Bat pluripotent stem cells reveal unusual entanglement between host and viruses.

Bats are distinctive among mammals due to their ability to fly, use laryngeal echolocation, and tolerate viruses. However, there are currently no reliable cellular models for studying bat biology or their response to viral infections. Here, we created induced pluripotent stem cells (iPSCs) from two species of bats: the wild greater horseshoe bat (Rhinolophus ferrumequinum) and the greater mouse-eared bat (Myotis myotis). The iPSCs from both bat species showed similar characteristics and had a gene expression profile resembling that of cells attacked by viruses. They also had a high number of endogenous viral sequences, particularly retroviruses. These results suggest that bats have evolved mechanisms to tolerate a large load of viral sequences and may have a more intertwined relationship with viruses than previously thought. Further study of bat iPSCs and their differentiated progeny will provide insights into bat biology, virus host relationships, and the molecular basis of bats' special traits.

N-Terminal Derivatization-Assisted Identification of Individual Amino Acids Using a Biological Nanopore Sensor.

Nanopore technology has been employed as a powerful tool for DNA sequencing and analysis. To extend this method to peptide sequencing, a necessary step is to profile individual amino acids (AAs) through their nanopore stochastic signals, which remains a great challenge because of the low signal-to-noise ratio and unpredictable conformational changes of AAs during their translocation through nanopores. We showed that the combination of an N-terminal derivatization strategy of AAs with nanopore technology could lead to effective in situ differentiation of AAs. Four different derivatization reactions have been tested with five selected AAs: Ala, Phe, Tyr, His, and Asp. Using an α-hemolysin nanopore, we demonstrated the feasibility of derivatization-assisted identification of AAs regardless of their charge composition and polarity. The method was further applied to discriminate each individual AA in testing data sets using their established nanopore profiles from training data sets. We envision that this proof-of-concept study will not only pave a way for identification of individual AAs but also lead to future applications in protein/peptide sequencing using the nanopore technology.

Sun protection education for adolescents: a feasibility study of a wait-list controlled trial of an intervention involving a presentation, action planning, and SMS messages and using objective measurement of sun exposure.

BACKGROUND: People increase their risk of melanoma unless they are protected from the harmful effects of sun exposure during childhood and adolescence. We aimed to assess the feasibility of a three-component sun protection intervention- presentation, action planning, and SMS messages - and trial parameters. METHODS: This feasibility wait-list trial was conducted in the United Kingdom in 2018. Students aged 13-15 years were eligible. Feasibility outcomes were collected for recruitment rates; data availability rates for objective measurements of melanin and erythema using a Mexameter and self-reported sunburn occurrences, severity and body location, tanning, sun protection behaviours and Skin Self-Examination (SSE) collected before (baseline) and after the school summer holidays (follow-up); intervention reach, adherence, perceived impact and acceptability. Quantitative data were analysed using descriptive statistics; qualitative data were analysed thematically. RESULTS: Five out of eight schools expressing an interest in participating with four allocated to act as intervention and one control. Four parents/carers opted their child out of the study. Four hundred and eighty-seven out of 724 students on the school register consented to the study at baseline (67%). Three hundred and eighty-five were in intervention group schools. Objective skin measurements were available for 255 (66%) of the intervention group at baseline and 237 (61%) of the group at follow up. Melanin increased; erythema decreased. Complete self-report data were available for 247 (64%) students in the intervention group. The number of students on the school register who attended the presentation and given the booklet was 379 (98%) and gave their mobile phone number was 155 (40%). No intervention component was perceived as more impactful on sun protection behaviours. Adolescents did not see the relevance of sun protection in the UK or for their age group. CONCLUSIONS: This is the first study to use a Mexameter to measure skin colour in adolescents. Erythema (visible redness) lasts no more than three days and its measurement before and after a six week summer holiday may not yield relevant or meaningful data. A major challenge is that adolescents do not see the relevance of sun protection and SSE. TRIAL REGISTRATION: International Standard Randomised Controlled Trial Number ISRCTN11141528. Date registered 0/2/03/2018; last edited 31/05/2018. Retrospectively registered.

Increasing participation of people with learning disabilities in bowel screening.

Learning disability nurses have a key role in addressing the health inequalities experienced by people with learning disabilities. People with learning disabilities are less likely to participate in bowel screening than other sectors of the population, despite there being evidence of this population being at an increased risk of developing bowel cancer. There are a range of barriers at individual and systemic levels that impact on participation in bowel screening by people with learning disabilities. Actions to address these barriers have been identified in the literature and learning disability nurses are a key agent of change in enabling people with learning disabilities to participate in the national screening programmes.

Methods for image guided and intensity modulated radiation therapy in high-risk abdominal neuroblastoma.

