An economic evaluation of restorative justice post-sentence in England and Wales.

Introduction: Participation in restorative justice interventions post-sentence has been shown to reduce reoffending and mitigate harm to victims. Investment in, and access to, restorative justice remains limited in England and Wales. An economic model was developed to synthesize the available evidence in order to develop contemporary and robust estimates of the economic impact of investment in restorative justice interventions. Methods: This research focused on direct and indirect restorative justice interventions for victims and offenders post-sentence in England and Wales. Included offences were those with an identifiable victim. A model was developed to estimate the social benefit-cost ratio of restorative justice, as well as the direct financial return to the criminal justice system. The modeled benefits of restorative justice included reductions in reoffending and direct wellbeing benefits for victims. It was not possible to incorporate direct wellbeing benefits for offenders due to evidence gaps. Results: In the model, 8% of referrals to restorative justice resulted in direct restorative justice interventions and 19% resulted in indirect Restorative justice interventions. The modeled cost of the restorative justice pathway per direct intervention was £3,394. The base case estimate for the social benefit-cost ratio of restorative justice was £14 per £1 invested, with a direct return to the criminal justice system of £4 as a result of substantial reductions in reoffending. Scenario analysis suggested a plausible range of £7 to £20 social benefit per £1 invested. Hypothetically, increasing the proportion of eligible cases referred for a restorative justice intervention from 15 to 40% could be associated with an increase in investment of £5 m, and benefits to the criminal justice system totaling £22 m, implying a net saving of £17 m. Conclusion: The research suggests that Restorative justice has the potential to yield a substantial social return on investment (SROI) and direct return on investment to the criminal justice system. The economic case for investment in restorative justice centers on identifying offenders with a high risk of offending and enabling them to participate in an intervention that has been repeatedly demonstrated to help them to change their behavior.

Cost-effectiveness of an insertable cardiac monitor in a high-risk population in the US.

BACKGROUND: Insertable cardiac monitors (ICMs) are a clinically effective means of detecting atrial fibrillation (AF) in high-risk patients, and guiding the initiation of non-vitamin K oral anticoagulants (NOACs). Their cost-effectiveness from a US clinical payer perspective is not yet known. The objective of this study was to evaluate the cost-effectiveness of ICMs compared to standard of care (SoC) for detecting AF in patients at high risk of stroke (CHADS2 ≥ 2), in the US. METHODS: Using patient data from the REVEAL AF trial (n = 393, average CHADS2 score = 2.9), a Markov model estimated the lifetime costs and benefits of detecting AF with an ICM or with SoC (specifically intermittent use of electrocardiograms and 24-h Holter monitors). Ischemic and hemorrhagic strokes, intra- and extra-cranial hemorrhages, and minor bleeds were modelled. Diagnostic and device costs, costs of treating stroke and bleeding events and medical therapy-specifically costs of NOACs were included. Costs and health outcomes, measured as quality-adjusted life years (QALYs), were discounted at 3% per annum, in line with standard practice in the US setting. One-way deterministic and probabilistic sensitivity analyses (PSA) were undertaken. RESULTS: Lifetime per-patient cost for ICM was $31,116 versus $25,330 for SoC. ICMs generated a total of 7.75 QALYs versus 7.59 for SoC, with 34 fewer strokes projected per 1000 patients. The model estimates a number needed to treat of 29 per stroke avoided. The incremental cost-effectiveness ratio was $35,528 per QALY gained. ICMs were cost-effective in 75% of PSA simulations, using a $50,000 per QALY threshold, and a 100% probability of being cost-effective at a WTP threshold of $150,000 per QALY. CONCLUSIONS: The use of ICMs to identify AF in a high-risk population is likely to be cost-effective in the US healthcare setting.

Pollinator foraging tactics have divergent consequences for the mating system of a tropical plant.

