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Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

In theory, pluripotent stem cells can give rise to all somatic cell types found in the human body. The ability to generate renewable sources of human cells has enormous potential to improve human health and wealth. One major obstacle to the routine deployment of stem cell-derived cells is their instability in culture. To tackle this issue a synthetic polymer surface is used., (© 2015 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One major obstacle to the routine deployment of stem cell-derived cells is their instability in culture. On page 1820 David C. Hay and co-workers describe the use of a synthetic polymer surface. The image shows stem cell-derived hepatocytes replated on this polyurethane surface. Importantly the cells express Zonal Occludin (green stain) at the cell surface, which indicates that the cells display elements of polarization. The blue stain is DAPI, which demarks the nucleus., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mammalian placentation is dependent upon the action of trophoblast cells at the time of implantation. Appropriate fetal growth, regulated by maternal nutrition and nutrient transport across the placenta, is a critical factor for adult offspring long-term health. We have demonstrated that a mouse maternal low-protein diet (LPD) fed exclusively during preimplantation development (Emb-LPD) increases offspring growth but programmes adult cardiovascular and metabolic disease. In this study, we investigate the impact of maternal nutrition on post-implantation trophoblast phenotype and fetal growth. Ectoplacental cone explants were isolated at day 8 of gestation from female mice fed either normal protein diet (NPD: 18% casein), LPD (9% casein) or Emb-LPD and cultured in vitro. We observed enhanced spreading and cell division within proliferative and secondary trophoblast giant cells (TGCs) emerging from explants isolated from LPD-fed females when compared with NPD and Emb-LPD explants after 24 and 48 h. Moreover, both LPD and Emb-LPD explants showed substantial expansion of TGC area during 24-48 h, not observed in NPD. No difference in invasive capacity was observed between treatments using Matrigel transwell migration assays. At day 17 of gestation, LPD- and Emb-LPD-fed conceptuses displayed smaller placentas and larger fetuses respectively, resulting in increased fetal:placental ratios in both groups compared with NPD conceptuses. Analysis of placental and yolk sac nutrient signalling within the mammalian target of rapamycin complex 1 pathway revealed similar levels of total and phosphorylated downstream targets across groups. These data demonstrate that early post-implantation embryos modify trophoblast phenotype to regulate fetal growth under conditions of poor maternal nutrition., (© 2015 The authors.)

Purpose: Fatigue is a distressing symptom occurring in more than 60% of patients with cancer. The CNS stimulants modafinil and methylphenidate are recommended for the treatment of cancer-related fatigue, despite a limited evidence base. We aimed to evaluate the efficacy and tolerability of modafinil in the management of fatigue in patients with non-small-cell lung cancer (NSCLC)., Patients and Methods: Adults with advanced NSCLC and performance status of 0 to 2, who were not treated with chemotherapy or radiotherapy within the last 4 weeks, were randomly assigned to daily modafinil (100 mg on days 1 to 14; 200 mg on days 15 to 28) or matched placebo. The primary outcome was change in Functional Assessment of Chronic Illness Therapy (FACIT) -Fatigue score from baseline to 28 days, adjusted for baseline fatigue and performance status. Secondary outcomes included safety and patient-reported measures of depression, daytime sleepiness, and quality of life., Results: A total of 208 patients were randomly assigned, and 160 patients (modafinil, n = 75; placebo, n = 85) completed questionnaires at both baseline and day 28 and were included in the modified intention-to-treat analysis. FACIT-Fatigue scores improved from baseline to day 28 (mean score change: modafinil, 5.29; 95% CI, 2.57 to 8.02; placebo, 5.09; 95% CI, 2.54 to 7.65), but there was no difference between treatments (0.20; 95% CI, -3.56 to 3.97). There was also no difference between treatments for the secondary outcomes; 47% of the modafinil group and 23% of the placebo group stated that the intervention was not helpful., Conclusion: Modafinil had no effect on cancer-related fatigue and should not be prescribed outside a clinical trial setting. Its use was associated with a clinically significant placebo effect., (© 2014 by American Society of Clinical Oncology.)

