Current Pharmaceutical Biotechnology - Volume 12, Issue 2, 2011

Over the last decade there has been an exponential rise in our understanding of the biochemical mechanisms controlling cell proliferation and differentiation. While the four transcription factors Oct4, Sox2, Klf4 and cMyc have shown to be sufficient to induce pluripotency in fibroblasts, there has in addition been much research into the mechanisms and pathways of cell differentiation and the specific properties of stem cells, namely their plasticity and capacity for trans-differentiation. These studies have allowed progress at very fundamental level, with the prospect of further progress - until recent years quite unimaginable - in the field of reparative, regenerative and transplant medicine. In fact, from the present time, the genetic engineering production of regulatory factors identified through such research, has allowed the production of new tissues and a new category of cell therapy products, in which the main biological action is carried out by cells or tissues, albeit in the presence of organic or inorganic matrices or coatings. Examples of this type of product are anti-tumor vaccines, in vitro cultivated skin, products made of structural and cellular elements for the reconstruction of bones, cartilages, teeth, etc. From the best, most analytical and detailed characterization of stem cells, then, it has become clear that some tumor cell behaviors - that have a crucial role in determining their malignity - can be attributed to the presence of cells with characteristics similar to those of stem cells. The field of cancer research is consequently also witnessing a surge in studies designed to identify the metabolic pathways common to tumor and stem cells. This will in turn cast light on which micro-environment factors can direct these pathways towards differentiation and induce cancerous cells to behave less aggressively. From this point of view, over recent years there has been a lively return to studies that were very significant in the 70's and 80's, on the role of the embryonic micro-environment in conditioning tumor cell behavior towards normal phenotypes. This research is now underway, and will in all probability lead to important results over the next few years. Against this background, this special issue on “Reprogramming of normal and cancer stem cells” focuses on research in terms of conditioning the fate of normal and tumor stem cells with a view to new prospects for therapies. The issue therefore begins with articles covering the possibility of reprogramming normal stem cells, including through use of biomaterials, and goes on to consider what characteristics of tumor stem cells can allow them to be identified and studied. This is followed by a series of further articles illustrating the role of the micro-environment in conditioning the fate of a tumor cell. A number of metabolic pathways characterizing and common to both stem and tumor cells are examined, in order to gain a better understanding of the possibilities of conditioning the fate of both cell types; in addition, the role played by infectious and inflammatory diseases in the genesis of tumor diseases is also considered. Today, in fact, we know that inflammatory processes can support rather than hinder tumor growth, and also that pro-inflammatory cytokines can promote tumor proliferation, inhibiting the cell pathways that are able to block the neoplastic growth. The special issue goes on with a series of articles taking a close look at the specific role played by the micro-environment in conditioning the destiny of the tumor stem cells present in some tumors, for example breast and retinoblastoma tumor, and the role played by the use of normal stem cells in treating disorders such as hematological diseases. One article also considers the risks run by some reprogramming techniques: for example, creating embryo cells via parthenogenesis can give rise to tumors. The issue continues with various articles illustrating in close detail the role of the embryonic micro-environment in conditioning the destiny of tumor cells. In this context, one review takes a look at general aspects, while others consider aspects that can help clarify the mechanisms underlying the capacity of factors of this type of microenvironment to reprogram a tumor cell. One mathematical model sets out from a description of the state of cell differentiation, making use of existing data from studies of tumor growth slowing, linked to the use of such factors, with the goal of shedding light on aspects such as fitness, dosage, and administration time for the differentiation factors on improvement in tumor inhibitory response. Other articles illustrate a number of clinical cases of full regression of hepatocellular carcinomas in intermediate-advanced stages observed following administration of stem cell differentiation factors, and describe the molecular mechanisms that might explain these inhibitory responses on the tumor growth. It should be noted that the randomized and controlled clinical studies launched to date using stem cell differentiation factors are limited to patients with intermediate-advanced stage of hepatocellular carcinomas where other therapies were no longer possible. These factors are at present used only for hepatocellular carcinomas, since it has been demonstrated that substances capable of slowing one tumor’s growth may be inefficacious for another type. Finally, it is important to note that research into the possibility of reprogramming normal and tumor stem cells requires a complex approach to the issue. In fact, the problem requires the study of networks of substances and genes involved in the reprogramming phase, demanding skills in a variety of different areas of research, not simply of medical/biological, but also mathematical/ computational and modeling, in view of the complexity and non-linearity of the processes being studied. A paradigm shift is underway, and the future will witness our engagement in increasing numbers of scientific studies requiring cross-disciplinary skills. The new paradigm and the new ideas were well understood many years ago by Professor John Klavins, who has been for a long time President of the International Academy of Tumor Marker Oncology. Professor Klavins has always sustained my studies on reprogramming cancer cells, though the possibility of controlling tumor growth by using reprogramming factors was not considered realistic at the time I began studying it. I wish to dedicate this work to my friend John Klavins. ACKNOWLEDGEMENTS Many thanks to Roberta Zorovini and Silvia Cefalo from Smile Tech srl, via Valdirivo 19 Trieste, Italy ([email protected]), for their accurate and thorough work as organizing and editing secretariat.