Resolving the consequences of pollinator foraging behaviour for plant mating systems is a fundamental challenge in evolutionary ecology. Pollinators may adopt particular foraging tactics: complete trapline foraging (repeated movements along a fixed route), sample-and-shift trapline foraging (a variable route that incorporates information from previous experiences) and territorial foraging (stochastic movements within a restricted area). Studies that integrate these pollinator foraging tactics with plant mating systems are generally lacking. We investigate the consequences of particular pollinator foraging tactics for Heliconia tortuosa. We combine parentage and sibship inference analysis with simulation modelling to: estimate mating system parameters; infer the foraging tactic adopted by the pollinators; and quantify the impact of pollinator foraging tactics on mating system parameters. We found high outcrossing rates, ubiquitous multiple paternity and a pronounced departure from near-neighbour mating. We also found that plants repeatedly receive pollen from a series of particular donors. We infer that the pollinators primarily adopt complete trapline foraging and occasionally engage in sample-and-shift trapline foraging. This enhances multiple paternity without a substantial increase in near-neighbour mating. The particular pollinator foraging tactics have divergent consequences for multiple paternity and near-neighbour mating. Thus, pollinator foraging behaviour is an important driver of the ecology and evolution of plant mating systems.

The Efficacy of Using Combination Therapy against Multi-Drug and Extensively Drug-Resistant Pseudomonas aeruginosa in Clinical Settings.

Pseudomonas aeruginosa is a Gram-negative bacterium which is capable of developing a high level of antibiotic resistance. It has been placed on the WHO's critical priority pathogen list and it is commonly found in ventilator-associated pneumonia infections, blood stream infections and other largely hospital-acquired illnesses. These infections are difficult to effectively treat due to their increasing antibiotic resistance and as such patients are often treated with antibiotic combination regimens. METHODS: We conducted a systematic search with screening criteria using the Ovid search engine and the Embase, Ovid Medline, and APA PsycInfo databases. RESULTS: It was found that in many cases the combination therapies were able to match or outperform the monotherapies and none performed noticeably worse than the monotherapies. However, the clinical studies were mostly small, only a few were prospective randomized clinical trials and statistical significance was lacking. CONCLUSIONS: It was concluded that combination therapies have a place in the treatment of these highly resistant bacteria and, in some cases, there is some evidence to suggest that they provide a more effective treatment than monotherapies.

The BlueScreen HC assay to predict the genotoxic potential of fragrance materials.

BlueScreen HC is a mammalian cell-based assay for measuring the genotoxicity and cytotoxicity of chemical compounds and mixtures. The BlueScreen HC assay has been utilized at the Research Institute for Fragrance Materials in a safety assessment program as a screening tool to prioritize fragrance materials for higher-tier testing, as supporting evidence when using a read-across approach, and as evidence to adjust the threshold of toxicological concern. Predictive values for the BlueScreen HC assay were evaluated based on the ability of the assay to predict the outcome of in vitro and in vivo mutagenicity and chromosomal damage genotoxicity assays. A set of 371 fragrance materials was assessed in the BlueScreen HC assay along with existing or newly generated in vitro and in vivo genotoxicity data. Based on a weight-of-evidence approach, the majority of materials in the data set were deemed negative and concluded not to have the potential to be genotoxic, while only a small proportion of materials were determined to show genotoxic effects in these assays. Analysis of the data set showed a combination of high positive agreement but low negative agreement between BlueScreen HC results, in vitro regulatory genotoxicity assays, and higher-tier test results. The BlueScreen HC assay did not generate any false negatives, thereby providing robustness when utilizing it as a high-throughput screening tool to evaluate the large inventory of fragrance materials. From the perspective of protecting public health, it is desirable to have no or minimal false negatives, as a false-negative result may incorrectly indicate the lack of a genotoxicity hazard. However, the assay did have a high percentage of false-positive results, resulting in poor positive predictivity of the in vitro genotoxicity test battery outcome. Overall, the assay generated 100% negative predictivity and 3.9% positive predictivity. In addition to the data set of 371 fragrance materials, 30 natural complex substances were evaluated for BlueScreen HC, Ames, and in vitro micronucleus assay, and a good correlation in all three assays was observed. Overall, while a positive result may have to be further investigated, these findings suggest that the BlueScreen HC assay can be a valuable screening tool to detect the genotoxic potential of fragrance materials and mixtures.

The Challenges of Outcomes-Based Contract Implementation for Medicines in Europe.