Purpose: Isotoxic dose escalation schedules such as IDEAL-CRT [isotoxic dose escalation and acceleration in lung cancer chemoradiation therapy] (ISRCTN12155469) individualize doses prescribed to lung tumors, generating a fixed modeled risk of radiation pneumonitis. Because the beam penumbra is broadened in lung, the choice of collimator margin is an important element of the optimization of isotoxic conformal radiation therapy for lung cancer., Methods and Materials: Twelve patients with stage I-III non-small cell lung cancer (NSCLC) were replanned retrospectively using a range of collimator margins. For each plan, the prescribed dose was calculated according to the IDEAL-CRT isotoxic prescription method, and the absolute dose (D99) delivered to 99% of the planning target volume (PTV) was determined., Results: Reducing the multileaf collimator margin from the widely used 7 mm to a value of 2 mm produced gains of 2.1 to 15.6 Gy in absolute PTV D99, with a mean gain ± 1 standard error of the mean of 6.2 ± 1.1 Gy (2-sided P<.001)., Conclusions: For NSCLC patients treated with conformal radiation therapy and an isotoxic dose prescription, absolute doses in the PTV may be increased by using smaller collimator margins, reductions in relative coverage being offset by increases in prescribed dose., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose/background: Although dynamic postural control is a prerequisite to the development of fundamental movement skills in children, few studies have examined the feasibility and reliability of assessment techniques that measure dynamic postural control in youth under 13 years of age. Therefore, the purpose of this study was to determine the feasibility and reliability of the Lower Quarter Y Balance Test (YBT-LQ) in children and to examine the reproducibility of these measures across developmental periods of childhood., Methods: 188 subjects in first through fifth grades (age = 6.9 to 12.1 yr) performed the YBT-LQ on two occasions in a field-based setting. Reach distances and cumulative score (sum of 3 directions) were measured and analyzed using intraclass correlation coefficients (ICC). Sub-cohorts of 14 and 8 subjects were used to assess inter-rater reliability within-session and between-session, respectively., Results: The overall ICC was moderate-to-good for the anterior (right=0.82; left=0.82), posteromedial (right=0.77; left=0.75), and posterolateral (right 0.80; left=0.77) reach directions. The combined ICC was also moderate-to-good for children in grades 1 (0.71), 2 (0.74), 3 (0.84), 4 (0.82), and 5 (0.79). Typical error values for right and left limbs were less than 10% of the mean for all reach measures across all grades. Interrater reliability within session (ICC > 0.995) and between sessions (0.907 ≤ ICC ≤ 0.974) were both excellent. No unexpected responses or injury occurred during testing., Conclusions: These findings indicate that the YBT-LQ is a feasible and reproducible measure of dynamic postural control in children in first through fifth grades., Level of Evidence: 2b.

Little is known about treatment for pregnant and postpartum women with co-occurring substance use and depression. Funded by the National Institute of Drug Abuse, we conducted three focus groups with 18 pregnant and postpartum women in 2011 at an urban substance use treatment clinic. A semi-structured discussion guide probed for factors impacting treatment outcomes and needs. Data were analyzed using grounded theory. Women identified motivational, family, friend, romantic, and agency characteristics as facilitative or challenging to their recoveries, and desired structure (group treatment, a safe environment, and transportation) and content (attention to mental health, family, and gender-specific issues) of treatment.