Generation of pluripotent stem cells (iPSCs) from adult fibroblasts starts a “new era” in stem cell biology, as it overcomes several key issues associated with previous approaches, including the ethical concerns associated with human embryonic stem cells. However, as the genetic approach for cell reprogramming has already shown potential safety issues, a chemical approach may be a safer and easier alternative. Moreover, a chemical approach could be advantageous not only for the de-differentiation phase, but also for inducing reprogrammed cells into the desired cell type with higher efficiency than current methodologies. Finally, a chemical approach may be envisioned to activate resident adult stem cells to proliferate and regenerate damaged tissues in situ, without the need for exogenous cell injections.

A stem cell is defined as a cell able to self-renew and at the same time to generate one or more specialized progenies. In the adult organism, stem cells need a specific microenvironment where to reside. This tissue-specific instructive microenvironment, hosting stem cells and governing their fate, is composed of extracellular matrix and soluble molecules. Cell-matrix and cell-cell interactions also contribute to the specifications of this milieu, regarded as a whole unitary system and referred to as “niche”. For many stem cell systems a niche has been identified, but only partially defined. In regenerative medicine and tissue engineering, biomaterials are used to deliver stem cells in specific anatomical sites where a regenerative process is needed. In this context, biomaterials have to provide informative microenvironments mimicking a physiological niche. Stem cells may read and decode any biomaterial and modify their behavior and fate accordingly. Any material is therefore informative in the sense that its intrinsic nature and structure will anyway transmit a signal that will have to be decoded by colonizing cells. We still know very little of how to create local microenvironments, or artificial niches, that will govern stem cells behavior and their terminal fate. Here we will review some characteristics identifying specific niches and some of the requirements allowing stem cells differentiation processes. We will discuss on those biomaterials that are being projected/engineered/manufactured to gain the informative status necessary to drive proper molecular cross-talk and cell differentiation; specific examples will be proposed for bone and cartilage substitutes.

Canonical WNT signaling activation leads to transcriptional up-regulation of FGF ligand, Notch ligand, noncanonical WNT ligand, WNT antagonist, TGFβ antagonist, and MYC. Non-canonical WNT signals inhibit canonical WNT signaling by using MAP3K7-NLK signaling cascade. Hedgehog up-regulates Notch ligand, WNT antagonist, BMP antagonists, and MYCN. TGFβ up-regulates non-canonical WNT ligand, CDK inhibitors, and NANOG, while BMP upregulates Hedgehog ligand. Based on these mutual regulations, WNT, FGF, Notch, Hedgehog, and TGFβ/BMP signaling cascades constitute the stem-cell signaling network, which plays a key role in the maintenance or homeostasis of pluripotent stem cells and cancer stem cells. Human embryonic stem cells (ESCs) are supported by FGF and TGFβ/Nodal/Activin signals, whereas mouse ESCs by LIF and canonical WNT signals. Combination of TGFβ inhibitor and canonical WNT activator alter the character of human induced pluripotent stem cells (iPSCs) from human ESC-like to mouse ESC-like. Fine-tuning of WNT, FGF, Notch, TGFβ/BMP, and Hedgehog signaling network by using small molecule compounds could open the door for regenerative medicine utilizing pluripotent stem cells without tumorigenic potential. Because FGF, Hedgehog, TGFβ, and non-canonical WNT signals synergistically induce EMT regulators, such as Snail (SNAI1), Slug (SNAI2), TWIST, and ZEB2 (SIP1), tumor-stromal interaction at the invasion front aids cancer stem cells to acquire more malignant phenotype. Cancer stem cells occur as mimetics of normal tissue stem cells based on germ-line variation, epigenetic change, and somatic mutation of stem-cell signaling components, and then acquire more malignant phenotype based on accumulation of additional epigenetic and genetic alterations, and tumor-stromal interaction at the invasion front.