OBJECTIVES: The aim was to outline the challenges of implementing outcomes-based contracts (OBCs) in Europe. METHODS: A scoping review was conducted, building on the searches of a previous systematic review and updating them for December 2017 until May 2021. The combined results were screened, based on inclusion and exclusion criteria. All identified studies published in the English language that described specific OBC schemes for medicines in European countries were included. Insights into the challenges of OBCs were extracted and analysed to develop a conceptual framework. RESULTS: Ten articles from the previous systematic review matched our inclusion criteria, along with 14 articles from electronic searches. Analysis of these 24 articles and classification of the challenges revealed that there are multiple barriers that must be overcome if OBCs that benefit all stakeholders are going to be adopted widely across Europe. These challenges were grouped according to five key themes: negotiation framework; outcomes; data; administration and implementation; and laws and regulation. CONCLUSIONS: If the promise of OBCs is to be fully realised in Europe, there remain major challenges that need to be overcome by all stakeholders working in partnership. The overlapping and interconnected nature of these challenges highlights the complexity of OBC arrangements.

Cost-effectiveness of an insertable cardiac monitor to detect atrial fibrillation in patients with cryptogenic stroke.

Background: We assessed cost-effectiveness of insertable cardiac monitors (ICMs) in a US cryptogenic stroke population. Materials & methods: We modelled lifetime costs and quality-adjusted life years for three monitoring strategies post cryptogenic stroke: ICM starting immediately, ICM starting after Holter monitoring (delayed ICM) and standard of care involving intermittent ECG and Holter monitoring. Patient characteristics and detection efficacy were based on the CRYSTAL-AF trial. AF detection altered the modelled anticoagulation therapy and subsequent stroke and bleed risks. Results & conclusion: Immediate ICM was found to be cost-effective versus standard of care and cost-saving versus delayed ICM. Results were robust to sensitivity analyses. ICMs are a cost-effective diagnostic tool for the prevention of recurrent stroke in a US cryptogenic stroke population.

Comparing Machine Learning Models for Aromatase (P450 19A1).

Aromatase, or cytochrome P450 19A1, catalyzes the aromatization of androgens to estrogens within the body. Changes in the activity of this enzyme can produce hormonal imbalances that can be detrimental to sexual and skeletal development. Inhibition of this enzyme can occur with drugs and natural products as well as environmental chemicals. Therefore, predicting potential endocrine disruption via exogenous chemicals requires that aromatase inhibition be considered in addition to androgen and estrogen pathway interference. Bayesian machine learning methods can be used for prospective prediction from the molecular structure without the need for experimental data. Herein, the generation and evaluation of multiple machine learning models utilizing different sources of aromatase inhibition data are described. These models are applied to two test sets for external validation with molecules relevant to drug discovery from the public domain. In addition, the performance of multiple machine learning algorithms was evaluated by comparing internal five-fold cross-validation statistics of the training data. These methods to predict aromatase inhibition from molecular structure, when used in concert with estrogen and androgen machine learning models, allow for a more holistic assessment of endocrine-disrupting potential of chemicals with limited empirical data and enable the reduction of the use of hazardous substances.

Comparison of Machine Learning Models for the Androgen Receptor.

The androgen receptor (AR) is a target of interest for endocrine disruption research, as altered signaling can affect normal reproductive and neurological development for generations. In an effort to prioritize compounds with alternative methodologies, the U.S. Environmental Protection Agency (EPA) used in vitro data from 11 assays to construct models of AR agonist and antagonist signaling pathways. While these EPA ToxCast AR models require in vitro data to assign a bioactivity score, Bayesian machine learning methods can be used for prospective prediction from molecule structure alone. This approach was applied to multiple types of data corresponding to the EPA's AR signaling pathway with proprietary software, Assay Central. The training performance of all machine learning models, including six other algorithms, was evaluated by internal 5-fold cross-validation statistics. Bayesian machine learning models were also evaluated with external predictions of reference chemicals to compare prediction accuracies to published results from the EPA. The machine learning model group selected for further studies of endocrine disruption consisted of continuous AC50 data from the February 2019 release of ToxCast/Tox21. These efforts demonstrate how machine learning can be used to predict AR-mediated bioactivity and can also be applied to other targets of endocrine disruption.

Machine Learning Models for Estrogen Receptor Bioactivity and Endocrine Disruption Prediction.