Background: Indigenous alcohol and other drug (AOD) workers' roles are often exhausting, poorly paid and under-recognised. There has been relatively little examination of work-related stressors on their health and well-being. This national study identified Indigenous AOD workers' experiences and perspectives on well-being, stress and burnout along with strategies to improve worker well-being., Methods: Focus groups were conducted with 121 participants (70 Indigenous, 20 non-Indigenous, 31 unspecified) from metropolitan, rural and remote locations around Australia, selected via a purposive sampling strategy. Audio files and interview notes were analysed to identify key themes., Results: Main themes identified included excessive workloads, extensive demands and expectations, workers' proximity to communities, loss and grief issues, lack of recognition, inadequate rewards, stigma and racism, and Indigenous ways of working. Stressors were compounded by workers' complex personal circumstances, profound levels of loss and grief, and lack of culturally safe working environments., Discussion and Conclusion: Indigenous workers' stress was exacerbated by close links and responsibilities to their communities and a 'dual accountability', being constantly on call, playing multiple roles, complex personal and professional lives, and needing to interact with multiple agencies. Many Indigenous AOD workers had developed mechanisms to deal with work-related pressures and received valued support from their communities. The study identified the importance of workforce strategies to improve Indigenous workers' well-being and reduce stress, including: mutual support networks, training in assertiveness and boundary setting, workloads that take account of Indigenous ways of working, adequate remuneration, supervision and mentorship, and cultural sensitivity training for non-Indigenous workers., (© 2013 Australasian Professional Society on Alcohol and other Drugs.)

We have generated a human feeder cell line from early second trimester Placental Stromal Fibroblasts (ihPSF) stably over-expressing the polycomb protein BMI-1. These feeder cells retain the ability to maintain human Embryonic Stem cells (hESc) over long-term culture whereas hTERT or BMI-1/hTERT immortalised feeder cell lines do not. ihPSFs were able to support the derivation of a new hESc line in near xenofree (free of non-human animal components) conditions and support continued culture of newly derived hESc and human induced Pluripotent Stem (hiPS) cell lines in complete xenofree conditions necessary for clinical use., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates. At this final stage, 74% (HUES1 cells) and up to 95-97% (HUES7 and HUES8 cells) express the chondrogenic transcription factor SOX9. The cell aggregates also express cell surface CD44 and aggrecan and deposit a sulfated glycosaminoglycan and cartilage-specific collagen II matrix, but show very low or no expression of genes and proteins associated with nontarget cell types. Our protocol should facilitate studies of chondrocyte differentiation and of cell replacement therapies for cartilage repair.

We report here the derivation of two new human embryonic stem cell lines, Man-1 and Man-2, and their full characterization as novel pluripotent stem cell lines. Man-1 was derived from an embryo surplus to requirement from routine IVF, while Man-2 was obtained from an oocyte classified as failed to fertilise and subsequently chemically activated. We report the characterisation of pluripotency and the differentiation potential of these lines. Work is in progress to establish novel methods of stem cell derivation and culture, which will avoid the use of xenobiotics and be relevant to clinical production of human embryonic stem cell lines. Both newly derived human embryonic stem cell lines will be available for the research community from the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk).

The role of individual supplements necessary for the self-renewal of human embryonic stem (hES) cells is poorly characterized, and furthermore we have found that previously reported feeder cell- and serum-free culture systems used for individual hES cell lines are unable to maintain HUES7 cells for more than one passage. We have therefore derived a feeder/serum-free culture system that can support the long-term (at least 10 passages) self-renewal of several euploid hES cell lines including MAN1, HUES7, and HUES1 with minimal spontaneous differentiation and without the need for manual propagation. This system contains fibroblast growth factor 2, activin A, neurotrophin 4, and the N2, B27 supplements together with a human fibronectin substrate. We demonstrate that these components exert distinct functions: both FGF2 and activin A were necessary to prevent differentiation of hES cells while NT4 promoted cell survival, FGF2 could not be substituted by IGFII, and the fibronectin substrate supported a rapid rate of hES culture expansion. Inhibition studies showed that β1 integrin-dependent attachment of hES cells to fibronectin was at least partially via the α5 subunit but independent of integrin αV.

