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It is well recognized that randomized controlled trials (RCTs) are a powerful tool to investigate causal relationships, and are considered the gold standard level of research evidence. However, RCTs can be expensive and time-consuming, and when they employ strict eligibility criteria, it results in an unrepresentative population and limited external validity. Recently, the registry-based randomized clinical trial (RRCT) has emerged as an alternative trial design. Utilizing registries to underpin such studies, RRCTs can have advantages including rapid recruitment, and enhanced generalizability. In Australia, legislated mandatory reporting of cancer diagnoses means that jurisdictional cancer registries are a rich source of systematically collected patient details, representing sound platforms for comprehensive data capture that can serve as a key tool for further research. We review the roles of cancer registries in Australia, discuss important considerations relevant to the design of RRCTs, and outline the opportunities provided by cancer registries to strengthen cancer research.

Extracellular vesicles (EVs) are a heterogeneous group of biological nano-sized vesicles that are released from cells and contribute to intercellular communication. Emerging knowledge about their biogenesis, composition, release, and uptake has resulted in broad interest in elucidating their potential roles in disease pathophysiology. The distinct biological properties of these biological nanoparticles emphasize several appealing advantages for potential therapeutic applications compared with the use of synthetic nanoparticles. When administered systemically, EVs are taken up and sequestered within the liver, further emphasizing opportunities for therapeutic use. Consequently, there is growing interest in their use for liver diseases. EVs can be used directly as therapeutics, and several studies have highlighted the intrinsic therapeutic properties of mesenchymal stem cell-derived EVs for chronic and acute liver diseases. Alternatively, EVs can be modified to facilitate their use for the delivery of therapeutic cargo. In this review, we discuss the cellular sources of EV, provide a concise overview of their potential use in diverse processes, and outline several promising applications for the use of EV-based therapeutics for liver diseases. The use of EV-based therapeutics provides a viable approach to target hepatic pathophysiology.

Summary Objective To determine the prevalence of neuropathic-like pain (NP) and pain sensitization (PS) defined by self-report questionnaires in knee and hip osteoarthritis, and whether prevalence is potentially explained by disease-severity or affected joint. Design MEDLINE, EMBASE, CINAHL were systematically searched (1990–April 2020) for studies describing the prevalence of NP and PS in knee and hip osteoarthritis using self-report questionnaires. Random-effects meta-analysis was performed. Statistical heterogeneity between studies and sub-groups (affected joint and population source as a proxy for disease severity) was assessed (I2 statistic and the Chi-squared test). Results From 2,706 non-duplicated references, 39 studies were included (2011–2020). Thirty-six studies reported on knee pain and six on hip pain. For knee osteoarthritis, the pooled prevalence of NP was: using PainDETECT, possible NP(score ≥13) 40% (95%CI 32–48%); probable NP(score >18) 20% (95%CI 15–24%); using Self-Report Leeds Assessment of Neuropathic Symptoms and Signs, 32% (95%CI 26–38%); using Douleur Neuropathique (DN4) 41% (95% CI 24–59%). The prevalence of PS using Central Sensitization Inventory (CSI) was 36% (95% CI 12–59%). For hip osteoarthritis, the pooled prevalence of NP was: using PainDETECT, possible NP 29% (95%CI 22–37%%); probable NP 9% (95%CI 6–13%); using DN4 22% (95%CI 12–31%) in one study. The prevalence of possible NP pain was higher at the knee (40%) than the hip (29%) (difference 11% (95% CI 0–22%), P = 0.05). Conclusions Using self-report questionnaire tools, NP was more prevalent in knee than hip osteoarthritis. The prevalence of NP in knee and hip osteoarthritis were similar for each joint regardless of study population source or tool used. Whether defining NP using self-report questionnaires enables more effective targeted therapy in osteoarthritis requires investigation.

The prevalence of potentially precarious cosmetic facial procedures appears to be on the rise. A significant amount of these cosmetic procedures are offered and performed by operators without formal medical training and anatomical knowledge, and with variable degrees of skill. Some of these procedures can result in devastating sight-threatening complications, and many of the individuals undergoing such treatments are relatively young and healthy. Patients need to be aware of the potential risks, including permanent visual loss, before embarking on any cosmetic facial procedure. Optometrists may be the first point of contact for patients with ocular complaints following these treatments. Hence, the authors present here a review on the various ocular injuries that may result from commercial cosmetic procedures.

Biologically derived nanoparticles such as extracellular vesicles are promising candidates for therapeutic applications. In vivo toxicity of biological nanoparticles can result in tissue or organ damage, immunological perturbations, or developmental effects but cannot be readily predicted from in vitro studies. Therefore, an essential component of the preclinical assessment of these particles for their use as therapeutics requires screening for adverse effects and detailed characterization of their toxicity in vivo. However, there are no standardized, comprehensive methods to evaluate the toxicity profile of nanoparticle treatment in a preclinical model. Here, we first describe a method to prepare bovine milk-derived nanovesicles (MNVs). These MNVs are inexpensive to isolate, have a scalable production platform, and can be modified to achieve a desired biological effect. We also describe two vertebrate animal models, mice and zebrafish, that can be employed to evaluate the toxicity profile of biologically derived nanoparticles, using MNVs as an example. Treatment-induced organ toxicity and immunological effects can be assessed in mice receiving systemic injections of MNVs, and developmental toxicity can be assessed in zebrafish embryos exposed to MNVs in embryo water. Utilizing these animal models provides opportunities to analyze the toxicity profiles of therapeutic extracellular vesicles in vivo. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of milk-derived nanovesicles Basic Protocol 2: In vivo screening for organ toxicity and immune cell profiling using mice Basic Protocol 3: In vivo developmental toxicity screening using zebrafish.

