A method is described for the rapid isolation and purification of bacterial genomic DNA. A total of 215 bacterial strains representing species of Campylobacter, Corynebacterium, Escherichia, Legionella, Neisseria, Staphylococcus and Streptococcus, were lysed with guanidium thiocyanate. DNA was prepared using just three other reagents and one high‐speed centrifugation step. The method, which was applicable to both Gram‐positive and Gram‐negative bacteria, eliminated endogenous nuclease activity and avoided the need for phenol, RNase and protease treatments. The DNA was of high purity, high molecular mass and double‐stranded.

Drug resistance continues to be a major barrier to the delivery of curative therapies in cancer. Historically, drug resistance has been associated with over-expression of drug transporters, changes in drug kinetics or amplification of drug targets. However, the emergence of resistance in patients treated with new-targeted therapies has provided new insight into the complexities underlying cancer drug resistance. Recent data now implicate intratumoural heterogeneity as a major driver of drug resistance. Single cell sequencing studies that identified multiple genetically distinct variants within human tumours clearly demonstrate the heterogeneous nature of human tumours. The major contributors to intratumoural heterogeneity are (i) genetic variation, (ii) stochastic processes, (iii) the microenvironment and (iv) cell and tissue plasticity. Each of these factors impacts on drug sensitivity. To deliver curative therapies to patients, modification of current therapeutic strategies to include methods that estimate intratumoural heterogeneity and plasticity will be essential.

The cellular and molecular microenvironment of epithelial stem and progenitor cells is poorly characterized despite well-documented roles in homeostatic tissue renewal, wound healing, and cancer progression. Here, we demonstrate that, in organotypic cocultures, dermal pericytes substantially enhanced the intrinsically low tissue-regenerative capacity of human epidermal cells that have committed to differentiate and that this enhancement was independent of angiogenesis. We used microarray analysis to identify genes expressed by human dermal pericytes that could potentially promote epidermal regeneration. Using this approach, we identified as a candidate the gene LAMA5, which encodes laminin α5, a subunit of the ECM component laminin-511/521 (LM-511/521). LAMA5 was of particular interest as we had previously shown that it promotes skin regeneration both in vitro and in vivo. Analysis using immunogold localization revealed that pericytes synthesized and secreted LAMA5 in human skin. Consistent with this observation, coculture with pericytes enhanced LM-511/521 deposition in the dermal-epidermal junction of organotypic cultures. We further showed that skin pericytes could also act as mesenchymal stem cells, exhibiting the capacity to differentiate into bone, fat, and cartilage lineages in vitro. This study suggests that pericytes represent a potent stem cell population in the skin that is capable of modifying the ECM microenvironment and promoting epidermal tissue renewal from non-stem cells, a previously unsuspected role for pericytes.