The idea of cancer stem cell (CSC) has recently moved to the forefront of cancer research. There is still a lack of a widespread consensus on the of these cells, description and definition. The increasing literature on CSCs has compelled researchers worldwide to rewrite the natural history of cancer including those cells as principal players as well as to revise their views on tumor formation and progression. CSCs are tumor cell components that can initiate a new tumor after an apparent therapeutic eradication. A functional definition of cancer stem cell or cancer initiating cell is that of a cell which, when transplanted in a mouse model, can give rise to a tumor recapitulating the original one or even a phenotypically diverse tumor related to the tumor of origin. Since the characteristic asymmetric division of stem cells is somewhat anomalous in cancer, it might be advisable to refer to them as “stemloids”. Stemness in cancer is not as much as an identity but rather a status. There is increasing evidence of the importance of the tumor and the host microenvironment in conditioning the stem cell status itself. The cancer stem cell microenvironment may be the key in the development of therapeutic strategies. We must think in terms of targeting “standard” tumor cells, cancer stem cells, and also their niche and tumor microenvironment. Here we discuss some features of cancer stem cells, and the role of the microenvironment, envisaging a choral view of cancer stem cell development and-or latency, towards development of specific therapeutic approaches. Here we propose models of replication and quiescence and the modulation by cells, genes and miRNAs. We also summarize in a table surface markers useful for the identification and isolation of CSCs.

The association of cancer with preceding parasitic infections has been observed for over 200 years. Some such cancers arise from infection of tissue stem cells by viruses with insertion of viral oncogenes into the host DNA (mouse polyoma virus, mouse mammary tumor virus). In other cases the virus does not insert its DNA into the host cells, but rather commandeers the metabolism of the infected cells, so that the cells continue to proliferate and do not differentiate (human papilloma virus and cervical cancer). Cytoplasmic Epstein Barr virus infection is associated with a specific gene translocation (Ig/c-myc) that activates proliferation of affected cells (Burkitt lymphoma). In chronic osteomyelitis an inflammatory reaction to the infection appears to act through production of inflammatory cytokines and oxygen radical formation to induce epithelial cancers. Infection with Helicobacter pylori leads to epigenetic changes in methylation and infection by a parasite. Clonorchis sinensis also acts as a promoter of cancer of the bile ducts of the liver (cholaniocarcinoma). The common thread among these diverse pathways is that the infections act to alter tissue stem cell signaling with continued proliferation of tumor transit amplifying cells.

Stem cells have an important role in cell biology, allowing tissues to be renewed by freshly created cells throughout their lifetime. The specific micro-environment of stem cells is called stem cell niche; this environment influences the development of stem cells from quiescence through stages of differentiation. Recent advance researches have improved the understanding of the cellular and molecular components of the micro-environment - or niche - that regulates stem cells. We point out an important trend to the study of niche activity in breast cancers. Breast cancer has long been known to conserve a heterogeneous population of cells. While the majority of cells that make up tumors are destined to differentiate and eventually stop dividing, only minority populations of cells, termed cancer stem cell, possess extensive self renewal capability. These cancer stem cells possess characteristics of both stem cells and cancer cells. Breast cancer stem cells reversal to breast somatic stem cells offer a new therapy, that not only can stop the spread of breast cancer cells, but also can differentiate breast cancer stem cells into normal breast somatic stem cells. These can replace damaged breast tissue. Nevertheless, the complexity of realizing this therapy approach needs further research.

About 20% of the total cells from primary breast tumors could generate palpable tumors in non-obese diabetic severe combined immunodeficient (NOD/SCID) immunocompromised mice. All the tumorigenic cells originate from a normal mammary stem cell. Human mammary stem cells are sensitive to oncogenic mutations and in mouse models they share similarities with breast cancer stem cells (BrCSCs). Tumorigenicity, invasion, progression and metastasization are further BrCSCs properties likely depending on their CD44+/CD24- phenotype. Local invasion and tumor metastasization seem to be facilitated by the epithelial to mesenchymal transition (EMT) program. This program may be reactivated from stable genetic alterations or through exposure of cancer cells to factors present in the surrounding micro-environment, or by an up-regulation of EMT-inducing transcription factors. One main explanation for resistance to treatment by cancer cells is that a rare subpopulation of cells in residual tumors with tumorigenic potential is intrinsically resistant to therapy. Consistent with this hypothesis, in human breast tumors, the subpopulation of tumor-initiating cancer cells with CD44high/CD24low cell surface-marker profile was found more resistant to cancer therapies (chemo, hormone and radiotherapy) than is the major population of more differentiated breast cancer cells. The reasons for CSC resistance to chemotherapy, hormonetherapy and radiotherapy also have been examined and they opened new scenarios for cancer therapy.