Histidyl dipeptides, are synthesized in the heartviaenzyme carnosine synthase (Carns), which facilitates glycolysis and glucose oxidation by proton buffering and attenuate ischemia and reperfusion injury. However, a composite understanding of the histidyl dipeptide mediated responses in the heart are lacking. We performed multilayer omics in the cardio specificCarnsoverexpressing mice, showing higher myocardial levels of histidyl dipeptides lead to extensive changes in microRNAs that could target the expression of contractile proteins and enzymes involved inβ-fatty acid oxidation and citric acid cycle (TCA). Similarly, global proteomics showed contractile function, fatty acid degradation and TCA cycle, pathways were enriched in the CarnsTg heart. Parallel with these changes, free fatty acids, and TCA intermediate-succinic acid were lower under aerobic and significantly attenuated under anaerobic conditions in the CarnsTg heart. Integration of multiomics data showsβ-fatty acid oxidation and TCA cycle exhibit correlative changes at all three levels in CarnsTg heart, suggesting histidyl dipeptides are critical regulators of myocardial structure, function and energetics.

Aberrant activation of β-catenin signaling is frequently observed in hepatocellular cancer. Although Wnt/β-catenin signaling can be targeted by vitamin D, therapeutic use of vitamin D for this purpose is not currently established. We evaluated the therapeutic use of vitamin D or its analogs using a synthetic transgenic mouse of hepatocarcinogenesis induced by mutant β-catenin, and MET overexpression in which 75% of mice develop well-differentiated HCC within 8 weeks in the absence of fibrosis. Vitamin D receptor expression was similar in both tumoral and nontumoral tissue. There was no significant difference in overall survival, or in tumor progression assessed by imaging, biochemical, or tumor cell burden assessments in mice receiving a vitamin D-supplemented diet containing 12.0 IU VD/g (HVD) compared with a standard diet (SD) containing 2.3 IU VD/g. Furthermore, systemic treatment with calcitriol [vitamin D analog 1α,25(OH)2D3] or EB1089 (synthetic vitamin D analog) by intraperitoneal injection for 4 weeks prolonged median survival but did not increase overall survival compared with controls. Although tumor formation was delayed in males compared with that in females, there was no difference in overall survival between males and females. In conclusion, although 1α,25(OH)2D3 is reported to inhibit β-catenin signaling, as well as proliferation, migration, and differentiation in cancer cells, neither dietary supplementation with vitamin D nor treatment with vitamin D analogs altered the formation or growth of HCC associated with β-catenin activation. These results conclusively demonstrate the lack of utility of targeting vitamin D for therapy of HCC in this setting.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Although HCC can respond to immune checkpoint inhibitors, such as monoclonal antibodies against programmed death 1 (PD-1), many patients fail to respond or develop secondary resistance. Activation of Wnt/β-catenin signaling can contribute to immune evasion. Mutations in β-catenin are among the most frequent mutations associated with HCC. Thus, our aim was to directly target β-catenin to enhance the therapeutic response to immune checkpoint inhibition. A synthetic transgenic mouse model of experimental HCC induced by tyrosine-protein kinase Met/β-catenin expression and extracellular vesicles (EVs) as a therapeutic delivery agent was used to evaluate the efficacy of directly targeting β-catenin on the response to anti-PD-1. These studies showed that (1) oncogenic β-catenin could be therapeutically targeted using a biological nanoparticle-based delivery approach, (2) targeting β-catenin using small interfering RNA (siRNA) delivered within EVs can reduce tumor growth, and (3) the therapeutic response to anti-PD-1 can be enhanced by concomitantly targeting β-catenin using therapeutic EVs. These preclinical studies establish the efficacy of the use of biological nanoparticles as an endogenous delivery vehicle for therapeutic RNA delivery and support the use of therapeutic strategies targeting tumor-intrinsic β-catenin as an adjunct to anti-PD-1-based therapy. Conclusion: Combination therapy with anti-PD-1 and β-catenin siRNA delivered using biological nanoparticles provides an effective strategy for the treatment of HCC. This strategy could be further exploited into targeted approaches for immune potentiation by countering oncogene-mediated resistance to immunotherapies.

BACKGROUND Clinical trials of allergen immunotherapy (AIT) may require up to 5 years to complete. These lengthy trials may be complicated by high and potentially differential dropouts, especially among participants who perceive that they are receiving placebo. We propose a three-stage design in which the placebo group in Stage 1 crosses over to receive active treatment in Stage 2. In Stage 3, AIT is discontinued to determine whether benefit is maintained post-treatment. We apply inferential statistics to support the three-stage design for clinical trials to determine clinical efficacy, treatment response over time, and sustained response to AIT. METHODS The proposed framework constitutes a series of hypothesis tests for comparing treatment responses at the end of each stage. A simulation study was performed to illustrate the statistical properties under varying statistical missing mechanisms and effect sizes. RESULTS The statistical properties in terms of bias and statistical power were consistent with what are expected from conventional analyses. Specifically, the extent of bias depended on the missing mechanism and magnitude. The statistical powers were largely driven by effect and sample sizes as well as prespecified success margins. As an illustration, assuming relative treatment differences of 25% and stagewise dropout rate of 15%, a sample size of 200 per group may achieve 93% power to demonstrate a treatment effect and 60% power to demonstrate a maintained response post-treatment. CONCLUSIONS Inferential statistics support our proposed study design for evaluating benefits of AIT over time and inform clinical understanding and decisions.