The U.S. Environmental Protection Agency (EPA) periodically releases in vitro data across a variety of targets, including the estrogen receptor (ER). In 2015, the EPA used these data to construct mathematical models of ER agonist and antagonist pathways to prioritize chemicals for endocrine disruption testing. However, mathematical models require in vitro data prior to predicting estrogenic activity, but machine learning methods are capable of prospective prediction from the molecular structure alone. The current study describes the generation and evaluation of Bayesian machine learning models grouped by the EPA's ER agonist pathway model using multiple data types with proprietary software, Assay Central. External predictions with three test sets of in vitro and in vivo reference chemicals with agonist activity classifications were compared to previous mathematical model publications. Training data sets were subjected to additional machine learning algorithms and compared with rank normalized scores of internal five-fold cross-validation statistics. External predictions were found to be comparable or superior to previous studies published by the EPA. When assessing six additional algorithms for the training data sets, Assay Central performed similarly at a reduced computational cost. This study demonstrates that machine learning can prioritize chemicals for future in vitro and in vivo testing of ER agonism.

Local adaptation to herbivory within tropical tree species along a rainfall gradient.

In tropical forests, insect herbivores exert significant pressure on plant populations. Adaptation to such pressure is hypothesized to be a driver of high tropical diversity, but direct evidence for local adaptation to herbivory in tropical forests is sparse. At the same time, herbivore pressure has been hypothesized to increase with rainfall in the tropics, which could lead to differences among sites in the degree of local adaptation. To assess the presence of local adaptation and its interaction with rainfall, we compared herbivore damage on seedlings of local vs. nonlocal populations at sites differing in moisture availability in a reciprocal transplant experiment spanning a rainfall gradient in Panama. For 13 native tree species, seeds collected from multiple populations along the rainfall gradient were germinated in a shadehouse and then transplanted to experimental sites within the species range. We tracked the proportion of seedlings attacked over 1.5 yr and quantified the percentage of leaf area damaged at the end of the study. Seedlings originating from local populations were less likely to be attacked and experienced lower amounts of herbivore damage than those from nonlocal populations, but only on the wetter end of the rainfall gradient. However, overall herbivore damage was higher at the drier site compared to wetter sites, contrary to expectation. Taken together, these findings support the idea that herbivory can result in local adaptation within tropical tree species; however, the likelihood of local adaptation varies among sites because of environmentally driven differences in investment in defense or herbivore specialization or both.

Cooperative Immune-Mediated Mechanisms of the HDAC Inhibitor Entinostat, an IL15 Superagonist, and a Cancer Vaccine Effectively Synergize as a Novel Cancer Therapy.

PURPOSE: Immunotherapy has demonstrated clinical efficacy in subsets of patients with solid carcinomas. Multimodal therapies using agents that can affect different arms of the immune system and/or tumor microenvironment (TME) might increase clinical responses. EXPERIMENTAL DESIGN: We demonstrate that entinostat, a class I histone deacetylase inhibitor, enhances the antitumor efficacy of the IL15 superagonist N-803 plus vaccine in 4T1 triple-negative breast and MC38-CEA colon murine carcinoma models. A comprehensive immune and gene-expression analysis was performed in the periphery and/or TME of MC38-CEA tumor-bearing mice. RESULTS: Although N-803 plus vaccine induced peripheral CD8+ T-cell activation and cytokine production, there was no reduction in tumor burden and poor tumor infiltration of CD8+ T cells with minimal levels of granzyme B. For the first time, we demonstrate that the addition of entinostat to N-803 plus vaccine promoted significant tumor control, correlating with increased expression of genes associated with tumor inflammation, enhanced infiltration of activated CD8+ T cells with maximal granzyme B, T-cell responses to multiple tumor-associated antigens, increased serum IFNγ, reduction of regulatory T cells in the TME, and decreased expression of the checkpoint V-domain Ig suppressor of T-cell activation (VISTA) on multiple immune subsets. CONCLUSIONS: Collectively, these data demonstrate that the synergistic combination of entinostat, N-803, and vaccine elicits potent antitumor activity by generating a more inflamed TME. These findings thus form the rationale for the use of this combination of agents for patients harboring poorly or noninflamed solid carcinomas.

The Landscape Genetic Signature of Pollination by Trapliners: Evidence From the Tropical Herb, Heliconia tortuosa.