PURPOSE: Our purpose was to determine methods for image guided intensity modulated radiation therapy (IMRT) in pediatric abdominal high-risk neuroblastoma and to quantify the degree of normal tissue dose reduction by using volumes compliant with International Commission on Radiation Units and Measurements (ICRU) Report 62. METHODS AND MATERIALS: Eight consecutive children with high-risk abdominal neuroblastoma (median age, 2.5 years; range, 20 months-5 years) were treated with IMRT using volumes accounting for physiologic motion (IMRT_phys) and daily pretreatment cone beam computed tomographic localization. Comparative IMRT planning using conventional volumes (IMRT_std) provided quantification for dose reduction to normal tissues. RESULTS: The IMRT_phys plan reduced the mean planning target volume from 668.8 ± 200.6 cc to 393.0 ± 132.5 cc (P < .001) and reduced mean body V50 from 1774.4 ± 383.9 cc to 1385.7 ± 365.7 cc (P < .001). The IMRT_phys plan reduced the percent mean dose to the ipsilateral kidney from 70.1% ± 4.3% to 66.0% ± 5.2% (P =.002); that to the contralateral kidney was reduced from 56.3% ± 7.0% to 40.7% ± 9.5% (P < .001), and that to the liver was reduced from 57.8% ± 16.0% to 22.1% ± 6.8% (P = .001). CONCLUSIONS: For IMRT planning, ICRU 62-compliant volume definition with image guidance in the pediatric abdomen enables volumetric reduction of the planning target volume and reduces normal tissue dose. These methods provide a framework for more conformal treatment planning in the pediatric abdomen.

Intensity-modulated arc therapy for pediatric posterior fossa tumors.

PURPOSE: To compare intensity-modulated arc therapy (IMAT) to noncoplanar intensity-modulated radiation therapy (IMRT) in the treatment of pediatric posterior fossa tumors. METHODS AND MATERIALS: Nine pediatric patients with posterior fossa tumors, mean age 9 years (range, 6-15 years), treated using IMRT were chosen for this comparative planning study because of their tumor location. Each patient's treatment was replanned to receive 54 Gy to the planning target volume (PTV) using five different methods: eight-field noncoplanar IMRT, single coplanar IMAT, double coplanar IMAT, single noncoplanar IMAT, and double noncoplanar IMAT. For each method, the dose to 95% of the PTV was held constant, and the doses to surrounding critical structures were minimized. The different plans were compared based on conformity, total linear accelerator dose monitor units, and dose to surrounding normal tissues, including the entire body, whole brain, temporal lobes, brainstem, and cochleae. RESULTS: The doses to the target and critical structures for the various IMAT methods were not statistically different in comparison with the noncoplanar IMRT plan, with the following exceptions: the cochlear doses were higher and whole brain dose was lower for coplanar IMAT plans; the cochleae and temporal lobe doses were lower and conformity increased for noncoplanar IMAT plans. The advantage of the noncoplanar IMAT plan was enhanced by doubling the treatment arc. CONCLUSION: Noncoplanar IMAT results in superior treatment plans when compared to noncoplanar IMRT for the treatment of posterior fossa tumors. IMAT should be considered alongside IMRT when treatment of this site is indicated.

Hypoxic preconditioning failure in aging hippocampal neurons: impaired gene expression and rescue with intracellular calcium chelation.

Hypoxic preconditioning in the brain (HPC), a phenomenon whereby noninjurious hypoxia induces resistance to cell death following ischemia, requires the expression of specific genes. Declines in signal transduction pathway activity with aging may decrease the genomic response to HPC and limit its neuroprotective efficacy. To test this, we determined how signal transduction gene expression, intracellular Ca(2+) levels, and phosphorylation of the survival-associated kinase Akt differ in hippocampal slice cultures (HSCs) made from postnatal day 7-10 (P7-10) and 2-year-old rats following HPC. HPC neuroprotection decreased with increasing source animal age, and HPC could not be demonstrated in HCSs made from animals >6 months of age, despite adjusting the duration of hypoxic exposure. Preconditioning protection required the survival kinase Akt in P10 hippocampal slices cultures. In P9 cultures, HPC increased Akt phosphorylation and the expression of prosurvival genes, including Bcl-2, heat shock proteins, protein kinases, c-jun, and NfκB. Lack of increased Akt phosphorylation and a greatly diminished signaling pathway gene response were found in HSCs from aging animals. Moderate and transient increases in [Ca(2+) ](i) during HPC occurred in P7-10 HSCs, but [Ca(2+) ](i) was persistently increased at 1 and 24 hr after preconditioning in HSCs from 2-year-old rats. The intracellular Ca(2+) chelator BAPTA-AM facilitated HPC neuroprotection in 2-year-old HSCs and restored the pattern of post-HPC gene expression seen in immature animals. We conclude that age-related loss of preconditioning may be due to altered intracellular Ca(2+) homeostasis (excess and sustained increase in [Ca(2+) ](i) ) and is a lesion that prevents critical elements of neuroprotective signal transduction.