Purpose/Objective(s) Identifying suspicious cancer metastasized lymph nodes (GTV is an essential task in esophageal cancer radiotherapy. Manual delineation requires high-level sophisticated clinical reasoning in multi-modality imaging, which is challenging and suffers from high subjectivity and inter-observer variability. We propose an automated esophageal GTV_LN detection method via integrating a distance-based decision stratification into a multi-task deep network, which is able to effectively learn the GTV_LN oncology features. This is inspired by the fact that lymph node involvements often follow certain distance patterns from the primary tumors. Materials/Methods We collected and curated 2 esophageal cancer datasets with PET and RTCT images. The 1st dataset contained 141 patients and 651 annotated GTV_LN for training and evaluating our deep network (60%, 10% and 30% for training, validation and testing), while the 2nd dataset contained 10 patients aiming for the multi-user agreement analysis. A distance-based decision stratification is designed to divide GTV_LN into “tumor-proximal” and “tumor-distal” categories and a multi-branch deep network is proposed to solve each of them. The multi-branch network has a shared encoder and separate decoders to detect and segment two GTV_LN subcategories, respectively. The distance-based decision stratification enables the automated multi-branch method to specialize in GTV_LN oncology features of each subcategory. We use both PET and RTCT as the network inputs to exploit the complementary information in each imaging modality. Results In the testing set of the 1st esophageal cancer dataset (34 patients with 138 GTV_LNs), our method achieves a sensitivity of 73.8% and 79.1% at 6 and 12 false positives (FPs) per patient as compared to 72.0% and 75.9% by MULAN, the state-of-the-art deep learning based universal lesion detection algorithm. On the 2nd dataset containing 10 patients, 3 radiation oncologists annotated 53 GTV_LNs in total, among which 42 GTV_LNs were agreed with all 3 physicians leading to 79.2% multi-user agreement. Our method achieves a sensitivity of 77.4% at 6 FPs per patient on the 2nd dataset. This shows that the sensitivity achieved by our automated deep learning approach can be close to the multi-user agreement. Conclusion We proposed a multi-task deep learning method with distance-based decision stratification to effectively detect and segment the GTV_LN in esophageal cancer radiotherapy. It significantly improves on previous state-of-the-art and the achieved performance has potential clinical values considering the large multi-user variability in this challenging task.

Background Recent genome-wide association meta-analysis for melanoma doubled the number of previously identified variants. We assessed the performance of an updated polygenic risk score (PRS) in a population of older individuals, where melanoma incidence and cumulative ultraviolet radiation exposure is greatest. Methods We assessed a PRS for cutaneous melanoma comprising 55 variants in a prospective study of 12 712 individuals in the ASPirin in Reducing Events in the Elderly Trial. We evaluated incident melanomas diagnosed during the trial and prevalent melanomas diagnosed preenrolment (self-reported). Multivariable models examined associations between PRS as a continuous variable (per SD) and categorical (low-risk [0%-20%], medium-risk [21%-80%], high-risk [81%-100%] groups) with incident melanoma. Logistic regression examined the association between PRS and prevalent melanoma. Results At baseline, mean participant age was 75 years; 55.0% were female, and 528 (4.2%) had prevalent melanomas. During follow-up (median = 4.7 years), 120 (1.0%) incident cutaneous melanomas occurred, 98 of which were in participants with no history. PRS was associated with incident melanoma (hazard ratio = 1.46 per SD, 95% confidence interval [CI] = 1.20 to 1.77) and prevalent melanoma (odds ratio [OR] = 1.55 per SD, 95% CI = 1.42 to 1.69). Participants in the highest-risk PRS group had increased risk compared with the low-risk group for incident melanoma (OR = 2.51, 95% CI = 1.28 to 4.92) and prevalent melanoma (OR = 3.66, 95% CI = 2.69 to 5.05). When stratifying by sex, only males had an association between the PRS and incident melanoma, whereas both sexes had an association between the PRS and prevalent melanoma. Conclusions A genomic risk score is associated with melanoma risk in older individuals and may contribute to targeted surveillance.

Summary Background Almost a third of those undergoing knee replacement for osteoarthritis have poor outcomes despite technically successful surgery. Preoperative neuropathic-like pain and/or pain sensitisation may increase the risk of pain following joint replacement. Objective To examine whether preoperative neuropathic-like pain and pain sensitisation predicts pain, function and satisfaction following joint replacement for knee osteoarthritis. Design Systematic review with meta-analysis. Methods Medline, EMBASE and CINAHL were systematically searched until March 2020. Studies detecting neuropathic-like pain and/or sensitisation using self-report questionnaires prior to knee replacement for osteoarthritis, and relating this to post-operative outcomes were identified. Data extraction, risk of bias assessment and meta-analysis were performed, where appropriate. Results Five manuscripts, including one preprint, examining six cohorts were included: four used painDETECT or modified painDETECT, one the Self-Report Leeds Assessment of Neuropathic Symptoms and Signs, and another the Central Sensitisation Inventory to identify preoperative characteristics. Three studies showed preoperative neuropathic-like pain or sensitisation was associated with more intense post-operative pain. All four studies examining the risk of significant pain after knee replacement suggested it was increased after > 3 months. The only study examining patient satisfaction and function found reduced satisfaction, but no difference in function in those with preoperative sensitisation. Meta-analysis found the relative risk of increased pain following knee replacement in those with neuropathic-like pain(painDETECT > 13) to be 2.05(95% confidence intervals 1.51, 2.79). Conclusion These results provide consistent but limited evidence that self-report tools detecting neuropathic-like pain and/or pain sensitisation, predict patients at higher risk of pain following knee replacement.

Hepatocellular carcinoma is highly resistant to chemotherapy. Here we evaluated the use and efficacy of milk-derived nanovesicles (MNV) as an approach to improve delivery of anticancer agents into HCC cells and intrahepatic tumors. We developed a protocol for isolation of MNVs from skim milk using ultracentrifugation, and characterized using nanoparticle tracking analysis (NTA) and electron microscopy. MNVs were loaded with doxorubicin (dox-MNV) or miR221 antisense oligonucleotides (anti-miR221-MNV), and further evaluated using spectrophotometry, NTA, and zeta potential measurements. HepG2, Hep3B, and PLC/PRF/5 HCC cells in culture were treated with dox-MNV and anti-miR221-MNV and evaluated with drug delivery and anticancer activity. The efficacy of dox-MNV and anti-miR221-MNV to arrest tumor growth in vivo was assessed on intrahepatic tumors induced in nude mice. Cellular uptake studies showed plain and dox-MNV attained saturation within 4 h of treatment. Cytotoxicity studies on HepG2, Hep3B, and PLC/PRF/5 HCC cells with dox-MNV at 1 µM resulted in 20% cell death at 24 h, 50% at 48 h, and 80% at 72 h. HepG2 cells treated with dox-MNV and anti-miR221-MNV exhibited nuclear disintegration, and apoptosis within 24 h. Combination treatment of intrahepatic tumors with dox-MNV and anti-miR221-MNV resulted in marked reduction of tumor size and increased survival rate in nude mice. Our studies demonstrated that MNVs can be effectively used for successful delivery of anticancer agents into HCC cells and intrahepatic tumors. MNV-mediated targeted delivery of anticancer agents could be an efficient modality for the treatment of malignant HCC and might produce a great impact on anticancer therapy.

