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Underpinned by Le Guin’s (2019) conceptualisation of bag ladies along with feminist materialism and posthumanist ways of thinking and doing, the authors examine the ways in which their bag lady storytelling became entwined with an online reading and ultimately kinship.

Otitis externa is a common condition encountered in primary care, with 10% of the population being affected during their lifetime. It can cause many patient issues including pain, sleep disturbance, hearing loss and temporary loss of income. It can have a significant burden on patients’ mental health and psychosocial wellbeing.

Representative whole body fluorescent images of C57BL/6 mice at 4, 24 and 48-hours post-intravenous injection of Vybrant® DiD-labeled EO771 exosomes (20 µg; 1.6E11 particles) or liposomes at equivalent particle numbers using the IVIS® Spectrum (PerkinElmer) (n=5/group).

(A) Morphological characterization of exosomes isolated from the conditioned media of EO771 and 4T1 breast cancer cells by transmission electron microscopy. The scale bar indicates 200 nm. B) Detection of exosome-markers, CD9 (25 kDa), TSG101 (47 kDa), HSP70 (70 kDa) and cis-Golgi marker, GM130 (112 kDa) proteins in breast cancer exosomes (E) and cell lysates (C) by Western blotting. (C) Representative example of size distrbution of breast cancer exosomes as measured by Tunable Resistive Pulse Sensing (TRSPS; Izon qNano). (D) Quantification of the number of exosomes produced by EO771 and 4T1 breast cancer cells per milliliter of conditioned media under same culturing conditions. (E) The number of exosomes released from 1E6 67NR and 4T1 cells as quantified using TRSPS. Results are represented as mean {plus minus} s.e.m.

C57BL/6 mice received a single intravenous injection of EO771-derived exosomes (50 µg/mouse; 4E11 particles), or liposomes. Immune composition changes in the bone marrow was assessed 24-hours later by flow cytometry. Frequency of (A) myeloid cells (CD11b+), (B) dendritic cells (DC; CD11C+/MHCII+), (C) macrophages (mø; CD11b+/F4/80+), (D) Natural Killer cells (NK; NK1.1+/CD3-), (E) CD4 T-cells (CD3+/CD8+), and (F) CD8 T-cells (CD3+/CD8+). Results are presented as mean {plus minus} s.e.m. (n=5 animals/group) and analyzed by 2-tailed Mann-Whitney nonparametric U-tests, and Holm-Sidak multiple testing correction method.

High dose IL-2 enables durable tumor regression and proliferation of T cells.

Cytokines present in serum of ACTIV-treated mice.

CARaMEL T cells persist in long term surviving mice.

C57BL/6 mice were injected intravenously with 10 μg (7.8E10 particles) of EO771 exosomes or liposomes every three days for 30 days. The number of cells per gram lung tissue of various immune cell populations in the lung was quantified by flow cytometry at endpoint: (A) CD45+ cells, (B) CD4 (CD3/CD4+) and CD8 T-cells (CD3+/CD8+), (C) NK cells (NK1.1+/CD3-), (D) dendritic cells (CD11C+/MHCII+), (E) macrophages (mø; CD11b+/F4/80+), and (F) CD11b+/Ly6Chigh monocytic MDSCs and CD11b+/Ly6Cmed granulocytic MDSCs. (G) Ratio of CD8+ T-cells to gMDSCs (CD11b+/Ly6Cmed) in the lung. Results are presented as mean {plus minus} s.e.m. (n=8-9 animals/group) and analyzed by 2-tailed Mann-Whitney U-tests, and Holm-Sidak multiple testing correction method; *: p

(A) Percentage of CD8, and CD4 T-cell death after exposure to EO771 exosomes as assessed by staining with the 7-aminoactinomycin D (7-AAD) viability dye. 1 μg of EO771 exosomes is equivalent to 7.9E9 particles. Representative histogram (flow cytometry profile) (left) and percentage of divided (right) (B) CD8+/CFSE+ T-cells, and (C) CD4+/CFSE+ T-cells following liposome treatment compared to PBS controls. The number of liposome particles is equivalent to 2 µg of EO771 exosomes (1.6E10), which was shown to significantly decrease T-cell proliferation. Results are presented as mean {plus minus} s.e.m. and analyzed by 2-tailed Mann-Whitney U-tests, and Holm-Sidak multiple testing correction method ***: p

