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Low-density polyethylene (LDPE) is one of the most important plastics, which is produced unfortunately under extreme conditions. In addition, it consists of robust aliphatic C─C bonds which are challenging to cleave for plastic recycling. A low-pressure and -temperature (p ethylene  = 2 bara, T = 70 °C) macromonomer-based synthesis of long chain branched polyethylene is reported. The introduction of recycle points permits the polymerization (grafting to) of the macromonomers to form the long chain branched polyethylene and its depolymerization (branch cleavage). Coordinative chain transfer polymerization employing ethylene and co-monomers is used for the synthesis of the macromonomers, permitting a high flexibility of their precise structure and efficient synthesis. The long chain branched polyethylene material matches key properties of low-density polyethylene., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Purpose: Low-risk early breast cancer rarely leads to the development of metastatic disease, and in these patients, additional imaging test is controversial. The aim of our study was to evaluate the conventional staging procedures in a bicentric German series of low-risk breast carcinoma patients., Methods: Retrospective evaluation of all patients diagnosed with early, low-risk breast cancer at Saarland University Hospital and Freiburg University Hospital in 2017 was performed. Clinical patient characteristics, the number and type of additional imaging examinations, follow-up examinations, and results were evaluated. The detection rate of metastases and the rate of false-positive findings were analyzed., Results: A total of 203 patients were included, with all patients received at least one additional imaging test. Initially, a total of 562 additional imaging examinations were performed: 166 chest X-rays, 169 upper abdominal ultrasounds, 199 bone scans, 27 computer tomographies (CT) chest and abdomen, and 1 CT abdomen. 6.8% of patients had abnormal findings reported, requiring 38 additional imaging examinations. One patient (0.5%) was found to have bone metastases. The rate of false-positive findings in the performed additional imaging procedures was 6.6%., Conclusion: Metastatic disease was detected in one of 203 patients with low-risk early breast cancer. A total of 562 examinations and additional 38 follow-up examinations were performed without detection of metastasis (this corresponds to approximately 3 examinations/patient). The rate of false-positive findings was 6.6%. The performance of additional imaging procedures for detection of distant metastases should be critically reconsidered in patients with low-risk early breast cancer., (© 2023. The Author(s).)

Numerous studies suggest that sleep benefits memory. A major theoretical question in this area is however if sleep does so by passively shielding memories from interference that arises during wakefulness or by actively stabilizing and strengthening memories. A key finding by Ellenbogen et al. Current Biology, 16, 1290-1294 (2006a) indicates that sleep can protect memories from retroactive interference, which suggests that sleep plays more than a passive role for memory consolidation. Sample size in this study was however small and subsequent reports in the literature provided mixed results. We therefore conducted an online study via Zoom to replicate Ellenbogen et al. Current Biology, 16, 1290-1294 (2006a). Subjects were asked to study paired associates. After a 12-h delay that included either nocturnal sleep or daytime wakefulness, half of all subjects were asked to study an additional list to elicit retroactive interference. All participants were then asked to complete a memory test for the studied list(s). The results were fully consistent with those reported by Ellenbogen et al. Current Biology, 16, 1290-1294 (2006a). We discuss this successful replication against the background of the mixed literature, with a focus on the possibly critical role of study-design features, like the use of high learning criteria that resulted in performance being at ceiling, or a confound between interference and the length of the retention interval. A collaborative replication effort may be needed to reach a straightfoward answer to the question if sleep protects memories from interference (and under what conditions)., (© 2023. The Author(s).)

Personalized spectacles customized according to an individual's facial anatomy were developed to provide enhanced visual performance and overall comfort when compared to standard spectacles. In this comparative crossover trial, each subject was randomly assigned to wear either personalized spectacles or standard spectacles for two weeks and then tried the second pair for another two weeks. Visual acuity and reading speed were measured, and visual quality and comfort were assessed using specific questionnaires. The correlation of the wearing parameters with the subjects' satisfaction was calculated. According to our results, the subjects wearing personalized glasses reported significantly less experience of swaying and significantly higher overall satisfaction compared to those wearing the control spectacles. At the end of the study, 62% of subjects preferred the personalized spectacles, and visual quality was the primary reason for their spectacle preference followed by wearing comfort. The difference from the ideal cornea-vertex distance was significantly lower when wearing the personalized spectacles compared to the control frames. In addition, the absolute value of the difference from the ideal cornea-vertex distance was significantly correlated with patient satisfaction. These results suggest that personalized spectacles, customized according to an individual's facial anatomy for the ideal wearing parameters, result in both visual and comfort advantages for wearers.

Background: Quercetin is an organic flavonoid present in several fruits and vegetables. The anti-inflammatory, antiviral, antioxidant, cardio-protective, anti-carcinogenic and neuroprotective properties demonstrated by this dietary supplement endorses it as a possible treatment for inflammatory diseases and cancer. Unfortunately, conflicting research has cast uncertainties on the toxicity of quercetin. The main purpose of this study was to determine if quercetin has any toxic properties in mice at doses that have shown efficacy in pre-clinical studies regarding cancer, cancer therapy, and their off-target effects., Methods: A sub-chronic toxicity study of quercetin was examined in male and female CD2F1 mice. Three different doses of quercetin (62, 125, and 250 mg/kg of diet) were infused into the AIN-76A purified diet and administered to mice ad libitum for 98 days. Body weight (BW), food consumption, water intake, body composition, blood count, behavior, and metabolic phenotype were assessed at various timepoints during the course of the experiment. Tissue and organs were evaluated for gross pathological changes and plasma was used to measure alkaline phosphatase (AP), aspartate transaminase (AST), and alanine transaminase (ALT)., Results: We found that low (62 mg/kg of diet), medium (125 mg/kg of diet), and high (250 mg/kg of diet) quercetin feeding had no discernible effect on body composition, organ function, behavior or metabolism., Conclusions: In summary, our study establishes that quercetin is safe for use in both female and male CD2F1 mice when given at ~ 12.5, 25, or 50 mg/kg of BW daily doses for 14 weeks (i.e. 98 days). Further studies will need to be conducted to determine any potential toxicity of quercetin following chronic ingestion., (© 2022. The Author(s).)