Uncorrected refractive errors are a leading cause of visual impairment across the world. In today's society, laser in situ keratomileusis (LASIK) has become the most commonly performed surgical procedure to correct refractive errors. However, regression of the initially achieved refractive correction has been a widely observed phenomenon following LASIK since its inception more than two decades ago. Despite technological advances in laser refractive surgery and various proposed management strategies, post-LASIK regression is still frequently observed and has significant implications for the long-term visual performance and quality of life of patients. This review explores the mechanism of refractive regression after both myopic and hyperopic LASIK, predisposing risk factors and its clinical course. In addition, current preventative strategies and therapies are also reviewed.

Extracellular vesicles are endogenous biological nanoparticles that have potential for use as therapeutic nanoparticles or as delivery vehicles for therapeutic agents. Milk nanovesicles (MNV) are extracellular vesicles isolated from bovine milk that have been explored for use as delivery vehicles for RNA therapeutics such as small interfering RNA (siRNA). We performed in vivo toxicological studies of MNV or therapeutic MNV (tMNV) loaded with siRNA as a prelude to their clinical use. Development toxicity was assessed in zebrafish embryos. Acute toxicity was assessed in both mice and zebrafish whereas safety, biochemical, histological and immune effects after multiple dosing were assessed in mice. Zebrafish embryo hatching was accelerated with MNV and tMNV. While acute toxicity or effects on mortality were not observed in zebrafish, developmental effects were observed at high concentrations of MNV. There was a lack of discernable toxicity, mortality and systemic inflammatory or immunological responses in mice following administration of either MNVs or tMNVs. The tolerability and lack of discernable developmental or systemic in vivo toxicity support their use as biological nano-therapeutics. Adoption of a standardized protocol for systematic analysis of in vivo safety and toxicity will facilitate preclinical assessment of EV based formulations for therapeutic use.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

By exploiting their biological functions, the use of biological nanoparticles such as extracellular vesicles can provide an efficient and effective approach for hepatic delivery of RNA‐based therapeutics for the treatment of liver cancers such as hepatocellular cancer (HCC). Targeting liver cancer stem cells (LCSC) within HCC provide an untapped opportunity to improve outcomes by enhancing therapeutic responses. Cells with tumor‐initiating capabilities such as LCSC can be identified by expression of markers such as epithelial cell adhesion molecule (EpCAM) on their cell surface. EpCAM is a target of Wnt/β‐catenin signaling, a fundamental pathway in stem‐cell growth. Moreover, mutations in the β‐catenin gene are frequently observed in HCC and can be associated with constitutive activation of the Wnt/β‐catenin pathway. However, targeting these pathways for the treatment of HCC has been challenging. Using RNA nanotechnology, we developed engineered biological nanoparticles capable of specific and effective delivery of RNA therapeutics targeting β‐catenin to LCSC. Extracellular vesicles isolated from milk were loaded with small interfering RNA to β‐catenin and decorated with RNA scaffolds to incorporate RNA aptamers capable of binding to EpCAM. Cellular uptake of these EpCAM‐targeting therapeutic milk‐derived nanovesicles in vitro resulted in loss of β‐catenin expression and decreased proliferation. The uptake and therapeutic efficacy of these engineered biological nanotherapeutics was demonstrated in vivo using tumor xenograft mouse models. Conclusion: β‐catenin can be targeted directly to control the proliferation of hepatic cancer stem cells using small interfering RNA delivered using target‐specific biological nanoparticles. Application of this RNA nanotechnology–based approach to engineer biological nanotherapeutics provides a platform for developing cell‐surface molecule–directed targeted therapeutics., By exploiting their biological functions, the use of biological nanoparticles such as extracellular vesicles can provide an efficient and effective approach for hepatic delivery of RNA‐based therapeutics for the treatment of hepatocellular cancer.

BackgroundLittle is known about long-term employment outcomes for patients with first-episode schizophrenia-spectrum (FES) disorders who received early intervention services.AimsWe compared the 10-year employment trajectory of patients with FES who received early intervention services with those who received standard care. Factors differentiating the employment trajectories were explored.MethodPatients with FES (N= 145) who received early intervention services in Hong Kong between 1 July 2001 and 30 June 2002 were matched with those who entered standard care 1 year previously. We used hierarchical clustering analysis to explore the 10-year employment clusters for both groups. We used the mixed model test to compare cluster memberships and piecewise regression analysis to compare the employment trajectories of the two groups.ResultsThere were significantly more patients who received the early intervention service in the good employment cluster (early intervention:N= 98 [67.6%]; standard care:N= 76 [52.4%];P= 0.009). In the poor employment cluster, there was a significant difference in the longitudinal pattern between early intervention and standard care for years 1–5 (P< 0.0001). The number of relapses during the first 3 years, months of full-time employment during the first year and years of education were significant in differentiating the clusters of the early intervention group.ConclusionsResults suggest there was an overall long-term benefit of early intervention services on employment. However, the benefit was not sustained for all patients. Personalisation of the duration of the early intervention service with a focus on relapse prevention and early vocational reintegration should be considered for service enhancement.