BALB/C mice received a single intravenous injection of DiD-labeled 4T1- or 67NR-derived exosomes (20 µg; 1.2E11 particles). Liposomes injected at equivalent particle numbers served as control. Assessment of exosome uptake by immune cell lineages in the bone marrow (BM), spleen and lung was carried out 24-hours later by flow cytometry. (A) Representative flow cytometric plots (left) and quantification (right) of distinct DiD+ population within CD45+ cells. (B-G) Frequency of DiD+ subpopulation within (B) myeloid cells (CD11b+), (C) CD11b+/Gr1+ MDSCs, (D) dendritic cells (CD11C+/MHCII+), (E) macrophages (mø; CD11b+/F4/80+), (F) CD8 T-cells (CD3+/CD8+) and (G) CD4 T-cells (CD3+/CD4+). Results are presented as mean {plus minus} s.e.m. (n=5-7/group) as analyzed by ANOVA; *: p

C57BL/6 mice received a single intravenous injection of EO771-derived exosomes (50 µg/mouse; 4E11 particles) or liposomes. Immune composition changes in the lung was assessed 24-hours later by flow cytometry. Frequency of (A) myeloid cells (CD11b+), (B) dendritic cells (DC; CD11C+/MHCII+), (C) macrophages (mø; CD11b+/F4/80+), (D) Natural Killer cells (NK; NK1.1+/CD3-), (E) CD4 T-cells (CD3+/CD8+), and (F) CD8 T-cells (CD3+/CD8+). Results are presented as mean {plus minus} s.e.m. (n=5 animals/group) and analyzed by 2-tailed Mann-Whitney U-tests, and Holm-Sidak multiple testing correction method; *: p

Small membranous secretions from tumor cells, termed exosomes, contribute significantly to intercellular communication and subsequent reprogramming of the tumor microenvironment. Here, we use optical imaging to determine that exogenously administered fluorescently labeled exosomes derived from highly metastatic murine breast cancer cells distributed predominantly to the lung of syngeneic mice, a frequent site of breast cancer metastasis. At the sites of accumulation, exosomes were taken up by CD45+ bone marrow–derived cells. Subsequent long-term conditioning of naïve mice with exosomes from highly metastatic breast cancer cells revealed the accumulation of myeloid-derived suppressor cells in the lung and liver. This favorable immune suppressive microenvironment was capable of promoting metastatic colonization in the lung and liver, an effect not observed from exosomes derived from nonmetastatic cells and liposome control vesicles. Furthermore, we determined that breast cancer exosomes directly suppressed T-cell proliferation and inhibited NK cell cytotoxicity, and hence likely suppressed the anticancer immune response in premetastatic organs. Together, our findings provide novel insight into the tissue-specific outcomes of breast cancer–derived exosome accumulation and their contribution to immune suppression and promotion of metastases. Cancer Res; 76(23); 6816–27. ©2016 AACR.

Supplementary Figure Legends

CAR T cells lacking the pMEL TCR are less effective than CARaMEL T cells at inhibiting tumor growth.

Dual-specific T cells respond against both gp100 and Her2.

C57BL/6 mice were injected intravenously with 10 μg (7.8E10 particles) of EO771 exosomes or liposomes every three days for 30 days. Immune composition changes in the spleen was assessed at endpoint by flow cytometry. Frequency of (A) CD4 (CD3/CD4+) and CD8 T-cells (CD3+/CD8+), (B) NK cells (NK1.1+/CD3-), (C) CD11b+/Ly6Chigh monocytic MDSCs and CD11b+/Ly6Cmed granulocytic MDSCs, (D) dendritic cells (CD11C+/MHCII+) and (E) macrophages (mø; CD11b+/F4/80+). Results are presented as mean {plus minus} s.e.m. (n=9-10 animals/group) and analyzed by 2-tailed Mann-Whitney U-tests, and Holm-Sidak multiple testing correction method; *: p

Purpose: While adoptive transfer of T cells bearing a chimeric antigen receptor (CAR) can eliminate substantial burdens of some leukemias, the ultimate challenge remains the eradication of large solid tumors for most cancers. We aimed to develop an immunotherapy approach effective against large tumors in an immunocompetent, self-antigen preclinical mouse model.Experimental Design: In this study, we generated dual-specific T cells expressing both a CAR specific for Her2 and a TCR specific for the melanocyte protein (gp100). We used a regimen of adoptive cell transfer incorporating vaccination (ACTIV), with recombinant vaccinia virus expressing gp100, to treat a range of tumors including orthotopic breast tumors and large liver tumors.Results: ACTIV therapy induced durable complete remission of a variety of Her2+ tumors, some in excess of 150 mm2, in immunocompetent mice expressing Her2 in normal tissues, including the breast and brain. Vaccinia virus induced extensive proliferation of T cells, leading to massive infiltration of T cells into tumors. Durable tumor responses required the chemokine receptor CXCR3 and exogenous IL2, but were independent of IFNγ. Mice were resistant to tumor rechallenge, indicating immune memory involving epitope spreading. Evidence of limited neurologic toxicity was observed, associated with infiltration of cerebellum by T cells, but was only transient.Conclusions: This study supports a view that it is possible to design a highly effective combination immunotherapy for solid cancers, with acceptable transient toxicity, even when the target antigen is also expressed in vital tissues. Clin Cancer Res; 23(10); 2478–90. ©2016 AACR.