Treatment planning and delivery of external beam radiotherapy for pediatric sarcoma: the St. Jude Children's Research Hospital experience.

PURPOSE: To describe and review the radiotherapy (RT) treatment planning and delivery techniques used for pediatric sarcoma patients at St. Jude Children's Research Hospital. The treatment characteristics serve as a baseline for future comparison with developing treatment modalities. PATIENTS AND METHODS: Since January 2003, we have prospectively treated pediatric and young-adult patients with soft-tissue and bone sarcomas on an institutional Phase II protocol evaluating local control and RT-related treatment effects from external-beam RT (conformal or intensity-modulated RT; 83.4%), low-dose-rate brachytherapy (8.3%), or both (8.3%). Here we describe the treatment dosimetry and delivery parameters of the initial 72 patients (median, 11.6 years; range, 1.4-21.6 years). RESULTS: Cumulative doses from all RT modalities ranged from 41.4 to 70.2 Gy (median, 50.4 Gy). Median D(95) and V(95) of the planning target volume of external-beam RT plans were, respectively, 93.4% of the prescribed dose and 94.6% of the target volume for the primary phase and 97.8% and 99.2% for the cone-down/boost phase. The dose-volume histogram statistics for 27 critical organs varied greatly. The spinal cord in 13 of 36 patients received dose >45 Gy (up to 52 Gy in 1 cc) because of tumor proximity. CONCLUSIONS: Planning and delivery of complex multifield external beam RT is feasible in pediatric patients with sarcomas. Improvements on conformity and dose gradients are still desired in many cases with sensitive adjacent critical structures. Long-term follow-up will determine the risk of local failure and the benefit of normal tissue avoidance for this population.

An integrated genomic-based approach to individualized treatment of patients with advanced-stage ovarian cancer.

PURPOSE: The purpose of this study was to develop an integrated genomic-based approach to personalized treatment of patients with advanced-stage ovarian cancer. We have used gene expression profiles to identify patients likely to be resistant to primary platinum-based chemotherapy and also to identify alternate targeted therapeutic options for patients with de novo platinum-resistant disease. PATIENTS AND METHODS: A gene expression model that predicts response to platinum-based therapy was developed using a training set of 83 advanced-stage serous ovarian cancers and tested on a 36-sample external validation set. In parallel, expression signatures that define the status of oncogenic signaling pathways were evaluated in 119 primary ovarian cancers and 12 ovarian cancer cell lines. In an effort to increase chemotherapy sensitivity, pathways shown to be activated in platinum-resistant cancers were subject to targeted therapy in ovarian cancer cell lines. RESULTS: Gene expression profiles identified patients with ovarian cancer likely to be resistant to primary platinum-based chemotherapy with greater than 80% accuracy. In patients with platinum-resistant disease, we identified expression signatures consistent with activation of Src and Rb/E2F pathways, components of which were successfully targeted to increase response in ovarian cancer cell lines. CONCLUSION: We have defined a strategy for treatment of patients with advanced-stage ovarian cancer that uses therapeutic stratification based on predictions of response to chemotherapy, coupled with prediction of oncogenic pathway deregulation, as a method to direct the use of targeted agents.

Topotecan combination chemotherapy in two new rodent models of retinoblastoma.

Chemotherapy combined with laser therapy and cryotherapy has improved the ocular salvage rate for children with bilateral retinoblastoma. However, children with late-stage disease often experience recurrence shortly after treatment. To improve the vision salvage rate in advanced bilateral retinoblastoma, we have developed and characterized two new rodent models of retinoblastoma for screening chemotherapeutic drug combinations. The first model is an orthotopic xenograft model in which green fluorescent protein- or luciferase-labeled human retinoblastoma cells are injected into the eyes of newborn rats. The second model uses a replication-incompetent retrovirus (LIA-E(E1A)) encoding the E1A oncogene. Clonal, focal tumors arise from mouse retinal progenitor cells when LIA-E(E1A) is injected into the eyes of newborn p53-/- mice. Using these two models combined with pharmacokinetic studies and cell culture experiments, we have tested the efficacy of topotecan combined with carboplatin and of topotecan combined with vincristine for the treatment of retinoblastoma. The combination of topotecan and carboplatin most effectively halted retinoblastoma progression in our rodent models and was superior to the current triple drug therapy using vincristine, carboplatin, and etoposide. Vincristine had the lowest LC50 in culture but did not reduce tumor growth in our preclinical retinoblastoma models. Taken together, these data suggest that topotecan may be a suitable replacement for etoposide in combination chemotherapy for the treatment of retinoblastoma.