Home healthcare (HHC) nurses are experiencing stress and burnout related to high workloads, isolation on the job, and COVID-19 restrictions. A literature review found numerous interventions effective in reducing nurse burnout through building resiliency and decreasing moral distress. The purpose of this evidence-based project was to improve resiliency and decrease the risk of burnout in HHC nurses through the introduction of a resiliency bundle. The following resiliency bundle interventions were implemented: a) gratitude strategies, b) connecting with co-workers, c) storytelling, and d) resiliency training. Duffy's Quality-Caring Model and Neal's Theory of Home Health Nursing Practice framed the project. The interventions were implemented in an HHC department at a large Midwestern pediatric hospital. Outcomes were measured using a pre- and postimplementation resiliency and burnout survey, which used a Likert scale to allow for quantitative analysis. Fourteen nurses completed the presurvey, 11 completed the postsurvey, and 10 completed both pre- and postsurveys. Results showed no statistically significant change in resiliency or burnout after implementation; however, nurses expressed desire to continue using the bundle pieces, especially the gratitude strategies, connecting with co-workers, and sharing stories. Leadership should take burnout risk in HHC nurses seriously and look for innovative ways to promote resiliency., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Cancer patients can develop visceral, somatic, and neuropathic pain, largely due to the malignancy itself and its treatments. Often cancer patients and survivors turn to the use of complementary and alternative medicine (CAM) to alleviate pain and fatigue. Thus, it is necessary to investigate how CAM therapies work as novel analgesics to treat cancer pain. Ojeok-san (OJS) is an herbal formula consisting of seventeen herbs. This herbal formula has been shown to possess anti-inflammatory, immunoregulatory, and analgesic properties. In this study, we examined the potential beneficial effects and mechanism of action of OJS in a preclinical model of colitis-associated colorectal cancer. Male and female C57BL/6J mice were exposed to the carcinogen, azoxymethane (AOM, 10 mg/kg) and a chemical inflammatory driver, dextran sulfate sodium (DSS1-2%), to promote tumorigenesis in the colorectum. OJS was given orally (500, 1000, and 2000 mg/kg) to determine its influence on disease activity, tumor burden, nociception, sedation, Erk signaling, and behavioral and metabolic outcomes. In addition, in vitro studies were performed to assess CT-26 cell viability, dorsal root ganglia (DRG) activation, and bone-marrow-derived macrophage (BMDM) inflammatory response to lipopolysaccharide stimulation after OJS treatment. We found that administration of 2000 mg/kg of OJS was able to mitigate mechanical somatic and visceral nociception via Erk signaling without affecting symptom score and polyp number. Moreover, we discovered that OJS has sedative properties and elicits prolonged total sleeping time in AOM/DSS mice. Our in vitro experiments showed that OJS has the capacity to reduce TNFα gene expression in LPS-stimulated BMDM, but no changes were observed in DRG spike number and CT-26 cell proliferation. Taken together, these data suggest that OJS ameliorates nociception in mice and warrants further examination as a potential CAM therapy to promote analgesia., Competing Interests: The authors have declared that no competing interests exist.

Purpose: This study explored the impact of a brief spiritual care training program upon the perceptions and self-reported practice of rehabilitation professionals working in traumatic injury., Methodology and Methods: A qualitative study. Semi-structured interviews were held with staff from a rehabilitation hospital in Sydney, Australia, between six and eight weeks after participation in spiritual care training. A thematic analysis was conducted., Results: Of the 41 rehabilitation professionals who attended the training (1 h online, 1.5 h face to face), 16 agreed to be interviewed. The majority worked in spinal cord injury and were female. Half reported holding a Christian affiliation. One overarching theme and six sub-themes were identified from the qualitative data. The overarching theme was "spirituality is everybody's business". The six sub-themes were: (i) increased awareness of the nature of spirituality, (ii) realisation of the importance of spirituality to clients, (iii) a desire to keep spirituality on the radar, (iv) identifying barriers to providing spiritual care (v) incorporating spirituality into practice, and, (vi) recognising spirituality as personally meaningful., Conclusions: A brief spiritual care training program can impact positively upon perceptions and practice of rehabilitation professionals. Ongoing training is needed to ensure that staff retain what was learnt.IMPLICATIONS FOR REHABILITATIONBrief spiritual care training can impact positively upon rehabilitation professionals' perceptions of spirituality and lead to practice change in the delivery of spiritual care across many clinical disciplines.The stories of patients and family members are powerful staff education tools in spiritual care training.Client spirituality is an under recognised resource that staff can draw upon in supporting and enhancing the rehabilitation process.

Background Prostate specific membrane antigen (PSMA) is a promising protein for breast cancer patients. It has not only been detected in prostate cancer but is also expressed by tumor cells and the endothelial cells of tumor vessels in breast cancer patients. PSMA plays a role in tumor progression and tumor angiogenesis. For this reason, a number of diagnostic and therapeutic methods to target PSMA have been developed. Method This paper provides a general structured overview of PSMA and its oncogenic potential, with a special focus on its role in breast cancer. This narrative review is based on a selective literature search carried out in PubMed and the library of Freiburg University Clinical Center. The following key words were used for the search: "PSMA", "PSMA and breast cancer", "PSMA PET/CT", "PSMA tumor progression". Relevant articles were explicitly read through, processed, and summarized. Conclusion PSMA could be a new diagnostic and therapeutic alternative, particularly for triple-negative breast cancer. It appears to be a potential predictive and prognostic marker., Competing Interests: Conflict of Interest/Interessenkonflikt The authors declare that they have no conflict of interest./Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commecial purposes, or adapted, remixed, transformed or built upon. ( https://creativecommons.org/licenses/by-nc-nd/4.0/ ).)

MIT's Emergency-Vent Project was launched in March 2020 to develop safe guidance and a reference design for a bridge ventilator that could be rapidly produced in a distributed manner worldwide. The system uses a novel servo-based robotic gripper to automate the squeezing of a manual resuscitator bag evenly from both sides to provide ventilation according to clinically specified parameters. In just one month, the team designed and built prototype ventilators, tested them in a series of porcine trials, and collaborated with industry partners to enable mass production. We released the design, including mechanical drawings, design spreadsheets, circuit diagrams, and control code into an open source format and assisted production efforts worldwide.Clinical relevance- This work demonstrated the viability of automating the compression of a manual resuscitator bag, with pressure feedback, to provide bridge ventilation support.

The functional relevance of preexisting cross-immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a subject of intense debate. Here, we show that human endemic coronavirus (HCoV)–reactive and SARS-CoV-2–cross-reactive CD4 + T cells are ubiquitous but decrease with age. We identified a universal immunodominant coronavirus-specific spike peptide (S816-830) and demonstrate that preexisting spike- and S816-830–reactive T cells were recruited into immune responses to SARS-CoV-2 infection and their frequency correlated with anti–SARS-CoV-2-S1-IgG antibodies. Spike–cross-reactive T cells were also activated after primary BNT162b2 COVID-19 messenger RNA vaccination and displayed kinetics similar to those of secondary immune responses. Our results highlight the functional contribution of preexisting spike–cross-reactive T cells in SARS-CoV-2 infection and vaccination. Cross-reactive immunity may account for the unexpectedly rapid induction of immunity after primary SARS-CoV-2 immunization and the high rate of asymptomatic or mild COVID-19 disease courses.