IntroductionHeterogeneous nuclear ribonucleoprotein particle proteins (hnRNPs) are abundant, multi-tasking proteins that play a central role in RNA metabolism. They are involved in packaging nascent hnRNA, alternative RNA splicing, mRNA export from the nucleus, and cytoplasmic trafficking, stability and translation (Dreyfuss et al., 2002;Krecic and Swanson, 1999;Shyu and Wilkinson, 2000;Weighardt et al., 1996). They also have an as yet poorly defined role in telomere maintenance.More than 20 hnRNPs, many of which are characterised by possession of RNA-recognition motifs (RRMs), have been identified. The hnRNPs A/B are the major components of the 40S particles that package hnRNA. This packaging was originally envisaged to mimic that of histones in nucleosomes but this analogy appears to be inappropriate as the levels of the core proteins are not stoichiometric: they differ markedly between cell types, and their association with RNA is dependent on the nucleic acid sequence (Dreyfuss et al., 1993). However, given the role of these core particles in hnRNA packaging one might anticipate a correlation between the abundance of their proteins and the transcriptional activity in the cell. This is borne out experimentally: there is a marked difference in the concentration of hnRNP A1 between resting or slowly dividing cells and rapidly dividing cells. In the latter, hnRNPs A1 and A2 have been proposed to be present at similar levels. But hnRNP A1, which is abundant in a range of human, hamster and mouse proliferating cells, is present at markedly lower levels in confluent or resting cells, whereas hnRNP A2 is less affected (Celis et al., 1986;LeStourgeon, 1978).The levels of hnRNPs A/B differ not only between proliferating and resting cells: some also fluctuate during the cell cycle (Leser and Martin, 1987;Minoo et al., 1989). In HeLa cells hnRNPs A2 and B1 are synthesized in the G1 phase, and their levels fall in G2 and M phases, with hnRNP B1 protein level falling more markedly . The relative and absolute levels of these two proteins also differ markedly between tissues, both being particularly abundant in rat brain, testis, lung, spleen and ovary (Kamma et al., 1999;Ma et al., 2002).Many genes show a correlation between strong expression in proliferating cancer cells and the fluctuations in the protein level across the cell cycle (Dreyfuss et al., 1993;Whitfield et al., 2002). The levels of hnRNP A/B proteins are of particular interest as it has been suggested that upregulation of some members of this protein family is associated, as either a cause or consequence, with cellular proliferation and cancer. The hnRNP A2 and its longer B1 isoform are expressed at an early stage in a variety of tumours and have been proposed as early markers for cancer, especially lung cancer (Fielding et al., 1999;Mulshine et al., 2002;Pino et al., 2003;Sueoka et al., 1999;Whitfield et al., 2002;Zhou et al., 1996) and possibly breast cancer (Zhou et al., 2001a). The upregulation of hnRNP A2/B1 in cancer parallels its expression in lung dev...

Previous studies have established that the regulation of gene expression is dependent upon the nucleosomal integrity of nuclear DNA. To a large extent, this integrity is dictated by the acetylation status of the core histone particles. The acetylation of histones is, in turn, controlled by the combined activity of specific acetylases and deacetylases. Moreover, disruption of histone acetylases and deacetylases has been linked to a wide variety of human cancers. For this reason, the recent availability of potent and specific histone deacetylase inhibitors has provoked a great deal of interest amongst cancer biologists, oncologists and pharmacologists. Within the past 2- 3 years, several novel histone deacetylase inhibitors have been reported, many of which have already been tested in vivo in mouse models of cancer. In this review we focus on the rationale behind the use of histone deacetylase inhibitors as anticancer agents. Moreover, we review some of the recent findings pertaining to the use of these compounds as anticancer agents.

In this study, we investigated the suppressive effect of a short hairpin RNA delivered by a lentiviral vector (LV-shRNA) against human papillomavirus (HPV) type 18 E6 on the expression of the oncogenes E6 and E7 in cervical cancer HeLa cells both in vitro and in vivo. The LV-shRNA effectively delivered the shRNA to HeLa cells and lead to a dose-dependent reduction of E7 protein and the stabilization of E6 target proteins, p53 and p21. Low-dose infection of HeLa cells with LV-shRNA caused reduced cell growth and the induction of senescence, whereas a high-dose infection resulted in specific cell death via apoptosis. Transplant of HeLa cells infected with a low dose of LV-shRNA into RagÀ/À mice significantly reduced the tumor weight, whereas transplant of cells infected with a high dose resulted in a complete loss of tumor growth. Systemic delivery of LV-shRNA into mice with established HeLa cell lung metastases led to a significant reduction in the number of tumor nodules. Our data collectively suggest that lentiviral delivery is an effective way to achieve stable suppression of E6/E7 oncogene expression and induce inhibition of tumor growth both in vitro and in vivo. These results encourage further investigation of this form of RNA interference as a promising treatment for cervical cancer.

Highlights d A strategy for improving the ADCC potential of therapeutic antibodies is presented d Temporary inhibition of endocytosis increases tumor cell antigen presentation d Prochlorperazine could be repurposed to enhance the efficacy of anti-tumor mAbs d Potential to reduce heterogeneity in tumor cell responses to many IgG1 antibodies

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