Extracellular vesicles (EVs) are membrane-bound vesicles released from cells into the extracellular environment. There is emerging interest in the use of EVs as potential therapeutic interventions. We sought to evaluate the safety of EVs that may be therapeutically used by performing in vitro toxicological assessments. EVs were obtained from mesenchymal stem cells (MSC-EV) or from bovine milk (BM-EV) by differential ultracentrifugation, and quantitated using nanoparticle tracking analysis. Genotoxic effects, hematological effects, immunological effects and endotoxin production were evaluated at two dose levels. Neither MSC-EVs nor BM-EVs elicited detectable genotoxic effects using either the alkaline comet assay or micronucleus assay. Hemolysis was observed with BM-EVs but not with MSC-EVs. MSC-EVs did not have any significant effect on either spontaneous or collagen-induced platelet aggregation. In contrast, BM-EVs were noted to increase collagen-induced platelet aggregation, even though no spontaneous increase in platelet aggregation was noted. Both types of EVs induced leukocyte proliferation, which was greater with BM-EV. Neither MSC-EVs nor BM-EVs induced HL-60 phagocytosis, although BM-EVs decreased zymosan-induced phagocytosis. Furthermore, neither MSC-EVs nor BM-EVs induced nitric oxide production. Unlike MSC-EVs, BM-EVs tested positive for endotoxin and induced complement activation. There are significant differences in toxicological profiles between MSC-EVs and BM-EVs that may reflect variations in techniques for EV isolation, EV content or cross-species differences. The safety of MSC-EV supports their use for disease therapeutics, whereas detailed safety and toxicological assessment will be necessary before the use of BM-EVs. Copyright © 2016 John Wiley & Sons, Ltd.

Trophopods are modified stipe bases that function as starch-storage organs in a wide variety of mainly temperate ferns. Ever since they were first observed, the presence of trophopods has been explained by reference to seasonality and they have been assumed to provide nutrition for the rapidly expanding fern leaves in spring. We present the results of an analysis of the annual variation in starch content in Matteuccia struthiopteris (L.) Todaro, cultivated in the Leiden botanical garden. Our results show a distinct seasonal variation in starch content, suggesting that the presence of trophopods is indeed functionally linked to seasonality, but we do not find a decrease in starch content corresponding to the period of leaf expansion that would clearly link the depletion of stored starch to the expansion of new leaves.

Radiomics is a newly emerging field that involves the extraction of a large number of quantitative features from biomedical images through the use of data-characterization algorithms. Radiomics provides a noninvasive approach for personalized therapy decision by identifying distinctive imaging features for predicting prognosis and therapeutic response. So far, many of the published radiomics studies utilize existing out of the box algorithms to identify the prognostic markers from biomedical images that are not specific to radiomics data. T o better utilize biomedical image, we propose a novel machine learning approach, stability selection supervised principal component analysis (SSSuperPCA) that identify a set of stable features from radiomics big data coupled with dimension reduction for right censored survival outcomes. In this paper, we describe stability selection supervised principal component analysis for radiomics data with right-censored survival outcomes. The proposed approach allows us to identify a set of stable features that are highly associated with the survival outcomes, control the per-family error rate, and predict the survival in a simple yet meaningful manner. We evaluate the performance of SSSuperPCA using simulations and real data sets for non-small cell lung cancer and head and neck cancer, and compare it with other machine learning algorithms. The results demonstrate that our method has a competitive edge over other existing methods in identifying the prognostic markers from biomedical big imaging data for the prediction of right-censored survival outcomes. An R package SSSuperPCA is available at the website: http://web.hku.hk/∼herbpang/SSSuperPCA.html

No Abstract available

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer related death worldwide. Although HCC can respond to immune checkpoint inhibitors such as monoclonal antibodies against PD-1, many patients fail to respond or develop secondary resistance. Activation of Wnt/β-catenin signaling can contribute to immune evasion. Mutations in β-catenin are amongst the most frequent mutations associated with HCC. Thus, our aim was to directly target β-catenin to enhance the therapeutic response to immune checkpoint inhibition. Methods: A synthetic transgenic mouse of experimental HCC induced by MET/ β-catenin expression, and extracellular vesicles as a therapeutic delivery agent were used to evaluate the efficacy of directly targeting β-catenin on response to anti-PD-1. Findings: These studies show that (1) oncogenic β-catenin could be therapeutically targeted using a biological nanoparticle based delivery approach, (2) targeting β-catenin using siRNA delivered within extracellular vesicles can reduce tumor growth, and (3) the therapeutic response to anti-PD-1 can be enhanced by concomitantly targeting β-catenin using therapeutic extracellular vesicles. These preclinical studies establish the efficacy of use of biological nanoparticles as an endogenous delivery vehicle for therapeutic RNA delivery and support the use of therapeutic strategies targeting tumor-intrinsic β-catenin as an adjunct to anti-PD-1 based therapy. Interpretation: Combination therapy with anti PD-1 and β-catenin siRNA delivered using biological nanoparticles provide an effective strategy for the treatment of HCC. This strategy could be further exploited into targeted approaches for immune potentiation by countering oncogenemediated resistance to immunotherapies. Funding Statement: Supported in part by the National Institutes of Health.TP is funded by NIH (UH3-TR000884). This publication is part of the NIH Extracellular RNA Communication Consortium paper package and was supported by the NIH Common Fund's exRNA Communication Program Declaration of Interests: There are no conflicts for any of the authors related to design, execution or analysis of the experiments presented in this manuscript. Ethics Approval Statement: All studies involving animals were performed in accordance with protocols approved by the Mayo Clinic Institutional Animal Care and Use committee.

The study of extracellular RNA has been recently reported as a tool for biomarker discovery. Extracellular vesicles can be isolated from different types of body fluids which contain protein, mRNA, and noncoding RNA. Extracellular RNA isolated from bile could be a useful tool for analyzing biliary tract diseases or cancer. Herein, we describe protocols based on modifications of commercially available kits for the collection, processing, and isolation of extracellular RNA from bile.

Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.

Objective:To study the clinical effect of lens cleaning paper patching on traumatic eardrum perforations.Methods:A total of 122 patients were divided into 2 groups, of which 56 patients were treated with lens cleaning paper patching and 66 acted as controls. The closure rate and healing time were compared between the two groups.Results:The healing rate of small perforations was 96.4 per cent (27 out of 28) in the patching group and 90 per cent (27 out of 30) in the control group. The difference was not statistically significant (p > 0.05). The healing rate of large perforations was 89.3 per cent (25 out of 28) and 80.6 per cent (29 out of 36) in the two groups, respectively. The difference was statistically significant (p < 0.05). The healing time of large perforations was shorter in the patching group than in the control group (p < 0.01).Conclusion:Patching with lens cleaning paper under an endoscope can accelerate the closure of large traumatic eardrum perforations.

Type 2 diabetes mellitus (T2DM) is a major public health challenge that affects countries across the world. The use of pharmacological therapy is often limited in some patients due to a loss of effect over time or development of adverse effects such as weight gain or hypoglycaemia. This has prompted searches into the role of non-pharmacological therapies in T2DM. The availability and use of vitamin supplements in developed countries have increased significantly and there is evidence that certain vitamins may have roles in the management of T2DM. This review examines the literature assessing the use of vitamins A, C, E, D, K and the B group vitamins (B1, B3, B7, B6, B9, B12) in the management of T2DM. No clear evidence supporting the beneficial role of any specific vitamin in the treatment of T2DM was found. Thus, it is recommended that until further studies are conducted to clarify the role of such vitamins in T2DM management, they should not be routinely recommended in clinical practice.

Hepatic ischemia/reperfusion injury (IRI) and associated inflammation contributes to liver dysfunction and complications after liver surgery and transplantation. Mesenchymal stem cells (MSCs) have been reported to reduce hepatic IRI because of their reparative immunomodulatory effects in injured tissues. Recent studies have highlighted beneficial effects of extracellular vesicles from mesenchymal stem cells (MSC-EV) on tissue injury. The effects of systemically administered mouse bone marrow-derived MSC-EV were evaluated in an experimental murine model of hepatic IRI induced by cross-clamping the hepatic artery and portal vein for 90 minutes followed by reperfusion for periods of up to 6 hours. Compared with controls, intravenous administration of MSC-EV 30 minutes prior to IRI dramatically reduced the extent of tissue necrosis, decreased caspase 3-positive and apoptotic cells, and reduced serum aminotransferase levels. MSC-EV increased hepatic messenger RNA (mRNA) expression of NACHT, LRR, and PYD domains-containing protein 12, and the chemokine (C-X-C motif) ligand 1, and reduced mRNA expression of several inflammatory cytokines such as interleukin 6 during IRI. MSC-EV increased cell viability and suppressed both oxidative injury and nuclear factor kappa B activity in murine hepatocytes in vitro. In conclusion, the administration of extracellular vesicles derived from bone marrow-derived MSCs may ameliorate hepatic IRI by reducing hepatic injury through modulation of the inflammatory response.Liver Transplantation 23 791-803 2017 AASLD.

Extracellular vesicles (EVs) are membrane-bound vesicles that are released by cells into their extracellular environment, have selective enrichment of specific proteins and RNA, and can mediate intercellular communication. In this review we highlight recent observations of the role of EVs in liver injury, viral hepatitis, alcoholic or nonalcoholic liver disease, biliary tract disease, and liver cancers. Potential applications as markers of diseases or for therapeutic applications are outlined to emphasize the new opportunities that are arising from the study of EVs.

Stem cell-based therapies have potential for treatment of liver injury by contributing to regenerative responses, through functional tissue replacement or paracrine effects. The release of extracellular vesicles (EV) from cells has been implicated in intercellular communication, and may contribute to beneficial paracrine effects of stem cell-based therapies. Therapeutic effects of bone-marrow derived mesenchymal stem cells (MSC) and vesicles released by these cells were examined in a lethal murine model of hepatic failure induced by d-galactosamine/tumor necrosis factor-α (TNF-α). Systemically administered EV derived from MSC accumulated within the injured liver following systemic administration, reduced hepatic injury, and modulated cytokine expression. Moreover, survival was dramatically increased by EV derived from either murine or human MSC. Similar results were observed with the use of cryopreserved mMSC-EV after 3 months. Y-RNA-1 was identified as a highly enriched noncoding RNA within hMSC-EV compared to cells of origin. Moreover, siRNA mediated knockdown of Y-RNA-1 reduced the protective effects of MSC-EV on TNF-α/ActD-mediated hepatocyte apoptosis in vitro. These data support a critical role for MSC-derived EV in mediating reparative responses following hepatic injury, and provide compelling evidence to support the therapeutic use of MSC-derived EV in fulminant hepatic failure.

Hepatocellular carcinoma (HCC) is characterized by a propensity for multifocality, growth by local spread, and dysregulation of multiple signaling pathways. These features may be determined by the tumoral microenvironment. The potential of tumor cells to modulate HCC growth and behavior by secreted proteins has been extensively studied. In contrast the potential for genetic modulation is poorly understood. We investigated the role and involvement of tumor derived nanovesicles capable of altering gene expression, and characterized their ability to modulate cell signaling and biological effects in other cells. We show that HCC cells can produce nanovesicles, exosomes, that differ in both RNA and protein content from their cells of origin. These can be taken up and internalized by other cells, and can transmit a functional transgene. The microRNA content of these exosomes was examined, and a subset that is highly enriched within exosomes was identified. A combinatorial approach to identify potential targets identified transforming growth factor β activated kinase-1 (TAK1) as the most likely candidate pathway that could be modulated by these miRNA. Loss of TAK1 has been implicated in hepatocarcinogenesis and is a biologically plausible target for inter-cellular modulation. We showed that HCC cell derived exosomes can modulate TAK1 expression and associated signaling and enhance transformed cell growth in recipient cells. Conclusion: Exosome mediated miRNA transfer is an important mechanism of inter-cellular communication in HCC cells. These observations identify a unique inter-cellular mechanism that could potentially contribute to local spread, intrahepatic metastases or multifocal growth in HCC.