Isoflurane neuroprotection in hypoxic hippocampal slice cultures involves increases in intracellular Ca2+ and mitogen-activated protein kinases.

BACKGROUND: The volatile anesthetic isoflurane reduces acute and delayed neuron death in vitro models of brain ischemia, an action that the authors hypothesize is related to moderate increases in intracellular calcium concentration ([Ca2+]i). Specifically, the authors propose that during hypoxia, moderate increases in [Ca2+]i in the presence of isoflurane stimulates the Ca2+-dependent phosphorylation of members of the mitogen-activated protein kinase (MAP) kinase Ras-Raf-MEK-ERK pathway that are critical for neuroprotective signaling and suppression of apoptosis. METHODS: Death of CA1, CA3, and dentate neurons in rat hippocampal slice cultures was assessed by propidium iodide fluorescence 48-72 h after 60-75 min of hypoxia. [Ca2+]i in CA1 neurons was measured with fura-2 and fura-2 FF. Concentrations of the survival-signaling proteins Ras, MEK, MAP kinase p42/44, and protein kinase B (Akt) were assessed by immunostaining, and specific inhibitors were used to ascertain the role of Ca2+ and MAP kinases in mediating survival. RESULTS: Isoflurane, 1%, decreased neuron death in CA1, CA3, and dentate gyrus neurons after 60 but not 75 min of hypoxia. Survival of CA1 neurons required an inositol triphosphate receptor-dependent increase in [Ca2+]i of 30-100 nm that activated the Ras-Raf-MEK-ERK (p44/42) signaling pathway. Isoflurane also increased the phosphorylation of Akt during hypoxia. CONCLUSIONS: Isoflurane stimulates the phosphorylation of survival signaling proteins in hypoxic neurons. The mechanism involves a moderate increase in [Ca2+]i from release of Ca from inositol triphosphate receptor-dependent intracellular stores. The increase in [Ca2+]i sets in motion signaling via Ras and the MAP kinase p42/44 pathway and the antiapoptotic factor Akt. Isoflurane neuroprotection thus involves intracellular signaling well known to suppress both excitotoxic and apoptotic/delayed cell death.

Rb regulates proliferation and rod photoreceptor development in the mouse retina.

The retinoblastoma protein (Rb) regulates proliferation, cell fate specification and differentiation in the developing central nervous system (CNS), but the role of Rb in the developing mouse retina has not been studied, because Rb-deficient embryos die before the retinas are fully formed. We combined several genetic approaches to explore the role of Rb in the mouse retina. During postnatal development, Rb is expressed in proliferating retinal progenitor cells and differentiating rod photoreceptors. In the absence of Rb, progenitor cells continue to divide, and rods do not mature. To determine whether Rb functions in these processes in a cell-autonomous manner, we used a replication-incompetent retrovirus encoding Cre recombinase to inactivate the Rb1(lox) allele in individual retinal progenitor cells in vivo. Combined with data from studies of conditional inactivation of Rb1 using a combination of Cre transgenic mouse lines, these results show that Rb is required in a cell-autonomous manner for appropriate exit from the cell cycle of retinal progenitor cells and for rod development.

Gene expression patterns that characterize advanced stage serous ovarian cancers.

OBJECTIVE: To identify gene expression patterns that characterize advanced stage serous ovarian cancers by using microarray expression analysis. METHODS: Using genome-wide expression analysis, we compared a series of 31 advanced stage (III or IV) serous ovarian cancers from patients who survived either less than 2 years or more than 7 years with three normal ovarian epithelial samples. Array findings were validated by analysis of expression of the insulin-like growth factor binding protein 2 (IGFBP2) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genes using quantitative real-time polymerase chain reaction (QRT-PCR). RESULTS: Hierarchical clustering identified patterns of gene expression that distinguished cancer from normal ovarian epithelium. We also identified gene expression patterns that distinguish cancers on the basis of patient survival. These genes include many that are associated with immune function. Expression of IGFBP2 and TRAIL genes measured by array and QRT-PCR analysis demonstrated correlation coefficients of 0.63 and 0.78, respectively. CONCLUSION: Global expression analysis can identify expression patterns and individual genes that contribute to ovarian cancer development and outcome. Many of the genes that determine ovarian cancer survival are associated with the immune response, suggesting that immune function influences ovarian cancer virulence. With the generation of newer arrays with more transcripts, larger studies are possible to fully characterize genetic signatures that predict survival that may ultimately be used to guide therapeutic decision-making.