Molecularly imprinted polymers (MIPs) come with the promise to be highly versatile, useful artificial receptors for sensing a wide variety of analytes. Despite a very large body of literature on imprinting, the number of papers addressing real-life biological samples and analytes is somewhat limited. Furthermore, the topic of MIP-based sensor design is still, rather, in the research stage and lacks wide-spread commercialization. This review summarizes recent advances of MIP-based sensors targeting biological species. It covers systems that are potentially interesting in medical applications/diagnostics, in detecting illicit substances, environmental analysis, and in the quality control of food. The main emphasis is placed on work that demonstrates application in real-life matrices, including those that are diluted in a reasonable manner. Hence, it does not restrict itself to the transducer type, but focusses on both materials and analytical tasks.

A one-dimensional (1D) chain compound [ Fe(en) 3 ] 3 ( FeSe 2 ) 4 Cl 2 ( en = ethylenediamine), featuring tetrahedral FeSe 2 chains separated by [Fe(en) 3 ] 2+ cations and Cl - anions, has been synthesized by a low temperature solvothermal method using simple starting materials. The degree of distortion in the Fe-Se backbone is similar to previously reported compounds with isolated 1D FeSe 2 chains. 57 Fe Mössbauer spectroscopy reveals the mixed-valent nature of [Fe(en) 3 ] 3 (FeSe 2 ) 4 Cl 2 with Fe 3+ centers in the [FeSe 2 ] - chains and Fe 2+ centers in the [Fe(en) 3 ] 2+ complexes. SQUID magnetometry indicates that [ Fe(en) 3 ] 3 ( FeSe 2 ) 4 Cl 2 is paramagnetic with a reduced average effective magnetic moment, μ eff = 9.51 μ B per formula unit, and a negative Weiss constant, θ = -10.9(4) K, indicating antiferromagnetic (AFM) nearest neighbor interactions within the [FeSe 2 ] - chains. Weak antiferromagnetic coupling between chains, combined with rather strong intra chain AFM coupling, leads to spin-glass behavior at low temperatures, as indicated by a frequency shift of the peak observed at 3 K in AC magnetic measurements. A combination of [Fe(en) 3 ] 2+ and Cl - ions is also capable of stabilizing mixed-valent 2D Fe-Se puckered layers in the crystal structure of [Fe(en) 3 ] 4 (Fe 14 Se 21 )Cl 2 , where Fe 14 Se 21 layers have a unique topology with large open pores. Property measurements of [Fe(en) 3 ] 4 (Fe 14 Se 21 )Cl 2 could not be performed due to the inability to either grow large crystals or synthesize this material in single-phase form.

Background: Researchers and practitioners continue to debate the most appropriate assessment, diagnostic, and treatment practices for ankyloglossia (tongue-tie). Health care workers struggle to provide evidence-based care in the absence of consistent standards., Research Aim: The aims of this pilot study were to qualitatively (a) evaluate the knowledge of, and attitudes toward tongue-tie and (b) describe how they shaped referral pathways and the establishment of practice patterns of frontline practitioners (pediatric dentists, speech-language pathologists, pediatric chiropractors, and International Board Certified Lactation Consultants)., Methods: We recruited clinicians ( N = 9) using nonprobability purposive sampling. Participants were interviewed using survey schedules adjusted to reflect their specialty area. Semistructured interviews were transcribed and coded using manual and inductive coding techniques common in grounded theory. Themes were iteratively developed using memoing techniques, in which observations and potential concepts were recorded using the aforementioned codes., Results: Participants were familiar with a variety of protocols and assessment tools, but did not consistently use them. No formal training about the management of tongue-tie was received through their degree programs. Instead they pursued self-guided study. Interprofessional consensus guided opinions about tongue-tie best practices, and referral pathways reflected these consensuses. International Board Certified Lactation Consultants were viewed as pivotal to the care of infants with tongue-tie while primary care physicians-primarily pediatricians-were omitted from referral pathways., Conclusion: Lack of formalized training, professional consensus about best practices, and insufficient resources for assessing and treating tongue-tie led participants to incomplete referral pathways and personal interpretations of the data through the lens of anecdotal evidence.

WNT2 acts as a pro-angiogenic factor in placental vascularization and increases angiogenesis in liver sinusoidal endothelial cells (ECs) and other ECs. Increased WNT2 expression is detectable in many carcinomas and participates in tumor progression. In human colorectal cancer (CRC), WNT2 is selectively elevated in cancer-associated fibroblasts (CAFs), leading to increased invasion and metastasis. However, if there is a role for WNT2 in colon cancer, angiogenesis was not addressed so far. We demonstrate that WNT2 enhances EC migration/invasion, while it induces canonical WNT signaling in a small subset of cells. Knockdown of WNT2 in CAFs significantly reduced angiogenesis in a physiologically relevant assay, which allows precise assessment of key angiogenic properties. In line with these results, expression of WNT2 in otherwise WNT2-devoid skin fibroblasts led to increased angiogenesis. In CRC xenografts, WNT2 overexpression resulted in enhanced vessel density and tumor volume. Moreover, WNT2 expression correlates with vessel markers in human CRC. Secretome profiling of CAFs by mass spectrometry and cytokine arrays revealed that proteins associated with pro-angiogenic functions are elevated by WNT2. These included extracellular matrix molecules, ANG-2, IL-6, G-CSF, and PGF. The latter three increased angiogenesis. Thus, stromal-derived WNT2 elevates angiogenesis in CRC by shifting the balance towards pro-angiogenic signals.

Damage to the sensory hair cells and the spiral ganglion neurons of the cochlea leads to deafness. Induced pluripotent stem cells (iPSCs) are a promising tool to regenerate the cells in the inner ear that have been affected by pathology or have been lost. To facilitate the clinical application of iPSCs, the reprogramming process should minimize the risk of introducing undesired genetic alterations while conferring the cells the capacity to differentiate into the desired cell type. Currently, reprogramming induced by synthetic mRNAs is considered to be one of the safest ways of inducing pluripotency, as the transgenes are transiently delivered into the cells without integrating into the genome. In this study, we explore the ability of integration-free human-induced pluripotent cell lines that were reprogrammed by mRNAs, to differentiate into otic progenitors and, subsequently, into hair cell and neuronal lineages. hiPSC lines were induced to differentiate by culturing them in the presence of fibroblast growth factors 3 and 10 (FGF3 and FGF10). Progenitors were identified by quantitative microscopy, based on the coexpression of otic markers PAX8, PAX2, FOXG1, and SOX2. Otic epithelial progenitors (OEPs) and otic neuroprogenitors (ONPs) were purified and allowed to differentiate further into hair cell-like cells and neurons. Lineages were characterised by immunocytochemistry and electrophysiology. Neuronal cells showed inward Na + ( I Na ) currents and outward ( I k ) and inward K + ( I K1 ) currents while hair cell-like cells had inward I K1 and outward delayed rectifier K + currents, characteristic of developing hair cells. We conclude that human-induced pluripotent cell lines that have been reprogrammed using nonintegrating mRNAs are capable to differentiate into otic cell types., Competing Interests: MNR is the founder of Rinri Therapeutics., (Copyright © 2020 Sarah L. Boddy et al.)