Introduction An EMR-generated individualized Spanish-language Asthma Action Plan (Sp-AAP) had been developed for use throughout a large pediatric health care system. This study completed the first quality assessment review of the Sp-AAP by comparing measures of Sp-AAP comprehensibility and user valuation to the comparator English-language AAP (E-AAP). Methods 53 caregivers (35 English, 18 Spanish-speaking) of children Results Both objective (English: 60.0% range 0-100%, Spanish: 32.5% range 0-80%, p = 0.009) and subjective AAP comprehension (p = 0.03) differed significantly between English and Spanish-language respondents. Sp-AAP users reported taking longer to answer questions using the AAP compared to E-AAP users (p 0.05) but maintained at higher educational levels. There was no significant difference in caregiver valuation of the Sp-AAP (9.2) vs. E-AAP (8.9) (p=0.50) and valuation did not associate with objective comprehension (p = 0.86). Conclusions Differences in comprehensibility between Sp-AAP and E-AAP appear to exist but this study is limited by the differences in educational level between groups. Comprehensibility of the AAP irrespective of language was lower than expected. Valuation occurs independent of objective comprehension.

Introduction Increasing the number of cancer patients that benefit from immunotherapies and maintain durable responses requires understanding of cellular mechanisms that govern anti-tumour immune responses. However, this is hampered by a lack of appropriate pre-clinical testing models that are both clinically relevant and practical. Therefore, we have set up a robust in vitro assay that allows image based high-throughput analysis of 3D cultures. Here, immune cells are co-cultured with cancer cells in a 3D environment which recapitulates the tumour micro-environment and its complex cellular interactions. Specifically, infiltration of immune cells into the organoids and their killing are visualised and measured, which enables a better understanding of the immune-modulatory profile of different immunotherapies. Material and methods Autologous colon organoids from normal and tumour tissue (Hubrecht Organoid Technology) from several patients were cultured in a 3D environment. Immune cells with and without activation were added and their infiltration into organoids and subsequent killing was visualised using high-content microscopy. Quantification of immune cell effects was achieved with morphometric analysis with OMinerTM software. Results and discussions 3D image-based analysis of organoid-immune cell interactions demonstrated differential responses to immune-modulation between normal and tumour tissues and highlighted patient related differences. These results elucidate the effect of immune cell targeting normal colon tissue and discriminate immune-tumour cell interactions depending on activation status of T cells. The 3D environment, both for the cell culture and image analysis, allows for measurement of spatially resolved information, not accessible by monolayer cultures or biochemical assays. Conclusion The 3D assay presented here allows the analysis of immuno-oncology drug effects on different cell types that engage in a more physiologically relevant spatial setting than when culturing them in traditional 2D cultures. Using image-based analysis, immune-tumour interactions can be visualised and quantified. Analysis of both healthy and tumour tissue from the same patient allows for better prediction of clinical outcome versus non-specific cytotoxic reactions. This new and innovative platform can empower immunotherapy drug developers with a tool to select the most promising candidates and understand their mechanism of action, which ultimately translate to better clinical performance.

Introduction Patient-derived xenograft (PDX) models emulate tumour complexity and heterogeneity, and reflect the variation in pathologic and genetic diversity within the population. As such, these models are a valuable source in preclinical drug development. We employ hydrogel embedding as a 3D culture technique to generate short term cultures of dissociated PDX material. This miniaturised setup allows economical screening of drug libraries, testing of drug combinations and pre-selection of relevant models for in vivo follow up. Material and methods In a high throughput approach, small amounts of tumour material are seeded in 384 well plates. Following exposure to multiple concentrations of standard-of-care chemotherapeutics, small molecules, targeted therapies, antibodies and antibody-drug-conjugate, the cultures are fixed and stained with cellular markers at the experimental endpoint. A 3D stack acquisition of each well is obtained, followed by high content image analysis using an in-house developed analysis platform, OminerTM. This enables measurement of relevant features such as tumour volume, tumour invasion, nucleus size and fraction of apoptotic cells; hence detailed tumour culture responses can be quantified, generating dose-dependent profiles for selected features. Results and discussions We successfully developed >50 cultures derived from breast, stomach, pancreatic, colon, bladder and lung cancer. Each model has unique growth characteristics and morphologic differences are prominent both between the different tissues of origin and similar pathologies. Histologic characterisation of the 3D tumour cultures show hallmarks of the original tumour, such as retention of relevant biomarkers. We present drug response data for various cancer indications, including multiple lung and breast cancer pathologies. Conclusion We demonstrate phenotypic variation amongst different PDX models and differential responses to anti-tumour therapies. This method facilitates the comparison and in depth interrogation of responses to both established, biosimilar and novel cancer drugs - and allows follow up in the same model in vivo.