Human parthenogenetic embryos have been recently proposed as an alternative, less controversial source of embryonic stem cells. However many aspects related to the biology of parthenogenetic cell lines are not fully understood and still need to be elucidated. These cells have great potentials; they possess most of the main features of bi-parental stem cells, show the typical morphology and express most of the pluripotency markers distinctive of ESC. They also have high telomerase activity, that disappears upon differentiation, and display great plasticity. When cultured in appropriate conditions, they are able to give rise to high specification tissues and to differentiate into mature cell types of the neural and hematopoietic lineages. However, their injection in immunodeficient mice has been reported to result in tumour formations. Aberrant levels of molecules related to spindle formation, cell cycle check points and chromosome segregation have also been detected in these cells, that are characterized by the presence of an abnormal number of centrioles and massive autophagy. All these observations indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferation versus differentiation in parthenogenetic stem cells. In this manuscript we summarize data related to these aberrant controls and describe experimental evidence indicating their uniparental origin as one of the possible cause. Finally we propose their use as an intriguing experimental tool where the pathways controlling potency, self renewal and cell plasticity are deeply interconnected with cell transformation, in an unstable, although highly controlled, equilibrium between pluripotency and malignacy.

Retinoblastoma (RB), an intraocular tumor of childhood, contains small subpopulation(s) of stem-like cells expressing the ABCG2 drug transporter that can efflux standard chemotherapies. Since chemo-resistant stem-like cells appear to be a driving force in tumor progression and metastasis for a variety of cancers, innovative treatment strategies are necessary to eradicate these rare cell populations. Terminal differentiation, as a means to deplete the pool of stem-like cells in RB, is an intriguing approach to cancer therapeutics. However, the full extent of RB differentiation remains unknown. Differentiation of RB cells has been examined in response to a variety of different agents, including retinoic acid/sodium butyrate, Pigment Epithelial-Derived Factor, as well as Succinylated Concanavalin A. RB cells exhibit morphologic and phenotypic responses to these differentiating agents, although the permanence of these effects is questionable due to reversibility. Further study of differentiation programs may lead to new approaches in the design of strategies to combat the initiation and progression of RB in vivo.

An increasing body of evidence has shown that hematologic malignancies, alike normal hematopoiesis, has a hierarchical structure including a stem cell compartment with self renewal capability, endowed in a neoplastic niche bearing resemblance to its normal hematopoietic counterpart. According to experimental data on NOD-SCID mice, leukemic stem cells are characterized by a CD34+/CD38- surface profile and account for 1 in 103 to 1 in 106 of the total amount of leukemic cells. The available knowledge about leukemic stem cells (LSC) has arisen the question as to whether some targeting of LSC is achieved by current treatments; the answer is dubitative at best, with the possible exception of arsenic trioxide in promyelocytic leukemia. On the other side, the unsatisfactory results in the treatment of many hematological neoplasms has prompted many research groups to find out whether direct targeting of LSC, possibly in its niche, would result in an improvement in cure rates. This approach implies the identification of LSC specific markers, clearly distinct from their normal counterpart in order to spare normal hematopoietic stem cells. Adhesion/surface antigens, metabolic pathways involved in LSC survival and renewal, telomerase, commonly mutated genes and epigenetic phenomena have been investigated as candidate targets for newer therapeutic strategies. So far, most of the possibly effective agents have been studied in experimental models only. FLT-3 inhibitors account for a notable exception since they have resulted effective in vivo in AML with mutated, but not over expressed, FLT-3. A main task for the future is to find out whether some common LSC specific markers would be identifiable in a substantial proportion of AML cases, or whether each AML case shows a unique fingerprint of markers. In the latter event, targeting of LSC could result in an arduous task.