Background: Spirituality may play an important role in neurorehabilitation, however research findings indicate that rehabilitation professionals do not feel well equipped to deliver spiritual care., Objective: To evaluate a spiritual care training program for rehabilitation professionals., Methods: An exploratory controlled trial was conducted. Participants enrolled in a two-module spiritual care training program. Spiritual care competency was measured with the Spiritual Care Competency Scale. Confidence and comfort levels were measured using the Spiritual Care Competency Scale domains. The Spirituality and Spiritual Care Rating Scale assessed participant attitudes and knowledge. Measures were administered three times: pre-program, post-program and six weeks follow-up., Results: The training (n = 41) and control (n = 32) groups comprised rehabilitation professionals working in spinal cord or traumatic brain injury units. No between-group differences were observed on the study variables at the pre-program time point. Multilevel models found that levels of spiritual care competency, confidence, comfort, and ratings on existential spirituality increased significantly for the training group (versus control) post-program (p < 0.05) and these significant differences were maintained at follow-up., Conclusions: A brief spiritual care training program can be effective in increasing levels of self-reported competency, confidence and comfort in delivery of spiritual care for rehabilitation professionals.

Background: Spirituality has been positively associated with key adjustment indicators for individuals affected by traumatic brain injury or spinal cord injury., Objective: To explore the perceptions of health professionals working in rehabilitation in relation to spirituality and spiritual care practice., Methods: An observational study. An adapted version of the Royal College of Nursing Spirituality Survey was emailed to specialty networks of rehabilitation health professionals across Australia., Results: The majority of the 125 participants were female (92.8%), from a nursing (67.2%) background, and selected 'Christian' as their religious affiliation (68.8%). A range of spiritual needs for rehabilitation clients were identified, including a source of hope and strength. Although 84% agreed that spirituality was a fundamental aspect of healthcare, 85% agreed that staff did not receive enough education or training. Thematic analysis identified three key ways participants felt their workplaces could better address spirituality: increasing staff knowledge and skills in providing spiritual care, incorporating spirituality into rehabilitation processes, and providing patients with access to spiritual resources., Conclusions: Spirituality is considered to play an important role after traumatic injury, but most staff do not feel well equipped to provide spiritual care. Training in spiritual care for rehabilitation professionals is warranted.

Purpose: In previous studies, we have shown that SEC62 has an essential function in cell migration, epithelial-to-mesenchymal transition, and endoplasmic reticulum stress tolerance of cancer cells. SEC62 expression correlated with distant and lymph node metastasis and poor outcome in different cancer entities. In this initial study, we investigated SEC62 expression and its possible role as a prognostic and predictive biomarker in breast cancer (BC)., Methods: Formalin-fixed, paraffin-embedded tissue samples of 53 BC patients were analyzed by immunohistochemistry. The immunoreactive score (IRS) according to Remmele and Stegner was evaluated and correlated with clinico-pathological findings and overall survival (OS)., Results: We found increased SEC62 protein levels in tumor tissue compared to tumor-free tissue samples from the same patients. Tumors with high SEC62 expression (IRS > 8), or containing isolated cells with high SEC62 staining intensity, independent of the IRS, had more frequently distant metastases (48.4% vs. 18.2%; p = 0.024 and 47.4 vs. 6.7%; p = 0.005, respectively). Overall survival was significantly worse in BC patients with high SEC62 expression (SEC62 IRS > 8) (54.8% vs. 81.8%; p = 0.011) and in cases with isolated high-intensity SEC62 staining cells independently of SEC62 IRS (55.3% vs 93.3%; p = 0.024)., Conclusions: We are the first to describe the SEC62 expression and its correlation to clinicopathological parameters in mammary carcinoma. Our results suggest that SEC62 expression may serve as a prognostic marker for patients with invasive ductal breast cancer.

Residual phenol, carried over from RNA purification, can alter RNA concentration measurements and is assumed to inhibit PCR. Here, we demonstrate that Impurities A260 values of spectral content profiling (SCP) UV/Vis measurements correlated with phenol concentration, whereas absorbance ratios of classical UV/Vis systems failed to reliably detect phenol in RNA samples. Phenol contamination led to over- or underestimation of RNA concentration on UV/Vis systems, whereas it had no influence on fluorometry quantification. Wrong RNA concentration results led to altered template input in qRT-PCR and consequently caused quantification cycle (Cq) shifts, although ≤ 0.01% phenol had no direct influence., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Many cell lines derived from solid cancers can form spheroids, which recapitulate tumor cell clusters and are more representative of the in vivo situation than 2D cultures. During spheroid formation, a small proportion of a variety of different colon cancer cell lines did not integrate into the sphere and lost cell-cell adhesion properties. An enrichment protocol was developed to augment the proportion of these cells to 100% purity. The basis for the separation of spheroids from non-spheroid forming (NSF) cells is simple gravity-sedimentation. This protocol gives rise to sub-populations of colon cancer cells with stable loss of cell-cell adhesion. SW620 cells lacked E-cadherin, DLD-1 cells lost α-catenin and HCT116 cells lacked P-cadherin in the NSF state. Knockdown of these molecules in the corresponding spheroid-forming cells demonstrated that loss of the respective proteins were indeed responsible for the NSF phenotypes. Loss of the spheroid forming phenotype was associated with increased migration and invasion properties in all cell lines tested. Hence, we identified critical molecules involved in spheroid formation in different cancer cell lines. We present here a simple, powerful and broadly applicable method to generate new sublines of tumor cell lines to study loss of cell-cell adhesion in cancer progression.

Objective: To identify the extent of research which has investigated spirituality or closely related meaning-making constructs after traumatic brain injury (TBI)., Design: A scoping review was employed to capture the broadest possible range of studies., Methods: Search terms 'spirituality', 'religion', 'beliefs', 'faith', 'hope', 'meaning', 'purpose in life', 'sense of coherence' and 'posttraumatic growth' were combined with search terms related to TBI. Findings were restricted to empirical studies published in English, in peer-reviewed journals and conducted over a 20-year period between 1997 and 2016., Results: Nine studies were identified, conducted in the USA, Canada and the UK. These included eight quantitative studies and one qualitative study. Definitions and measurement of spirituality varied widely among the studies. Findings revealed that spirituality was closely related to a number of positive outcomes following TBI including psychological coping, physical health, mental health, productivity, life satisfaction, functional independence and posttraumatic growth., Conclusions: The limited research conducted into spirituality following TBI suggests it can play an important role in the recovery process. Further research is necessary to identify the particular spiritual needs of this population, and how clinical staff may be supported to address such needs.