Bax is a member of the Bcl-2 family that, together with Bak, is required for permeabilisation of the outer mitochondrial membrane (OMM). Bax differs from Bak in that it is predominantly cytosolic in healthy cells and only associates with the OMM after an apoptotic signal. How Bax is targeted to the OMM is still a matter of debate, with both a C-terminal tail anchor and an N-terminal pre-sequence being implicated. We now show definitively that Bax does not contain an N-terminal import sequence, but does have a C-terminal anchor. The isolated N terminus of Bax cannot target a heterologous protein to the OMM, whereas the C terminus can. Furthermore, if the C terminus is blocked, Bax fails to target to mitochondria upon receipt of an apoptotic stimulus. Zebra fish Bax, which shows a high degree of amino-acid homology with mammalian Bax within the C terminus, but not in the N terminus, can rescue the defective cell-death phenotype of Bax/Bak-deficient cells. Interestingly, we find that Bax mutants, which themselves cannot target mitochondria or induce apoptosis, are recruited to clusters of activated wild-type Bax on the OMM of apoptotic cells. This appears to be an amplification of Bax activation during cell death that is independent of the normal tail anchor-mediated targeting.

Until recent years, public interest and political investment in malaria prevention, control, and research have been stagnant. The global malaria agenda is now experiencing an unprecedented time of public and political will and momentum. At the heart of this favorable period lies a nascent, but increasingly sophisticated, global advocacy effort that has contributed to new and expanded malaria funding, programs, and technology. This paper reviews the elements of malaria's rise to political and public prominence, tracks the increase in funding and policy commitments to malaria over the past decade, and comments on an evolving policymaking progress, increasing transparency and accountability in program governance, and the impact of philanthropic investments in malaria advocacy. In addition, the principles of sound advocacy are described along with the mechanisms that will underlie sustained pro-political momentum for malaria research, resources, and results. "Today, we have begun to write the final chapter in the history of malaria. We have raised hopes and expectations of our people--we must not let them down. We cannot afford to let them down." --His Excellency Olusegan Obasanjo, President of Nigeria, Abuja Summit 2000.

Permethrin is a pyrethroid insecticide used in dermally applied spot-on flea treatments for dogs. Permethrin-based spot-on preparations are contraindicated in cats because of the high risk of toxicosis. The Veterinary Poisons Information Service (VPIS) is a 24-h access telephone service that provides veterinary professionals in the United Kingdom with information on the management of poisoned animals. In a review of 286 cases reported to the VPIS regarding inappropriate feline exposure to permethrin spot-on (PSO) preparations, 96.9% were symptomatic. Increased muscular activity (as evidenced by twitching, tremor, muscle fasciculations or convulsions) was common and occurred in 87.8% of cases. The duration of increased muscle activity was long, with convulsions lasting on average 38.9 h and tremors 32 h. Recovery typically occurred within 2 to 3 days but in some cases took 5 to 7 days. Death occurred in 10.5% of cases.

Objectives: There is an increased risk of myeloid malignancy in individuals with Down's syndrome (DS), which is associated with a mutation in exon 2 of the transcription factor GATA-1. It is recognized that there is accelerated telomere shortening in blood cells of children with DS similar to that in conditions such as Fanconi anemia and dyskeratosis congenita. The latter conditions are associated with stem cell deficiency and clonal change, including acute myeloid leukemia. In this study we address the questions 1) whether the accelerated telomere shortening is associated with progenitor/stem cell deficiency in individuals with DS, predisposing to clonal change and 2) whether the occurrence of reduced numbers of stem/progenitor cells precede the incidence of mutations in exon 2 of GATA-1., Material and Methods: Peripheral blood from fetuses (23-35 weeks gestation) and/or bone marrow from children affected by DS and age-matched hematologically healthy controls were analyzed for telomere length, content of stem/progenitor cells, and mutations in exon 2 of GATA-1., Results: We found that hematopoietic stem/progenitor cell deficiency and telomere shortening occurs in individuals with DS in fetal life. Moreover, the presence of a low number of progenitor cells was not associated with mutations in exon 2 of GATA-1., Conclusions: We propose that stem cell deficiency may be a primary predisposing event to DS leukemia development.