Introduction Drug treatment response prediction for cancer patients is still a challenge. It compromises clinical trials due to suboptimal selection of patients and leads to disappointing response rates for drug treatment in the clinic. Functional testing using 3D cultures of patient tumour biopsies offers the opportunity to test drug sensitivity upfront, and subsequently optimise treatment strategies for patients. Our technology based on 3D image analysis of tumour cultures accommodates accurate evaluation of drug sensitivity with small amounts of heterogeneous tumour samples. We have initiated a clinical trial to compare drug sensitivity of tumour cultures from pancreatic cancer patients, and clinical response on standard of care treatment. Material and methods 3D cultures embedded in an extracellular matrix protein hydrogel are generated from tumour tissue of pancreatic cancer patients and exposed to standard-of-care therapies including FOLFIRINOX (combination of5-fluorouracil, oxaliplatin, irinotecan) and gemcitabine. An automated high content screening platform measures morphologic features of the tissue, cells and nuclei. Drug responses are reported by parameters such as tumour cell death, apoptosis, growth arrest, and local invasion. In addition, genetic characterisation of the tumour will allow further identification of relevant biomarkers. Advanced bio-informatic analysis will establish the correlation between drug sensitivity of tumour cultures with treatment response in the clinic. Results and discussions We present interim results of the trial (approved by LUMC ethical review board) with 3D cultured tumour material from pancreatic cancer patients. Differentiated drug responses are identified for treatment schedules including standard-of-care therapies and novel drugs that are not (yet) considered standard of care (targeting Wnt signalling pathway, PARP inhibitors). Conclusion Functional testing is gaining interest for the development of personalised prediction of treatment outcome. Our technology enables drug sensitivity testing with standard-of-care and novel agents in 3D tumour cultures. Patient specific treatment responses can be measured per drug. Ongoing trials will establish the correlation with treatment response in the clinic. Our study results could accelerate (pre) clinical development of novel drugs. In addition, it offers opportunities to develop predictive testing for patients that are candidates for clinical trials, and to improve selection of existing drug treatment options for patients in the clinic.

Importance Patients with schizophrenia have a substantially higher suicide rate than the general public. Early intervention (EI) services improve short-term outcomes. However, little is known about the association of EI with suicide reduction in the long term. Objective To examine the association of a 2-year EI service with suicide reduction in patients with first-episode schizophrenia-spectrum (FES) disorders during 12 years and the risk factors for early and late suicide. Design, Setting, and Participants This historical control study compared 617 consecutive patients with FES who received the 2-year EI service in Hong Kong between July 1, 2001, and June 30, 2003, with 617 patients with FES who received standard care (SC) between July 1, 1998, and June 30, 2001, matched individually. Clinical information was systematically retrieved for the first 3 years of clinical care for both groups. The details of death were collected up to 12 years from presentation to the services. Data analysis was performed from October 30, 2016, to August 18, 2017. Main Outcomes and Measures Suicide rate during 12 years was the primary measure. The association of the EI service with the suicide rates during years 1 through 3 and years 4 through 12 were explored separately. Results The main analysis included 1234 patients, with 617 in each group (mean [SD] age at baseline, 21.2 [3.4] years in the EI group and 21.3 [3.4] years in the SC group; 318 male [51.5%] in the EI group and 322 [52.2%] in the SC group). The suicide rates were 7.5% in the SC group and 4.4% in the EI group (McNemar χ 2 = 5.55, P = .02). Patients in the EI group had significantly better survival (propensity score–adjusted hazard ratio, 0.57; 95% CI, 0.36-0.91; P = .02), with the maximum association observed in the first 3 years. The number of suicide attempts was an indicator of early suicide (1-3 years). Premorbid occupational impairment, number of relapses, and poor adherence during the initial 3 years were indicators of late suicide (4-12 years). Conclusions and Relevance This study suggests that the EI service may be associated with reductions in the long-term suicide rate. Suicide at different stages of schizophrenia was associated with unique risk factors, highlighting the importance of a phase-specific service.

In order to validate the results of a thorough QT/QTc clinical trial, ICH E14 recommended that a concurrent positive control treatment be included in the trial. Zhang (2008) recommended that the study results are validated if the positive control establishes assay sensitivity, i.e., has an effect on the mean QT/QTc interval of 5 ms or more. Zhang (2008) and Tsong et al. (2008) discussed the intersection-union test approach and an alternative global average test approach for testing assay sensitivity during the validation process. In this article, we further discuss the multiple comparison issues of the repeatedly measured QT difference between positive control treatment and placebo in the validation test. We describe and discuss several approaches for type I error rate adjustment that are applicable to the situation.

Moxifloxacin has been the most commonly used positive control in "thorough" QTc (TQT) studies. In a TQT study, the assay sensitivity is often considered to be established if the baseline corrected mean difference in QTc between moxifloxacin and placebo is greater than 5 ms in common practice at one or more prespecified time points and the observed moxifloxacin induced QTc effect over time follows the proper pharmacokinetics profile. To better understand the statistical characteristics of moxifloxacin-induced QTc prolongation and to provide guidance for future studies, 20 TQT studies that involved moxifloxacin have been evaluated. We study the QTc profile of the baseline adjusted mean difference in QTc between moxifloxacin and placebo over time. Zhang (2008) proposed that the moxifloxacin induced QTc effect can be evaluated between 1 and 4 h after a single dose (400 mg) administration near the time (T(max)) of peak concentration instead of all time points (typically 9-12 time points) at which QT was measured for the study drug evaluation. After evaluating 20 TQT studies, we confirm that the maximum moxifloxacin effect occurs in the time window between 1 and 4 h post dose. We also investigate the variability of the data as well as correlations between time points and between regimens. These findings and results can be used as a reference for future studies.

To generate new breed lines of bermudagrass and reduce management costs of turfgrass, gamma-ray irradiation was used for induction of dwarf mutants from a native bermudagrass (Cynodon dactylon) germplasm. Three dwarf-type mutant lines (7-9, 10-5 and 10-12) were isolated from 3000 irradiated stolons. The data from 3-year greenhouse tests and 2-year field tests indicated that the dwarf mutant lines had lower canopy height, shorter internodes and shorter leaves. The line 7-9 had a similar turf coverage rate to the wild-type control, while the lines 10-5 and 10-12 had a slower turf coverage rate than the wild-type control. Under drought stress, all three dwarf mutant lines maintained higher relative water content and lower ion leakage than the wild type in the greenhouse tests. The field tests showed that the dwarf mutant lines had lower leaf firing during a progressive drying of soil. Our results indicated that bermudagrass mutants induced by gamma radiation exhibited dwarf characteristics and improved drought resistance, which was probably associated with unbalance of plant hormones in vivo.