The present study was undertaken to investigate the expression of prostate-specific membrane antigen (PSMA) in normal breast tissues, in cancerous breast tissues and in distant metastases from patients with breast cancer. Immunohistochemical analysis was performed to determine PSMA expression and angiogenic activity using anti-PSMA mAb and anti-CD31 mAb respectively. Immunofluorescence staining was applied to confirm the exact co-localization of PSMA and CD31. We observed different patterns of PSMA expression between normal and cancerous tissues. Normal breast tissues showed PSMA expression only in normal glandular cells. However, primary breast tumors and distant metastases showed PSMA expression in tumor cells and in tumor-associated neovasculature. PSMA score group status in primary breast tumors was significantly associated with histologic type and tumor grade (p = 0.026 and p = 0.004 respectively). Distant metastases showed higher PSMA expression in tumor-associated neovasculature comparing with primary tumors. Moreover, brain tumor-associated neovasculture had significantly higher expression of PSMA comparing with bone tumor-associated neovasculture. The localized binding of PSMA mAb to the neovasculature endothelium was confirmed with the double Immunofluorescence staining. 68 Ga-PSAM imaging of a patient with metastatic breast cancer showed strong tracer uptake in all known skeletal metastases. To the best of our knowledge, this study is the second one that has assessed PSMA expression in a large number of breast cancer patients. Our findings showed that PSMA is particularly expressed in tumor-associated neovasculature of breast tumors and its distant metastases, thus enhancing the evidence on the potential usefulness of PSMA as a therapeutic vascular target.

Defects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The canonical WNT signaling pathway is crucial for intestinal stem cell renewal and aberrant WNT signaling is an early event in colorectal cancer (CRC) development. Here, we show for the first time that WNT2 is one of the most significantly induced genes in CRC stroma as compared to normal stroma. The impact of stromal WNT2 on carcinoma formation or progression was not addressed so far. Canonical WNT/β-catenin signaling was assessed using a 7TGP-reporter construct. Furthermore, effects of WNT2 on fibroblast migration and invasion were determined using siRNA-mediated gene silencing. Tumor cell invasion was studied using organotypic raft cultures and in vivo significance was assessed via a xenograft mouse model. We identified cancer-associated fibroblasts (CAFs) as the main source of WNT2. CAF-derived WNT2 activated canonical signaling in adenomatous polyposis coli/β-catenin wild-type colon cancer cells in a paracrine fashion, whereas no hyperactivation was detectable in cell lines harboring mutations in the adenomatous polyposis coli/β-catenin pathway. Furthermore, WNT2 activated autocrine canonical WNT signaling in primary fibroblasts, which was associated with a pro-migratory and pro-invasive phenotype. We identified FZD8 as the putative WNT2 receptor in CAFs. Three-dimensional organotypic co-culture assays revealed that WNT2-mediated fibroblast motility and extracellular matrix remodeling enhanced cancer cell invasion of cell lines even harboring mutations in the adenomatous polyposis coli/β-catenin pathway. Thus, suggesting a tumor-promoting influence on a broad range of CRC. In line, WNT2 also promotes tumor growth, invasion and metastasis in vivo. Moreover, high WNT2 expression is associated with poor prognosis in human CRC. The identification of the pro-malignant function of stromal derived WNT2 in CRC classifies WNT2 and its receptor as promising stromal targets to confine cancer progression in combination with conventional or targeted therapies.

The insulin-like growth factor (IGF)2/IGF1 receptor (IGF1R) signaling axis has an important role in intestinal carcinogenesis and overexpression of IGF2 is an accepted risk factor for colorectal cancer (CRC) development. Genetic amplifications and loss of imprinting contribute to the upregulation of IGF2, but insufficiently explain the extent of IGF2 expression in a subset of patients. Here, we show that IGF2 was specifically induced in the tumor stroma of CRC and identified cancer-associated fibroblasts (CAFs) as the major source. Further, we provide functional evidence that stromal IGF2, via the paracrine IGF1R/insulin receptor axis, activated pro-survival AKT signaling in CRC cell lines. In addition to its effects on malignant cells, autocrine IGF2/IGF1R signaling in CAFs induced myofibroblast differentiation in terms of alpha-smooth muscle actin expression and contractility in floating collagen gels. This was further augmented in concert with transforming growth factor-β (TGFβ) signaling suggesting a cooperative mechanism. However, we demonstrated that IGF2 neither induced TGFβ/smooth muscle actin/mothers against decapentaplegic (SMAD) signaling nor synergized with TGFβ to hyperactivate this pathway in two dimensional and three dimensional cultures. IGF2-mediated physical matrix remodeling by CAFs, but not changes in extracellular matrix-modifying proteases or other secreted factors acting in a paracrine manner on/in cancer cells, facilitated subsequent tumor cell invasion in organotypic co-cultures. Consistently, colon cancer cells co-inoculated with CAFs expressing endogenous IGF2 in mouse xenograft models exhibited elevated invasiveness and dissemination capacity, as well as increased local tumor regrowth after primary tumor resection compared with conditions with IGF2-deficient CAFs. In line, expression of IGF2 correlated with elevated relapse rates and poor survival in CRC patients. In agreement with our results, high-level coexpression of IGF2 and TGFβ was predicting adverse outcome with higher accuracy than increased expression of the individual genes alone. Taken together, we demonstrate that stroma-induced IGF2 promotes colon cancer progression in a paracrine and autocrine manner and propose IGF2 as potential target for tumor stroma cotargeting strategies.

Background: Like many other cancer patients, most pancreatic carcinoma patients suffer from severe weight loss. As shown in numerous studies with fish oil (FO) supplementation, a minimum daily intake of 1.5 g n-3-fatty acids (n-3-FA) contributes to weight stabilization and improvement of quality of life (QoL) of cancer patients. Given n-3-FA not as triglycerides (FO), but mainly bound to marine phospholipids (MPL), weight stabilization and improvement of QoL has already been seen at much lower doses of n-3-FA (0,3 g), and MPL were much better tolerated. The objective of this double-blind randomized controlled trial was to compare low dose MPL and FO formulations, which had the same n-3-FA amount and composition, on weight and appetite stabilization, global health enhancement (QoL), and plasma FA-profiles in patients suffering from pancreatic cancer., Methods: Sixty pancreatic cancer patients were included into the study and randomized to take either FO- or MPL supplementation. Patients were treated with 0.3 g of n-3-fatty acids per day over six weeks. Since the n-3-FA content of FO is usually higher than that of MPL, FO was diluted with 40% of medium chain triglycerides (MCT) to achieve the same capsule size in both intervention groups and therefore assure blinding. Routine blood parameters, lipid profiles, body weight, and appetite were measured before and after intervention. Patient compliance was assessed through a patient diary. Quality of life and nutritional habits were assessed with validated questionnaires (EORTC-QLQ-C30, PAN26). Thirty one patients finalized the study protocol and were analyzed (per-protocol-analysis)., Results: Intervention with low dose n-3-FAs, either as FO or MPL supplementation, resulted in similar and promising weight and appetite stabilization in pancreatic cancer patients. MPL capsules were slightly better tolerated and showed fewer side effects, when compared to FO supplementation., Conclusion: The similar effects between both interventions were unexpected but reliable, since the MPL and FO formulations caused identical increases of n-3-FAs in plasma lipids of included patients after supplementation. The effects of FO with very low n-3-FA content might be explained by the addition of MCT. The results of this study suggest the need for further investigations of marine phospholipids for the improvement of QoL of cancer patients, optionally in combination with MCT.