A universal material tester has proved invaluable during the development of one company's needle-free injection system.

The anatomy of the anal canal is complex but well demonstrated by MRI. Understanding the anatomy is a prerequisite for determining the true site and the extent of pathology, especially for surgical workup. In this article, the MRI anatomy of the anal canal has been displayed using highlighted MRI images and line diagrams.

Purpose: HER-2/neu oncogene is overexpressed in 10-30% of epithelial ovarian cancers and is associated with a poor prognosis. The E1A gene product of adenovirus type 5 down-regulates HER-2/neu and causes tumor regression in animal models. In the current study, we sought to determine the toxicity and biological activity of E1A-lipid complex in ovarian cancer patients., Experimental Design: A Phase I trial involving intraperitoneal (i.p.) administration of E1A-lipid complex was initiated in ovarian cancer patients to assess biological activity (E1A gene transfer/transcription/translation and HER-2/neu expression) and to determine the maximum tolerated dose. Successive cohorts received E1A-lipid complex at doses of 1.8, 3.6, and 7.2 mg DNA/m(2), given as weekly i.p. infusions for 3 of 4 weeks (each cycle) up to a maximum of six cycles. Peritoneal fluid was sampled at baseline and twice monthly for cellularity, cytology, CA-125, and biological activity, Results: Fifteen patients, with a median age of 57 years (range, 43-81) were recruited. Three (1.8 mg DNA/m(2)), 4 (3.6 mg DNA/m(2)), and 8 patients (7.2 mg DNA/m(2)) received i.p. E1A. A total of 91 infusions (range, 1-18) was administered. Abdominal pain was the dose-limiting toxicity, and the maximum-tolerated dose was 3.6 mg DNA/m(2). E1A gene transfer and expression was observed in all of the patients and at all of the dose levels. HER-2/neu down-regulation could be demonstrated in the tumor cells of 2 patients (18%). There was no correlation between dose and biological activity., Conclusions: I.P. EIA-lipid complex gene therapy is feasible and safe. Future studies, either alone or in combination with chemotherapy, particularly in patients with minimal residual disease, should be evaluated.

Background: The topoisomerase I inhibitor exatecan mesylate (DX-8951f ) is a water-soluble hexacyclic analogue of camptothecin that does not require enzymatic activation. This study determined the toxicity, maximum tolerated dose (MTD), pharmacokinetics and pharmacodynamics of a weekly intravenous (i.v.) schedule of DX-8951f., Patients and Methods: Thirty-five patients with advanced solid malignancies, stratified as minimally (MP) or heavily (HP) pre-treated, received escalating doses of DX-8951f as 30-min i.v. infusions for three out of every 4 weeks. Pharmacokinetics were described after the first infusion of DX-8951f., Results: Infusions (244) of DX-8951f were administered with a median of two cycles (range 1-10). The main toxicity observed was haematological. There was no significant gastrointestinal toxicity. Two patients (6%) had confirmed partial responses. Twelve patients (39%) had stable disease. DX-8951f had a terminal elimination half-life of approximately 8 h and a clearance of 2 l/h/m(2). The area under the plasma concentration versus time curve (AUC( infinity )) and the maximum plasma concentration (C(max)) increased linearly with the dose. A linear relationship was present for the percentage decrease in neutrophil counts or platelet counts and AUC( infinity ) as well as C(max)., Conclusions: The dose-limiting toxicity of DX-8951f is neutropenia for MP patients and neutropenia and thrombocytopenia for HP patients. Evidence for clinical activity was seen, suggesting phase II study of the drug is indicated. Using this schedule the recommended dose is 2.75 mg/m(2)/week for MP patients and 2.10 mg/m(2)/week for HP patients.