Animal-mediated pollination is essential for the maintenance of plant reproduction, especially in tropical ecosystems, where pollination networks have been thought to have highly generalized structures. However, accumulating evidence suggests that not all floral visitors provide equally effective pollination services, potentially reducing the number of realized pollinators and increasing the cryptic specialization of pollination networks. Thus, there is a need to understand how different functional groups of pollinators influence pollination success. Here, we examined whether patterns of contemporary pollen-mediated gene flow in Heliconia tortuosa are consistent with the foraging strategy of its territorial or traplining hummingbird pollinators. Territorial hummingbirds defend clumps of flowers and are expected to transfer pollen locally. In contrast, traplining hummingbirds forage across longer distances, thereby increasing pollen flow among forest fragments, and are thought to repeatedly visit particular plants. If trapliners indeed visit the same plants repeatedly along their regular routes, this could lead to a situation where neighboring plants sample genetically distinct pollen pools. To test this hypothesis, we genotyped 720 seeds and 71 mother plants from 18 forest fragments at 11 microsatellite loci. We performed TwoGener analysis to test pollen pool differentiation within sites (among neighboring plants within the same forest fragment: Φ SC ) and between sites (among forest fragments: Φ CT ). We found strong, statistically significant pollen pool differentiation among neighboring mother plants (Φ SC = 0.0506), and weaker, statistically significant differentiation among sites (Φ CT = 0.0285). We interpret this pattern of hierarchical pollen pool differentiation as the landscape genetic signature of the foraging strategy of traplining hummingbirds, where repeatable, long-distance, and high-fidelity routes transfer pollen among particular plants. Although H. tortuosa is also visited by territorial hummingbirds, our results suggest that these pollinators do not contribute substantially to successful pollination, highlighting differences in realized pollination efficiency. This cryptic reduction in the number of realized pollinators potentially increases the vulnerability of pollination success to the decline of populations of traplining hummingbirds, which have been shown to be sensitive to forest fragmentation. We conclude that maintaining habitat connectivity to sustain the foraging routes of trapliners may be essential for the maintenance of pollen-mediated gene flow in human-modified landscapes.

Adenoviral vector-based vaccine is fully protective against lethal Lassa fever challenge in Hartley guinea pigs.

Lassa virus (LASV), the causative agent of Lassa fever (LF), was first identified in 1969. Since then, outbreaks in the endemic countries of Nigeria, Liberia, and Sierra Leone occur on an annual basis resulting in a case-fatality rate of 15-70% in hospitalized patients. There is currently no licensed vaccine and there are limited animal models to test vaccine efficacy. An estimated 37.7 million people are at risk of contracting LASV; therefore, there is an urgent need for the development of a safe, effective vaccine against LASV infection. The LF endemic countries are also inflicted with HIV, Ebola, and malaria infections. The safety in immunocompromised populations must be considered in LASV vaccine development. The novel adenovirus vector-based platform, Ad5 (E1-,E2b-) has been used in clinical trial protocols for treatment of immunocompromised individuals, has been shown to exhibit high stability, low safety risk in humans, and induces a strong cell-mediated and pro-inflammatory immune response even in the presence of pre-existing adenovirus immunity. To this nature, our lab has developed an Ad5 (E1-,E2b-) vector-based vaccine expressing the LASV-NP or LASV-GPC. We found that guinea pigs vaccinated with two doses of Ad5 (E1-,E2b-) LASV-NP and Ad5 (E1-,E2b-) LASV-GPC were protected against lethal LASV challenge. The Ad5 (E1-,E2b-) LASV-NP and LASV-GPC vaccine represents a potential vaccine candidate against LF.

Identification of New Mono/Dihydroxynaphthoquinone as Lead Agents That Inhibit the Growth of Refractive and Triple-Negative Breast Cancer Cell Lines.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in nearly 20-30% of breast cancers and is associated with metastasis resulting in poor patient survival and high recurrence. The dual EGFR/HER2 kinase inhibitor lapatinib has shown promising clinical results, but its limitations have also led to the resistance and activation of tumor survival pathways. Following our previous investigation of quinones as HER2 kinase inhibitors, we synthesized several naphthoquinone derivatives that significantly inhibited breast tumor cells expressing HER2 and trastuzumab-resistant HER2 oncogenic isoform, HER2Δ16. Two of these compounds were shown to be more effective than lapatinib at the inhibition of HER2 autophosphorylation of Y1248. Compounds 7 (5,8-dihydroxy-2-methylnaphthalene-1,4-dione) and 9 (2-(bromomethyl)-5,8-dihydroxynaphthalene-1,4-dione) inhibited HER2-expressing MCF-7 cells (IC50 0.29 and 1.76 µM, respectively) and HER2Δ16-expressing MCF-7 cells (IC50 0.51 and 1.76 µM, respectively). Compound 7 was also shown to promote cell death in multiple refractory breast cancer cell lines with IC50 values ranging from 0.12 to 2.92 µM. These compounds can function as lead compounds for the design of a new series of nonquinonoid structural compounds that can maintain a similar inhibition profile.