ACTIV therapy induces cytokine secretion and apoptosis of tumor cells.

It is critically important that early foal handling and training takes into account foals cognitive abilities and ability to cope with intense work without becoming overly aroused and fearful. Foals may experience days off during planned training programs due to owner/trainers circumstances and not as an intended part of the training schedule. Recent research has suggested that days off within training schedules may be beneficial to training outcomes. This study aimed to determine how the incorporation of days off during initial training influences foal learning outcomes. Twenty Thoroughbred foals underwent a 6-7 session training program. Foals were blocked by age ( 8 weeks) and randomly allocated to either training condition A (trained for 2 consecutive days followed by a rest day, 2 more training days, rest day, finishing with up to 3 more consecutive training days) or training condition B (trained for 4 consecutive days, 2 rest days, finishing with up to 3 more consecutive training days). Qualified Foal NZ Ltd trainers used negative reinforcement to train foals to ‘Go’ (cranial pressure on the halter until foal steps forward) and ‘Stop’ (caudal pressure until the foal stops) from first, the left and second, the right side. Session length was determined by progress made and did not exceed 25 minutes. Training success was assessed by number of signals required (recall) for the foal to perform the task in the next training day. Biting behaviors exhibited by foals were instantaneously recorded during training sessions. Recall data for all tasks were non-parametric (left ‘Go’ Anderson Darling = 6.32; ‘Stop’ Anderson Darling = 2.89; right ‘Go’ Anderson Darling = 4.24; and ‘Stop’ Anderson Darling =6.10; all P 0.05). Younger foals ( 0.05). On the basis of the findings of this study, it is recommended that young foal initial training programs incorporate a day off within the first 4 days in order to decrease the development and occurrence of adverse behaviors such as biting.

Background Stroke is a leading cause of disability worldwide and the cardiovascular fitness levels of stroke survivors are diminished to an extent that impairs functioning and activities of daily living performance. While cardiovascular training seems an empirically appropriate intervention, the optimal dosage and intensity of cardiovascular training in stroke survivors remains unclear. The aim was to determine the safety and feasibility of moderate-intensity cardiovascular training following stroke, including measurement of adherence to training. Methods A pilot, prospective, patient- and assessor-blinded randomised controlled trial conducted in a tertiary, metropolitan hospital-based community rehabilitation centre. Eligibility criteria included ambulant (> 100 m), 6 weeks-12 months post stroke. Moderate-intensity fitness training or control (low-intensity) exercise was offered biweekly for 12 weeks. Outcome measures included adverse events, peak oxygen uptake (VO2), functional exercise capacity (6-Minute Walk Test, 10-m Walk Test) and health-related quality of life (Short Form-36) and mood (Patient Health Questionnaire, PHQ9). Results Feasibility: Seventy-one (50%) of 141 screened participants were eligible (29% did not agree to participate). Twenty participants (10 intervention, 10 control) were recruited. The median (%; IQR) supervised sessions was 19.5 (81%; 12, 20); and 20 (83%; 19, 22) in the intervention and control groups, respectively. Progression of duration and intensity was limited; mean of 10 sessions to achieve target duration (30 min). There were no adverse events. Baseline peak oxygen uptake (VO2) levels were low (15.94 ml/kg/min). Significant improvements in VO2 peak in both groups were observed (p Conclusions Moderate-intensity fitness training was safe but achievement of target duration and intensity was challenging for stroke survivors. A definitive adequately-powered randomised trial is required. Alternative fitness training protocols may need to be explored. Trial registration The trial protocol was prospectively registered on the Australian New Zealand Clinical Trials Registry (ACTRN 12613000822785) on 25/07/2013.

Background Intrinsic and acquired drug resistance represent fundamental barriers to the cure of high-grade serous ovarian carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. Defects in homologous recombination (HR) DNA repair are key determinants of sensitivity to chemotherapy and poly-ADP ribose polymerase inhibitors. Restoration of HR is a common mechanism of acquired resistance that results in patient mortality, highlighting the need to identify new therapies targeting HR-proficient disease. We have shown promise for CX-5461, a cancer therapeutic in early phase clinical trials, in treating HR-deficient HGSC. Methods Herein, we screen the whole protein-coding genome to identify potential targets whose depletion cooperates with CX-5461 in HR-proficient HGSC. Results We demonstrate robust proliferation inhibition in cells depleted of DNA topoisomerase 1 (TOP1). Combining the clinically used TOP1 inhibitor topotecan with CX-5461 potentiates a G2/M cell cycle checkpoint arrest in multiple HR-proficient HGSC cell lines. The combination enhances a nucleolar DNA damage response and global replication stress without increasing DNA strand breakage, significantly reducing clonogenic survival and tumour growth in vivo. Conclusions Our findings highlight the possibility of exploiting TOP1 inhibition to be combined with CX-5461 as a non-genotoxic approach in targeting HR-proficient HGSC.