Food supplementation with the conditionally essential amino acid arginine (Arg) has been shown to have nutritional benefits. Degradation of cyanophycin (CGP), a peptide polymer used for nitrogen storage by cyanobacteria, requires cyanophycinase (CGPase) and results in the release of β-aspartic acid (Asp)-Arg dipeptides. The simultaneous production of CGP and CGPase in plants could be a convenient source of Arg dipeptides. Different variants of the cphB coding region from Thermosynechococcus elongatus BP-1 were transiently expressed in Nicotiana benthamiana plants. Translation and enzyme stability were optimized to produce high amounts of active CGPase. Protein stability was increased by the translational fusion of CGPase to the green fluorescent protein (GFP) or to the transit peptide of the small subunit of RuBisCO for peptide production in the chloroplasts. Studies in mice showed that plant-expressed CGP fed in combination with plant-made CGPase was hydrolysed in the intestine, and high levels of ß-Asp-Arg dipeptides were found in plasma, demonstrating dipeptide absorption. However, the lack of an increase in Asp and Arg or its metabolite ornithine in plasma suggests that Arg from CGP was not bioavailable in this mouse group. Intestinal degradation of CGP by CGPase led to low intestinal CGP content 4 h after consumption, but after ingestion of CGP alone, high CGP concentrations remained in the large intestine; this indicated that intact CGP was transported from the small to the large intestine and that CGP was resistant to colonic microbes., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Three-dimensional (3D) cancer models are used as preclinical systems to mimic physiologic drug responses. We provide evidence for strong changes of proliferation and metabolic capacity in three dimensions by systematically analyzing spheroids of colon cancer cell lines. Spheroids showed relative lower activities in the AKT, mammalian target of rapamycin (mTOR) and S6K (also known as RPS6KB1) signaling pathway compared to cells cultured in two dimensions. We identified spatial alterations in signaling, as the level of phosphorylated RPS6 decreased from the spheroid surface towards the center, which closely coordinated with the tumor areas around vessels in vivo These 3D models displayed augmented anti-tumor responses to AKT-mTOR-S6K or mitogen-activated protein kinase (MAPK) pathway inhibition compared to those in 2D models. Inhibition of AKT-mTOR-S6K resulted in elevated ERK phosphorylation in 2D culture, whereas under these conditions, ERK signaling was reduced in spheroids. Inhibition of MEK1 (also known as MAP2K1) led to decreased AKT-mTOR-S6K signaling in 3D but not in 2D culture. These data indicate a distinct rewiring of signaling in 3D culture and during treatment. Detached tumor-cell clusters in vessels, in addition to circulating single tumor cells, play a putative role in metastasis in human cancers. Hence, the understanding of signaling in spheroids and the responses in the 3D models upon drug treatment might be beneficial for anti-cancer therapies., (© 2017. Published by The Company of Biologists Ltd.)

BACKGROUND Ciprofloxacin is a commonly used fluoroquinolone antibiotic. It is occasionally associated with benign elevations in liver enzymes. Few reports in the literature correlate ciprofloxacin with significant liver injury. We present a fatal case of ciprofloxacin-induced liver failure. CASE REPORT  A 74-year-old female was successfully treated with ciprofloxacin for a urinary tract infection (UTI), but immediately began having new-onset symptoms, including fatigue and nausea. This continued for two months, at which time she presented to the hospital; she was found to have elevated liver enzymes and another UTI. She was treated with ciprofloxacin again for UTI and discharged three days later, following mild improvement. One week later, she returned to another hospital and was found to have more significantly elevated liver function tests and jaundice. Extensive viral and autoimmune panels were unremarkable. Liver biopsy showed cholestatic hepatitis of unclear etiology. The patient was discharged again following a mild decline in liver enzymes. Soon after, the patient was admitted to our institution with similar complaints. Serum transaminases remained elevated, with an increase in alkaline phosphatase and bilirubin. The Council for International Organizations of Medical Sciences/the Roussel Uclaf Causality Assessment Method (CIOMS/RUCAM) scale was found to be 8, outlining a high or definite probability that the ciprofloxacin was the cause of the patient's hepatotoxicity. A one-week course of prednisone for possible hypersensitivity reaction was tried; however, it proved unsuccessful. Palliative care was consulted, and the patient passed away shortly thereafter. CONCLUSIONS This case demonstrates the potential for liver failure from ciprofloxacin. Clinicians should evaluate the possibility of ciprofloxacin-induced hepatotoxicity in a patient presenting with liver injury of unknown etiology. Similarly, it is important to consider this significant effect when a practitioner considers antibiotic choice.

Ustilago tritici causes loose smut, which is a seed-borne fungal disease of wheat, and responsible for yield losses up to 40%. Loose smut is a threat to seed production in developing countries where small scale farmers use their own harvest as seed material. The killer protein 4 (KP4) is a virally encoded toxin from Ustilago maydis and inhibits growth of susceptible races of fungi from the Ustilaginales. Enhanced resistance in KP4 wheat to stinking smut, which is caused by Tilletia caries, had been reported earlier. We show that KP4 in genetically engineered wheat increased resistance to loose smut up to 60% compared to the non-KP4 control under greenhouse conditions. This enhanced resistance is dose and race dependent. The overexpression of the transgene kp4 and its effect on fungal growth have indirect effects on the expression of endogenous pathogen defense genes.

Objective: The purpose of this study was to compare professional rating and patient satisfaction of nasal appearance after secondary cleft rhinoplasty., Method: We used a cross-sectional study design and enrolled German adults with nonsyndromic unilateral or bilateral cleft lip and/or palate (UCLP and BCLP, respectively) undergoing secondary cleft rhinoplasty from January 2001 to December 2013. The predictor variable was professional rating. The outcome variable was patient satisfaction with postoperative nasal aesthetics. Other study variables included patient age and gender, type of surgery, and patient rating of nasal function. Appropriate descriptive and univariate statistics were computed, and a P value of <0.05 was regarded as statistically significant. Inter-rater reliability was assessed by the use of Cohen's kappa coefficient., Results: The study sample consisted of 242 adult cleft patients of mean age of 22.1 ± 9.2 years (range 14-64), including 97 females (40 %) and 176 unilateral clefts (73 %). Most of the patients reported good function (82 %) and good aesthetics (74 %). The main professional rating was good aesthetics (65 %). Analysis of inter-observer reliability revealed significant differences between patient satisfaction and professional assessment (κ = 0.385; P < 0.0001)., Conclusions: Although most of the patients were satisfied with the functional and aesthetic results of secondary cleft rhinoplasty, patient self-assessment of nasal appearance differed from professional assessment.

