The function of the proteasome is essential for maintenance of cellular homeostasis, and in pluripotent stem cells this has been extended to the removal of nascent proteins in a manner that restricts differentiation. In this study we show enhanced expression of genes encoding subunits of the 20s proteasome in human embryonic stem cells (hESCs) coupled to their downregulation as the cells progress into differentiation. The decrease in expression is particularly marked for the alternative catalytic subunits of the 20s proteasome variant known as the immunoproteasome indicating the possibility of a hitherto unknown function for this proteasome variant in pluripotent cells. The immunoproteasome is normally associated with antigen presenting cells where it provides peptides of an appropriate length for antibody generation: however our data suggests that it may be involved in maintaining the pluripotency in human embryonic stem cells (hESC). Selective inhibition of two immunoproteasome subunits (PSMB9 and PSMB8) results in downregulation of cell surface and transcriptional markers that characterise the pluripotent state, subtle cell accumulation in G1 at the expense of S phase and upregulation of various markers characterising the differentiated primitive and definitive lineages arising from hESC. Our data also support a different function for each of these two subunits in hESC that may be linked to their selectivity in driving proteasome mediated degradation of cell cycle regulatory components and/or differentiation inducing factors.

Objectives In the United States, access to hormonal contraception commonly requires medical procedures, including Pap smears, breast and pelvic examinations. Over the past 15 years, these procedures, which may constitute barriers to effective contraception, have been declared unnecessary by several organisations. This study aims to evaluate the usefulness of the hormonal contraception with optional pelvic exam (HOPE) programme offered in Southeastern Virginia. Methods A cross-sectional survey was conducted among women participating in the HOPE programme over a four-month period. Questionnaire items included demographics, reproductive history and perceptions concerning physical examinations and access to contraception. Results The HOPE programme was perceived as enhancing unintended pregnancy prevention by 73% of respondents. Most of them valued the opportunity to obtain contraception without Pap smears, breast or pelvic examinations. Conclusions The HOPE programme, which provides access to contraception without the requirement for invasive examinations, increases and maintains effective levels of pregnancy prevention. HOPE patients were mostly satisfied with the programme. Lower expense and prompt appointment appear to weigh more than the optional pelvic examination programme in the choice of the modality of contraceptive guidance.

The therapeutic potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is vast, allowing disease modelling, drug discovery and testing and perhaps most importantly regenerative therapies. However, problems abound; techniques for cultivating self-renewing hESCs tend to give a heterogeneous population of self-renewing and partially differentiated cells and general include animal-derived products which can be cost-prohibitive for large scale production and effective lineage specific differentiation protocols also still remain relatively undefined and are inefficient in producing large amounts of cells for therapeutic use. Further, the mechanisms and signalling pathways which mediate pluripotency and differentiation are still to be fully appreciated. However, over the recent years, the development/discovery of a range of effective small molecule inhibitors/activators has had a huge impact in hESC biology. Large scale screening techniques, coupled with greater knowledge of the pathways involved, have generated pharmacological agents which can boost hESC pluripotency/self-renewal and survival and has allowed great increases in the efficiency of various differentiation protocols, while also aiding the delineation of several important signalling pathways. Within this review, we hope to describe the current uses of small molecule inhibitors/activators in hESC biology and their potential uses in the future. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

BACKGROUND: Combination therapy with interferon alpha (IFN) is correlated with improved survival in patients with pancreatic ductal adenocarcinoma (PDAc) but frequently presents side effects. We designed a novel targeted adenovirus with replication restricted to cyclooxygenase 2 (Cox2)-overexpressing PDAcs and hypothesize that the locally delivered therapeutic gene IFN can augment oncolytic effects while minimizing systemic toxicity. METHODS: IFN-expressing vectors were tested in vitro with the use of 4 PDAc cell lines with cytocidal effect measured by crystal violet and colorimetrically and IFN production assayed by ELISA. Cox2 promoter activity was checked by a luciferase reporter assay. In vivo, subcutaneous tumor xenografts with 2 PDAc cell lines in nude mice were treated with a single intratumoral viral dose. RESULTS: All PDAc cell lines were Cox2-positive. Oncolysis from the novel Cox2-controlled virus was comparable or superior to Adwt, the wild-type virus without safety features. The absence of cytocidal effect in Cox2-negative cells with the novel virus indicated cancer specificity. In vivo, stronger tumor suppression from the novel virus was seen when compared with nonreplicating IFN-expressing vectors. CONCLUSION: We demonstrated the potent therapeutic effects of a novel tumor-specific conditionally replicative IFN-expressing adenovirus. With potential to locally deliver IFN and avoid systemic toxicity, this strategy may therefore expand the application of this robust and promising therapy.