The U@Uni Academy provides a case study of a composite preparation and access programme, integrating key elements of widening participation, alternate entry and enabling schemes. It is offered to s...

There is now evidence that gene fusions activating the MAPK pathway are relatively common in pancreatic acinar cell carcinoma with potentially actionable BRAF or RET fusions being found in ~30%. We sought to investigate the incidence of RAF1 fusions in pancreatic malignancies with acinar cell differentiation. FISH testing for RAF1 was undertaken on 30 tumors comprising 25 'pure' acinar cell carcinomas, 2 mixed pancreatic acinar-neuroendocrine carcinomas, 1 mixed acinar cell-low grade neuroendocrine tumor and 2 pancreatoblastomas. RAF1 rearrangements were identified in 5 cases and confirmed by DNA and RNA sequencing to represent oncogenic fusions (GATM-RAF1, GOLGA4-RAF1, PDZRN3-RAF1, HERPUD1-RAF1 and TRIM33-RAF1) and to be mutually exclusive with BRAF and RET fusions, as well as KRAS mutations. Large genome-wide copy number changes were common and included 1q gain and/or 1p loss in all five RAF1 FISH-positive acinar cell carcinomas. RAF1 expression by immunohistochemistry was found in 3 of 5 (60%) of fusion-positive cases and no FISH-negative cases. Phospho-ERK1/2 expression was found in 4 of 5 RAF1-fusion-positive cases. Expression of both RAF1 and phospho-ERK1/2 was heterogeneous and often only detected at the tumor-stroma interface, thus limiting their clinical utility. We conclude that RAF1 gene rearrangements are relatively common in pancreatic acinar cell carcinomas (14.3% to 18.5% of cases) and can be effectively identified by FISH with follow up molecular testing. The combined results of several studies now indicate that BRAF, RET or RAF1 fusions occur in between one third and one-half of these tumors but are extremely rare in other pancreatic malignancies. As these fusions are potentially actionable with currently available therapies, a strong argument can be made to perform FISH or molecular testing on all pancreatic acinar cell carcinomas.

Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease., Acquired resistance limits the efficacy of PARP inhibitors (PARPi) in high grade serous ovarian cancer (HGSOC). Here, the authors show that inhibition of RNA polymerase I transcription using CX-5461 increases the therapeutic efficacy of PARPi and overcomes PARPi resistance in PDX models of HGSOC.

The global prevalence of overweight and obesity in pregnancy is rising and this represents a significant challenge for the management of pregnancy and delivery. Women who have a pre‐pregnancy body mass index greater than 25 kg m–2 are more likely than those with a body mass index in the ideal range (20–24.99 kg m–2) to have problems conceiving a child and are at greater risk of miscarriage and stillbirth. All pregnancy complications are more likely with overweight, obesity and excessive gestational weight gain, including those that pose a significant threat to the lives of mothers and babies. Labour complications arise more often when pregnancies are complicated by overweight and obesity. Pregnancy is a stage of life when women have greater openness to messages about their lifestyle and health. It is also a time when they come into greater contact with health professionals. Currently management of pregnancy weight gain and the impact of overweight tends to be poor, although a number of research studies have demonstrated that appropriate interventions based around dietary change can be effective in controlling weight gain and reducing the risk of pregnancy complications. The development of individualised and flexible plans for avoiding adverse outcomes of obesity in pregnancy will require investment in training of health professionals and better integration into normal antenatal care.

The increasing coincidence of obesity with heart failure may preclude eligibility for orthotopic heart transplantation, requiring continuous-flow left ventricular assist devices (LVADs) as destination therapy. This report describes intraoperative considerations for patients who underwent LVAD implantation with concurrent laparoscopic sleeve gastrectomy (LSG) to promote weight loss. In particular, right ventricular dysfunction associated with acute left ventricular unloading may be compounded by pneumoperitoneum for LSG due to the difficulty in ventilating patients with obesity, hypercarbia-mediated increase in pulmonary vascular resistance, and variable cardiac loading conditions. We identify specific anesthetic challenges and discuss methods of monitoring and management.

RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine–rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.

The stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional Scl knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl. These Pf4Sclc-KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4Sclc-KO mice generated on an Lyl1-null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of Gata1, Fli1, Nfe2, and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets, and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations in inherited thrombocytopenia.