A New Approach to an Old Technique-The S.U.T.R. First Technique.

Ventral and incisional hernias of the abdominal wall are common problems treated by surgeons around the globe. Incisional hernias are common postoperative complications of abdominal laparotomies with a reported incidence of up to 20 per cent. The increasing use of prosthetic mesh in open ventral hernia repairs necessitated the development of different operative techniques used in the repairs. It also required that surgeons become facile with placement of the mesh in different anatomical positions on the abdominal wall. One of the most common locations is placement of the mesh in the underlay position. Many surgeons who use the underlay technique have expressed significant concerns. Among these are fear of an inadvertent bowel injury while placing the mesh, poor visualization during mesh placement, and the inability to use the underlay technique for difficult hernias. We present a very useful, if not, novel technique of open hernia repair using mesh in the underlay position that helps to 1) prevent complications, 2) facilitate easier mesh fixation, 3) simplify open repair of atypical ventral hernias, and 4) reduce total operative time while still adhering to the important fundamental principles of a tension-free hernia repair. This technique as we describe it has been compared with the old parachute technique, but we think this is a significant improvement of that seldom used technique. We believe the use of this technique for the underlay position makes open ventral hernia repair safer, faster, and easier; however, our goal for this article is to describe the procedure in detail. In addition, we recently have started using this technique to fix the mesh when doing the retrorectus approach as well.

Ablating ErbB4 in PV neurons attenuates synaptic and cognitive deficits in an animal model of Alzheimer's disease.

Accumulation of amyloid ß (Aß) induces neuronal, synaptic, and cognitive deficits in patients and animal models of Alzheimer's disease (AD). The underlying mechanisms, however, remain to be fully elucidated. In the present study, we found that Aß interacted with ErbB4, a member of the receptor tyrosine kinase family and mainly expressed in GABAergic interneurons. Deleting ErbB4 in parvalbumin-expressing neurons (PV neurons) significantly attenuated oligomeric Aß-induced suppression of long term potentiation (LTP). Furthermore, specific ablation of ErbB4 in PV neurons via Cre/loxP system greatly improved spatial memory and synaptic plasticity in the hippocampus of hAPP-J20 mice. The deposition of Aß detected by 3D6 and Thioflavin S staining and the proteolytic processing of hAPP analyzed by western blotting were not affected in the hippocampus of hAPP-J20 mice by deleting ErbB4 in PV neurons. Our data suggested that ErbB4 in PV neurons mediated Aß-induced synaptic and cognitive dysfunctions without affecting Aß levels.

Identification and characterization of enhancer agonist human cytotoxic T-cell epitopes of the human papillomavirus type 16 (HPV16) E6/E7.

Human papillomavirus (HPV) is associated with the etiology of cervical carcinoma, head and neck squamous cell carcinoma, and several other cancer types. Vaccines directed against HPV virus-like particles and coat proteins have been extremely successful in the prevention of cervical cancer through the activation of host HPV-specific antibody responses; however, HPV-associated cancers remain a major public health problem. The development of a therapeutic vaccine will require the generation of T-cell responses directed against early HPV proteins (E6/E7) expressed in HPV-infected tumor cells. Clinical studies using various vaccine platforms have demonstrated that both HPV-specific human T cells can be generated and patient benefit can be achieved. However, no HPV therapeutic vaccine has been approved by the Food and Drug Administration to date. One method of enhancing the potential efficacy of a therapeutic vaccine is the generation of agonist epitopes. We report the first description of enhancer cytotoxic T lymphocyte agonist epitopes for HPV E6 and E7. While the in silico algorithm revealed six epitopes with potentially improved binding to human leukocyte antigen-A2 allele (HLA-A2)-Class I, 5/6 demonstrated enhanced binding to HLA-Class I in cell-based assays and only 3/6 had a greater ability to activate HPV-specific T cells which could lyse tumor cells expressing native HPV, compared to their native epitope counterparts. These agonist epitopes have potential for use in a range of HPV therapeutic vaccine platforms and for use in HPV-specific adoptive T- or natural killer-cell platforms.