Synthetic mRNA transfection enables efficient and controlled gene expression in human cells, without genome integrations. Further, modifications to the mRNA and transfection protocol now allow for repeated transfection and long-term gene expression of an otherwise short-lived mRNA expression. This is mainly achieved through introducing modified nucleosides and interferon suppression. In this study we provide an overview and details of the advanced synthesis and modifications of mRNA originally developed for reprogramming. This mRNA allows for very efficient transfection of fibroblasts enabling the generation of high quality human iPS cells with a six-factor mRNA cocktail in 9 days. Furthermore, we synthesised and transfected modified MYOD1 mRNA to transdifferentiate human fibroblasts into myoblast-like cells without a transgene footprint. This efficient and integration-free mRNA technology opens the door for safe and controlled gene expression to reverse or redirect cell fate., (Copyright © 2015 Elsevier Inc. All rights reserved.)

After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts., (Copyright © 2016 John Wiley & Sons, Inc.)

Background: Human induced pluripotent stem (hiPS) cells have the ability to undergo self-renewal and differentiation similarly to human embryonic stem (hES) cells. We have recently shown that hES cells under replication stress fail to activate checkpoint kinase 1 (CHK1). They instead commit to apoptosis, which appears to be a primary defense mechanism against genomic instability. It is not known whether the failure of CHK1 activation and activation of apoptosis under replication stress is solely a feature of hES cells, or if it is a feature that can be extended to hiPS cells., Methods: Here we generated integration-free hiPS cell lines by mRNA transfection, and characterised the cell lines. To investigate the mechanism of S phase checkpoint activation, we have induced replication stress by adding excess thymidine to the cell culture medium, and performed DNA content analysis, apoptosis assays and immunoblottings., Results: We are showing that hiPS cells similarly to hES cells, fail to activate CHK1 when exposed to DNA replication inhibitors and commit to apoptosis instead. Our findings also suggest the Ataxia Telangiectasia Mutated pathway might be responding to DNA replication stress, resulting in apoptosis., Conclusion: Together, these data suggest that the apoptotic response was properly restored during reprogramming with mRNA, and that apoptosis is an important mechanism shared by hiPS and hES cells to maintain their genomic integrity when a replication stress occurs.

Solid cancers are not simple accumulations of malignant tumor cells but rather represent complex organ-like structures. Despite a more chaotic general appearance as compared to the highly organized setup of healthy tissues, cancers still show highly differentiated structures and a close interaction with and dependency on the interwoven connective tissue. This complexity within cancers is not known in detail at the molecular level so far. The first part of this article will shortly describe the technology and strategies to quantify and dissect the heterogeneity in human solid cancers. Moreover, there is urgent need to better understand human cancer biology since the development of novel anti-cancer drugs is far from being efficient, predominantly due to the scarcity of predictive preclinical models. Hence, in vivo and in vitro models were developed, which better recapitulate the complexity of human cancers, by their intrinsic three-dimensional nature and the cellular heterogeneity and allow functional intervention for hypothesis testing. Therefore, in the second part 3D in vitro cancer models are presented that analyze and depict the heterogeneity in human cancers. Advantages and drawbacks of each model are highlighted and their suitability to preclinical drug testing is discussed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The analytical and clinical validity of analyses of RNA samples destined for clinical diagnosis and therapeutic management is directly impacted by RNA quality. RNA is affected by heat, enzymatic degradation, and Ultraviolet (UV) light. RNA from three eukaryotic cell lines was degraded by heat, RNase, or UV light. RNA integrity values obtained with the benchmark Agilent Bioanalyzer 2100 system were compared with those from the more recent QIAxcel Advanced system. The application of this novel method has allowed us to unravel differences between RNA biophysical and biochemical degradation modes. Agilent RNA integrity number (RIN) and QIAxcel RIS were comparable in heat-degraded and RNase III-degraded RNA. Agilent RIN and QIAxcel RIS were comparable at a RIN decision level of 7 in UV-degraded RNA but not overall. The QIAxcel RIS method was more precise than Agilent RIN for RIN<8, while the inverse was true for RIN≥8. Greater degradation of mRNA and rRNA in UV-damaged samples hampered the Agilent RIN calculation algorithm. Overall, RIS was more robust than RIN for assessing RNA integrity. The ΔΔCt-values for heat- and UV-degraded RNA samples showed slightly higher correlation with RIS than with RIN. RNA integrity can be used to categorize RNA samples for suitability for downstream gene expression analyses, independently of the RNA degradation mechanism. The new method QIAxcel is more robust and therefore allows more accurate categorization of compromised RNA samples., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The t(2;5)(p23;q35) chromosomal translocation results in the expression of the fusion protein NPM/ALK that when expressed in T-lymphocytes gives rise to anaplastic large cell lymphomas (ALCL). In search of new therapy options the dichloromethane extract of the ethnomedicinal plant Neurolaena lobata (L.) R.Br. ex Cass was shown to inhibit NPM/ALK expression., Purpose: Therefore, we analysed whether the active principles that were recently isolated and found to inhibit inflammatory responses specifically inhibit growth of NPM/ALK+ ALCL, leukaemia and breast cancer cells, but not of normal cells, and the intravasation through the lymphendothelial barrier., Methods: ALCL, leukaemia and breast cancer cells, and normal peripheral blood mononuclear cells (PBMCs) were treated with isolated sesquiterpene lactones and analysed for cell cycle progression, proliferation, mitochondrial activity, apoptosis, protein and mRNA expression, NF-κB and cytochrome P450 activity, 12(S)-HETE production and lymphendothelial intravasation., Results: In vitro treatment of ALCL by neurolenin B suppressed NPM/ALK, JunB and PDGF-Rβ expression, inhibited the growth of ALCL cells late in M phase, and induced apoptosis via caspase 3 without compromising mitochondrial activity (as a measure of general exogenic toxicity). Moreover, neurolenin B attenuated tumour spheroid intravasation probably through inhibition of NF-κB and CYP1A1., Conclusion: Neurolenin B specifically decreased pro-carcinogenic NPM/ALK expression in ALK+ ALCL cells and, via the inhibition of NF-kB signalling, attenuated tumour intra/extravasation into the lymphatics. Hence, neurolenin B may open new options to treat ALCL and to manage early metastatic processes to which no other therapies exist., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Background: Hypoxic and necrotic regions that accrue within solid tumors in vivo are known to be associated with metastasis formation, radio- and chemotherapy resistance, and drug metabolism. Therefore, integration of these tumor characteristics into in vitro drug screening models is advantageous for any reliable investigation of the anticancer activity of novel drug candidates. In general, usage of cell culture models with in vivo like characteristics has become essential in preclinical drug studies and allows evaluation of complex problems such as tumor selectivity and anti-invasive properties of the drug candidates., Materials and Methods: In this study, we investigated the anticancer activity of clinically approved, investigational and experimental drugs based on platinum (cisplatin, oxaliplatin and KP1537), gallium (KP46), ruthenium (KP1339) and lanthanum (KP772) in different cell culture models such as monolayers, multicellular spheroids, as well as invasion and metastasis models. Results Application of the Alamar Blue assay to multicellular spheroids and a spheroid-based invasion assay resulted in an altered rating of compounds with regard to their cytotoxicity and ability to inhibit invasion when compared with monolayer-based cytotoxicity and transwell assays. For example, the gallium-based drug candidate KP46 showed in spheroid cultures significantly enhanced properties to inhibit protrusion formation and fibroblast mediated invasiveness, and improved cancer cell selectivity., Conclusion: Taken together, our results demonstrate the advantages of spheroid-based assays and underline the necessity of using different experimental models for reliable preclinical investigations assessing and better predicting the anticancer potential of new compounds.

