assay and western blotting by AZD1775 treatment. Results: AZD1775 effectively inhibit GC cell proliferation. The TP53 gene status or p-Wee1 expression levels were not associated with AZD1775 sensitivity. AZD1775 effectively down-regulated p-Wee1 and p-cdc2 expressions in SNU-601, a sensitive cell line. However, these effects were not significant in KATO-III cells, which is less sensitive to AZD1775. Cell cycle analysis revealed that the cells had different responses by AZD1775 treatment. SNU-601 cells were enriched in S phase when we decide cell cycle only by its DNA contents. However, KATO-III cells were arrested in G2/M phase. The sub-G1 population was dramatically increased only in SNU-601 cells. A population of BrdU-positive and p-HH3 positive cells was significantly increased in the SNU-601 cells which indicate that premature mitosis was increased. However, no differences were observed in KATO-III cells. Moreover, due to premature mitotic entry, multi-nucleated cells were observed only in SNU-601 cells. Conclusion: AZD1775 effectively inhibited the growth of GC cell lines. The inhibition of cdc2 by AZD1775 was observed only in a sensitive cell line. Moreover, early mitotic entry was induced by AZD1775, and it led the induction of mitotic catastrophe. This result indicates that AZD1775 have an antitumor effect on GC and the possibility of adoption to the clinical trial design. Citation Format: Seongyeong Kim, Ahrum Min, So Hyeon Kim, Dong Hyeon Ha, Hyemin Jang, Yu JIn Kim, Hee Jun Kim, Kyung-Hun Lee, Tae-Yong Kim, Do-Youn Oh, Yung-Jue Bang, Seock-Ah Im. Antitumor effect of Wee1 inhibitor in gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 306. doi:10.1158/1538-7445.AM2017-306

Abstract 23: Antibody targeting of ADAM8 for treatment of triple-negative breast cancer

Abstract Triple-Negative Breast Cancer (TNBC) accounts for 25% of breast cancer deaths. Current therapeutic options are restricted to chemotherapy (CT) and radiation, which fail to block disease progression. Recently, we identified the cell surface protein ADAM8 as a critical driver of TNBC tumor growth and of metastasis in half of all breast cancers (EMBO Mol. Med. 6:278, 2014). Elevated ADAM8 levels correlate with poor breast cancer patient outcome. One third of TNBC patient samples have high ADAM8 levels, while it is undetectable in normal breast tissue. ADAM8 Metalloproteinase (MP) and Disintegrin (DI) domains promote angiogenesis needed for tumor growth, and cancer cell adhesion to and invasion through the endothelium needed for dissemination, respectively. Using a prototype reagent in mice, we validated a unique antibody-based strategy that simultaneously targets ADAM8 MP/DI activities as an effective therapeutic intervention in TNBC. Our group has now prepared a panel of pre-clinical, highly specific anti-human ADAM8 mouse monoclonal antibodies (ADPs) of either IgG1 or IgG2 subclass with dual MP/DI antagonist activity. The top 6 ADPs bind ADAM8 with low dissociation constants of 1.3 x 10-9 M to 7.23 x 10-8 M. The 4 most effective inhibitory ADPs in cell-based assays were screened in a single-dose efficacy study (10 mg/kg 3x/wk) against pre-existing MDA-MB-231 cell-derived tumors (50-75 mm3). ADP2 and ADP13 treatment resulted in significant tumor volume reduction, of 47% and 52%, respectively. Dose-response curves are in progress. ADP13 was also tested in a neo-adjuvant model for its ability to reduce metastases. Antibody treatment was initiated as above and continued for 6 weeks after tumors were resected at a volume of ~200 mm3. Metastases in dissected organs were examined using biophotonic imaging. ADP13 substantially decreased metastasis to multiple organs. Lastly, a neo-adjuvant survival experiment was performed, as above, except that following surgical resection of tumors mice were treated for 12 weeks. Recurrence at the primary site was assessed using palpation. Mice were sacrificed when recurrent tumors reached 900 mm3. ADP13 treatment resulted in significantly improved disease-free survival (P=0.05) and overall survival (P=0.03). More recently, we noted that ADAM8 regulates miRNAs associated with resistance to CT and showed that ADAM8 inhibition sensitizes TNBC cells to Cisplatin, Paclitaxel, and Doxorubicin. In vivo testing of a combinatorial anti-ADAM8 + CT approach is in progress and may present a promising strategy for early entry into the clinic. Conclusions: A new anti-ADAM8 antibody therapeutic shows efficacy against TNBC tumor growth and dissemination, and improves survival in pre-clinical testing. We hypothesize that addition of an anti-ADAM8 antibody to current CT standards of care will enhance tumor killing, reduce metastasis, and increase survival of patients with ADAM8-expressing TNBC. Citation Format: Sonia G. Das, Stefania Pianetti, Gail E. Sonenshein, Nora D. Mineva. Antibody targeting of ADAM8 for treatment of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 23. doi:10.1158/1538-7445.AM2017-23