Background/aims: This study investigated the effect of specific, commonly used diaper types on scrotal temperatures in normal healthy, young boys. These included both modern disposable and reusable diapers as well as various types of protective outer coverings that are in common use in both North America and Europe, Methods: Scrotal and skin surface temperatures were continuously monitored in healthy, young males using a computerized data-logging system based on temperature probes specifically designed for paediatric studies. These systems could be used either tethered to the PC or made completely portable depending upon the age and activity of the child being measured. Based on our results from several pilot studies, it became clear that the best way to determine if disposable and reusable diapers differ with regard to their impact on scrotal temperatures is to run these comparisons under controlled laboratory conditions where "diaper type" was the primary variable. A 2-h time period was chosen to ensure that sufficient time had elapsed for thermal equilibrium to be established under the diapers. We also felt it necessary to study the impact of urination and simulated this condition over the last 15 min using standardized methods. In addition to the skin surface temperatures, we also measured the temperature of the tympanic membrane using an infrared thermometer as an estimate of "core" temperature for each individual at various times during the session., Results and Conclusions: In this study, we have clearly shown that scrotal temperatures are the same whether the child is wearing disposable or reusable cloth diapers with a protective cover. The only situation in which scrotal temperatures were found to be lower is when the cloth diaper is used alone without a protective cover but this is not representative of how these products are actually used. We also found that on average scrotal temperatures are significantly lower than core for each diaper type. Occasionally, we did see individuals in which the maximal scrotal temperatures approached core temperatures but in every case the thermal sensors were soiled by a bowel movement. We also found that skin surface temperatures increased not only when covered by a diaper but also due to the thermal insulation provided by outer garments and blankets.

A 6-year-old boy developed respiratory distress, metabolic acidosis, severe esophageal and gastric burns, and a coagulopathy after ingestion of an unknown volume of methyl ethyl ketone peroxide (MEKP) in dimethyl phthalate. He was discharged from the pediatric intensive care unit 19 days postingestion but subsequently developed a stricture of the gastroesophageal junction and complete fibrosis of the middle third of the stomach, necessitating gastric resection and reconstruction. He was discharged 93 days postingestion on a program of dilation for the residual esophageal stricture. MEKP acts by initiating lipid peroxidation via free radical production that results in cellular dysfunction and death. Acetylcysteine, a glutathione precursor and possible free radical scavenger, may be of use in severe MEKP poisoning. This case demonstrates the severe effects that some industrial chemicals can have both systemically and locally at the point of contact with the gastrointestinal tract, as well as the long-term management required to ensure good quality of life.

Overexpression of the ERBB2 proto-oncogene in breast tumours, which occurs in 25-30% of patients, correlates with poor prognosis. In oestrogen receptor (ER) positive breast epithelial cells oestrogens reduce ERBB2 mRNA and protein levels, an effect that is reversed in the presence of anti-oestrogens such as tamoxifen and ICI 182780. Our previous studies have shown that the major effect of oestrogen on ERBB2 expression is at the level of transcription and that this is mediated through a region within the ERBB2 first intron which can act as an oestrogen-suppressible enhancer in ER positive breast cells. In vitro footprinting of the smallest DNA fragment that retained full activity revealed four transcription factor binding sites. We report here that two of these sites are recognized by AP-2 proteins and the other two are bound by a variety of bZIP factors, including CREB and ATFI, with a major complex containing ATFa/ JunD. However, by using ER mutants it is clear that repression occurs essentially off the DNA. Indeed, the essential domain of the ER responsible for repression of the ERBB2 enhancer is a region termed AF2 which is required for the ligand-dependent association of non-DNA binding cofactors. We further demonstrate that one of these ER cofactors, SRC-1, can relieve oestrogen repression of the ERBB2 enhancer and conclude that these data fit with a model whereby the ER and the ERBB2 enhancer compete for this limiting, non-DNA binding cofactor. Thus, in oestrogenic conditions SRC-1 preferentially binds to the ER which effectively sequesters it thereby reducing enhancer activity, but in antioestrogenic media the cofactor is released from the ER and is therefore available to activate the ERBB2 enhancer.