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.

Whole-organism compound sensitivity assays are a valuable strategy in infectious diseases to identify active molecules. In schistosomiasis drug discovery, larval-stage Schistosoma allows the use of a certain degree of automation in the screening of compounds. Unfortunately, the throughput is limited, as drug activity is determined by manual assessment of Schistosoma viability by microscopy. To develop a simple and quantifiable surrogate marker for viability, we targeted glucose metabolism, which is central to Schistosoma survival. Lactate is the end product of glycolysis in human Schistosoma stages and can be detected in the supernatant. We assessed lactate as a surrogate marker for viability in Schistosoma drug screening assays. We thoroughly investigated parameters of lactate measurement and performed drug sensitivity assays by applying schistosomula and adult worms to establish a proof of concept. Lactate levels clearly reflected the viability of schistosomula and correlated with schistosomulum numbers. Compounds with reported potencies were tested, and activities were determined by lactate assay and by microscopy. We conclude that lactate is a sensitive and simple surrogate marker to be measured to determine Schistosoma viability in compound screening assays. Low numbers of schistosomula and the commercial availability of lactate assay reagents make the assay particularly attractive to throughput approaches. Furthermore, standardization of procedures and quantitative evaluation of compound activities facilitate interassay comparisons of potencies and, thus, concerted drug discovery approaches., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Cell reprogramming promises to make characterization of the impact of human genetic variation on health and disease experimentally tractable by enabling the bridging of genotypes to phenotypes in developmentally relevant human cell lineages. Here we apply this paradigm to two disorders caused by symmetrical copy number variations of 7q11.23, which display a striking combination of shared and symmetrically opposite phenotypes--Williams-Beuren syndrome and 7q-microduplication syndrome. Through analysis of transgene-free patient-derived induced pluripotent stem cells and their differentiated derivatives, we find that 7q11.23 dosage imbalance disrupts transcriptional circuits in disease-relevant pathways beginning in the pluripotent state. These alterations are then selectively amplified upon differentiation of the pluripotent cells into disease-relevant lineages. A considerable proportion of this transcriptional dysregulation is specifically caused by dosage imbalances in GTF2I, which encodes a key transcription factor at 7q11.23 that is associated with the LSD1 repressive chromatin complex and silences its dosage-sensitive targets.

An apolar extract of the traditional medicinal plant Neurolaena lobata inhibited the expression of the NPM/ALK chimera, which is causal for the majority of anaplastic large cell lymphomas (ALCLs). Therefore, an active principle of the extract, the furanoheliangolide sesquiterpene lactone lobatin B, was isolated and tested regarding the inhibition of ALCL expansion and tumour cell intravasation through the lymphendothelium. ALCL cell lines, HL-60 cells and PBMCs were treated with plant compounds and the ALK inhibitor TAE-684 to measure mitochondrial activity, proliferation and cell cycle progression and to correlate the results with protein- and mRNA-expression of selected gene products. Several endpoints indicative for cell death were analysed after lobatin B treatment. Tumour cell intravasation through lymphendothelial monolayers was measured and potential causal mechanisms were investigated analysing NF-κB- and cytochrome P450 activity, and 12(S)-HETE production. Lobatin B inhibited the expression of NPM/ALK, JunB and PDGF-Rβ, and attenuated proliferation of ALCL cells by arresting them in late M phase. Mitochondrial activity remained largely unaffected upon lobatin B treatment. Nevertheless, caspase 3 became activated in ALCL cells. Also HL-60 cell proliferation was attenuated whereas PBMCs of healthy donors were not affected by lobatin B. Additionally, tumour cell intravasation, which partly depends on NF-κB, was significantly suppressed by lobatin B most likely due to its NF-κB-inhibitory property. Lobatin B, which was isolated from a plant used in ethnomedicine, targets malignant cells by at least two properties: I) inhibition of NPM/ALK, thereby providing high specificity in combating this most prevalent fusion protein occurring in ALCL; II) inhibition of NF-κB, thereby not affecting normal cells with low constitutive NF-κB activity. This property also inhibits tumour cell intravasation into the lymphatic system and may provide an option to manage this early step of metastatic progression., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Anti-cancer drug development is inefficient, mostly due to lack of efficacy in human patients. The high fail rate is partly due to the lack of predictive models or the inadequate use of existing preclinical test systems. However, progress has been made and preclinical models were improved or newly developed, which all account for basic features of solid cancers, three-dimensionality and heterotypic cell interaction. Here we give an overview of available in vivo and in vitro models of cancer, which meet the criteria of being 3D and mirroring human tumor-stroma interactions. We only focus on drug response models without touching models for pharmacokinetic and dynamic, toxicity or delivery aspects., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Purpose: Neurolaena lobata is a Caribbean medicinal plant used for the treatment of several conditions including inflammation. Recent data regarding potent anti-inflammatory activity of the plant and isolated sesquiterpene lactones raised our interest in further pharmacological studies. The present work aimed at providing a mechanistic insight into the anti-inflammatory activity of N. lobata and eight isolated sesquiterpene lactones, as well as a structure-activity relationship and in vivo anti-inflammatory data., Methods: The effect of the extract and its compounds on the generation of pro-inflammatory proteins was assessed in vitro in endothelial and monocytic cells by enzyme-linked immunosorbent assay. Their potential to modulate the expression of inflammatory genes was further studied at the mRNA level. In vivo anti-inflammatory activity of the chemically characterized extract was evaluated using carrageenan-induced paw edema model in rats., Results: The compounds and extract inhibited LPS- and TNF-α-induced upregulation of the pro-inflammatory molecules E-selectin and interleukin-8 in HUVECtert and THP-1 cells. LPS-induced elevation of mRNA encoding for E-selectin and interleukin-8 was also suppressed. Furthermore, the extract inhibited the development of acute inflammation in rats., Conclusions: Sesquiterpene lactones from N. lobata interfered with the induction of inflammatory cell adhesion molecules and chemokines in cells stimulated with bacterial products and cytokines. Structure-activity analysis revealed the importance of the double bond at C-4-C-5 and C-2-C-3 and the acetyl group at C-9 for the anti-inflammatory activity. The effect was confirmed in vivo, which raises further interest in the therapeutic potential of the compounds for the treatment of inflammatory diseases., (Copyright © 2014 Elsevier GmbH. All rights reserved.)