Trastuzumab stimulation of ribosomal protein S6 kinase 1 (S6K1) predicts de novo trastuzumab resistance.

Breast cancer is a complex disease with at least five different molecular subtypes identified. The breast tumor molecular subtypes guide stratification of patients for specific targeted therapy regimens and each subtype is associated with significantly different patient outcomes. For example, patients with the HER2 positive molecular subtype benefit from the HER2 targeted therapy trastuzumab. Unfortunately, women with the HER2 positive molecular subtype have the worst overall prognosis and nearly 70% of women with HER2 positive breast cancer exhibit de novo or acquired resistance to trastuzumab. Identification of tumor markers predicting trastuzumab response can be used to further stratify patients for life-saving personalized therapeutic options. The aim of this study was to identify clinically useful tumor markers predicting de novo tumor cell resistance to trastuzumab treatment. To identify oncogenic signaling pathways activated in response to trastuzumab treatment, we performed a Human Phospho-Kinase Proteome Profiler Array analysis comparing trastuzumab sensitive MCF-7/HER2.2 and trastuzumab resistant MCF-7/HER2Δ16H cells following acute treatment with 20 µg/ml of trastuzumab for 2 h. We found that of the 43 phosphorylation activated human kinases represented on the array, S6K1 was the only kinase altered greater than 1.5-fold in response to trastuzumab treatment of the trastuzumab resistant MCF-7/HER2Δ16H cells. Trastuzumab activation of S6K1 was confirmed in the two trastuzumab resistant SUM190 and SUM225 cell lines. Significantly, trastuzumab failed to stimulate S6K1 activation in the trastuzumab sensitive MCF-7/HER2.2, BT474, and SKBR3 cell lines suggesting that trastuzumab activation of S6K1 is a tumor cell marker for trastuzumab resistance. Consistent with a role for mTORC1/S6K1 signaling promoting trastuzumab resistance, all cell lines were sensitive to S6K1 inactivation with significant growth inhibition following treatment with the mTORC1 inhibitor rapamycin. In conclusion, characterizing rapid trastuzumab induced molecular alterations resulted in the identification of activated S6K1 as an early breast tumor cell marker for trastuzumab resistance. Our results further suggest that trastuzumab resistant breast tumor cells are addicted to mTORC1/S6K1 oncogenic signaling and targeting mTORC1 with rapamycin reverses trastuzumab resistance.

Intrinsic HER4/4ICD transcriptional activation domains are required for STAT5A activated gene expression.

The epidermal growth factor receptor family member HER4 undergoes proteolytic processing at the cell surface to release the HER4 intracellular domain (4ICD) nuclear protein. Interestingly, 4ICD directly interacts with STAT5 and functions as an obligate STAT5 nuclear chaperone. Once in the nucleus 4ICD binds with STAT5 at STAT5 target genes, dramatically potentiating STAT5 transcriptional activation. These observations raise the possibility that 4ICD directly coactivates STAT5 gene expression. Using both yeast and mammalian transactivation reporter assays, we performed truncations of 4ICD fused to a GAL4 DNA binding domain and identified two independent 4ICD transactivation domains located between residues 1022 and 1090 (TAD1) and 1192 and 1225 (TAD2). The ability of the 4ICD DNA binding domain fusions to transactivate reporter gene expression required deletion of the intrinsic tyrosine kinase domain. In addition, we identified the 4ICD carboxyl terminal TVV residues, a PDZ domain binding motif (PDZ-DBM), as a potent transcriptional repressor. The transactivation activity of the HER4 carboxyl terminal domain lacking the tyrosine kinase (CTD) was significantly lower than similar EGFR or HER2 CTD. However, deletion of the HER4 CTD PDZ-DBM enhanced HER4 CTD transactivation to levels equivalent to the EGFR and HER2 CTDs. To determine if 4ICD TAD1 and TAD2 have a physiologically relevant role in STAT5 transactivation, we coexpressed 4ICD or 4ICD lacking TAD2 or both TAD1 and TAD2 with STAT5 in a luciferase reporter assay. Our results demonstrate that each 4ICD TAD contributes additively to STAT5A transactivation and the ability of STAT5A to transactivate the ß-casein promoter requires the 4ICD TADs. Taken together, published data and our current results demonstrate that both 4ICD nuclear chaperone and intrinsic coactivation activities are essential for STAT5 regulated gene expression.