Chronic granulomatous disease (CGD) is an inherited disorder of phagocytes in which NADPH oxidase is defective in generating reactive oxygen species. In this study, we reprogrammed three normal unrelated patient's fibroblasts (p47(phox) and gp91(phox)) to pluripotency by lentiviral transduction with defined pluripotency factors. These induced pluripotent stem cells (iPSC) share the morphological features of human embryonic stem cells, express the key pluripotency factors, and possess high telomerase activity. Furthermore, all the iPSC lines formed embryoid bodies in vitro containing cells originating from all three germ layers and were capable of teratoma formation in vivo. They were isogenic with the original patient fibroblasts, exhibited normal karyotype, and retained the p47(phox) or gp91(pho)(x) mutations found in the patient fibroblasts. We further demonstrated that these iPSC could be differentiated into monocytes and macrophages with a similar cytokine profile to blood-derived macrophages under resting conditions. Most importantly, CGD-patient-specific iPSC-derived macrophages showed normal phagocytic properties but lacked reactive oxygen species production, which correlates with clinical diagnosis of CGD in the patients. Together these results suggest that CGD-patient-specific iPSC lines represent an important tool for modeling CGD disease phenotypes, screening candidate drugs, and the development of gene therapy.

Eighteen months of 7-hourly analyses of rainfall and stream water chemistry are presented, spanning a wide range of chemical determinands and building on over 20years of weekly records for the moorland headwaters of the river Severn. The high-frequency time series data show that hydrochemical responses to major hydrological and biological drivers of short-term variability in rainfall and rivers are not captured by conventional low-frequency monitoring programmes. A wealth of flow related, flow independent, diurnal, seasonal and annual fluctuations indicate a cacophony of interactions within the catchment and stream. The complexity of the chemical dynamics is visually obvious, although there appears to be no clear way of translating this complexity into a simple algorithm. The work provides a proof of concept for the complex structure of catchment functioning revealed by extensive high-frequency measurements coupled with high analytical sensitivity and reproducibility. It provides new insights into hydrogeochemical functioning and a novel resource for catchment modelling.

To celebrate thirty years of peer-reviewed publication of cutting edge stem cell research in STEM CELLS, the first journal devoted to this promising field, we pause to review how far we have come in the three-decade lifetime of the Journal. To do this we will present our views of the ten most significant developments that have advanced stem cell biology where it is today. With the increasing rate of new data, it is natural that the bulk of these developments will have occurred in recent years but we must not think that stem cell biology is a young science. The idea of a stem cell has actually been around for quite a long time having appeared in the scientific literature as early as 1868 with Haeckels' concept of a stamzelle as an uncommitted or undifferentiated cell responsible for producing many types of new cells to repair the body (1), but it took many years to obtain hard evidence in support of this theory. Not until the work of James Till and Ernest McCulloch in the 1960's did we have proof of the existence of stem cells and until the derivation of embryonal carcinoma cells in the 1960's - 1970's and the first ESC in 1981, such adult or tissue specific stem cells were the only known class. The first issue of STEM CELLS was published in 1981; no small wonder that most of its papers were devoted to hematopoietic progenitors. More recently induced pluripotent stem cells (iPSC) have been developed and this is proving to be a fertile area of investigation as shown by the volume of publications appearing not only inSTEM CELLS, but in other journals over the last five years. The reader will note that many of the articles in this special issue are concerned with iPSC; however, this reflects the current surge of interest in the topic rather than any deliberate attempt to ignore other areas of stem cell investigation.

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.

Nurses in developing countries are frequently exposed to infectious tuberculosis (TB) patients, and have a high prevalence of TB infection. To estimate the incidence of new TB infection, we recruited a cohort of young nursing trainees at the Christian Medical College in Southern India. Annual tuberculin skin testing (TST) was conducted to assess the annual risk of TB infection (ARTI) in this cohort. 436 nursing students completed baseline two-step TST testing in 2007 and 217 were TST-negative and therefore eligible for repeat testing in 2008. 181 subjects completed a detailed questionnaire on exposure to tuberculosis from workplace and social contacts. A physician verified the questionnaire and clinical log book and screened the subjects for symptoms of active TB. The majority of nursing students (96.7%) were females, almost 84% were under 22 years of age, and 80% had BCG scars. Among those students who underwent repeat testing in 2008, 14 had TST conversions using the ATS/CDC/IDSA conversion definition of 10 mm or greater increase over baseline. The ARTI was therefore estimated as 7.8%(95%CI: 4.3-12.8%). This was significantly higher than the national average ARTI of 1.5%. Sputum collection and caring for pulmonary TB patients were both high risk activities that were associated with TST conversions in this young nursing cohort. Our study showed a high ARTI among young nursing trainees, substantially higher than that seen in the general Indian population. Indian healthcare providers and the Indian Revised National TB Control Programme will need to implement internationally recommended TB infection control interventions to protect its health care workforce.