Liver failure results in impairment of many functions and dependent organs such as brain and kidneys begin to fail, reducing the chance of recovery even further. Orthotopic liver transplantation (OLTx) is the only treatment that improves the survival rate in patients with liver failure. Liver Transplantation (LT), including orthologous liver transplantation (OLT), cadaveric LT, split LT, living donor LT (LDLT) brings hopes to patients with these diseases. Globally, 1.4 million deaths occur annually as a result of chronic liver diseases. The reasons for this high death toll include unavailability of healthy liver donor and highly expensive liver transplantation treatment. Furthermore, some other factors such as operative risks and post-transplant rejection are major limitation of OLT. Isolated hepatocyte transplantation is emerging as alternative bridge support till the healthy donor is arranged. Mature hepatocytes have several drawbacks such as low proliferation both in vitro and in vivo, low viability after cryopreservation, and requirement of large number of cells for infusion. The studies on isolation of hepatic progenitors have shown promising results to overcome these limitations. These cells possess higher proliferative capacity, are less immunogenic and more resistant to cryopreservation, and ischemic injury; properties that could enhance their engraftment within the recipient liver. The hepatic progenitors have been isolated from the intra-hepatic sources and extra-hepatic sources. Fetal cells are one of the ideal sources of hepatic stem/progenitor cells. Autologous bone marrow stem cell transplantation in patients with cirrhosis has shown promising result.

The recent tumor research has lead scientists to recognize the central role played by cancer stem cells in sustaining malignancy and chemo-resistance. A model of cancer presented by one of us describes the mechanisms that give rise to the different kinds of cancer stem-like cells and the role of these cells in cancer diseases. The model implies a shift in the conceptualization of the disease from reductionism to complexity theory. By exploiting the link between the agentbased simulation technique and the theory of complexity, the medical view is here translated into a corresponding computational model. Two main categories of agents characterize the model, 1) cancer stem-like cells and 2) stem cell differentiation stage factors. Cancer cells agents are then distinguished based on the differentiation stage associated with the malignancy. Differentiation factors interact with cancer cells and then, with varying degrees of fitness, induce differentiation or cause apoptosis. The model inputs are then fitted to experimental data and numerical simulations carried out. By performing virtual experiments on the model's choice variables a decision-maker (physician) can obtains insights on the progression of the disease and on the effects of a choice of administration frequency and or dose. The model also paves the way to future research, whose perspectives are discussed.

Cancer cells introduced into developing embryos can be committed to a complete reversion of their malignant phenotype. It is unlikely that such effects could be ascribed to only few molecular components interacting according to a simple linear-dynamics model, and they claim against the somatic mutation theory of cancer. Some 50 years ago, Needham and Waddington speculated that cancer represents an escape from morphogenetic field like those which guide embryonic development. Indeed, disruption of the morphogenetic field of a tissue can promote the onset as well as the progression of cancer. On the other hand, placing tumor cells into a “normal” morphogenetic field - like that of an embryonic tissue - one can reverse malignant phenotype, “reprogramming” tumor into normal cells. According to the theoretical framework provided by the thermodynamics of dissipative systems, morphogenetic fields could be considered as distinct attractors, to which cell behaviors are converging. Cancer-attractors are likely positioned somewhat close to embryonicattractors. Indeed, tumors share several morphological and ultra-structural features with embryonic cells. The recovering of an “embryonic-like” cell shape might enable the gene regulatory network to reactivate embryonic programs, and consequently to express antigenic and biochemical embryonic characters. This condition confers to cancer an unusual sensitivity to embryonic regulatory cues. Thus, it is not surprising that cancer cells exposed to specific embryonic morphogenetic fields undergoes significant modifications, eventually leading to a complete phenotypic reversion.

Hepatocellular carcinoma (HCC) represents the third cause of cancer-related death. Because HCC is multicentric with time, excluding the few transplanted patients, sooner or later it becomes untreatable with loco-regional therapies and, until some years ago, it was not responsive to systemic therapies. In 2005 a randomized trial indicated the efficacy of a product containing stem cell differentiation stage factors (SCDSF) taken from zebra fish embryos during the stage in which the totipotent stem cells are differentiating into the pluripotent adult stem cells. In such a trial the patients, with “intermediate” and “advanced” HCC according to BCLC/AASLD guidelines, presented benefit in terms of performance status (PS) and objective tumoral response, with some cases (2.4%) of complete response (CR). The aim of this cohort study is to report the experience of a tertiary referral center on the evidence of cases of CR in patients with “advanced” stage HCC treated with SCDSF as supportive care. CR was regarded as sustained disappearance of the neoplastic areas or blood supply therein, accompanied by normalization of AFP levels. Out of 49 patients consecutively recruited and retrospectively evaluated, 38 had “advanced” stage and 11 “terminal” stage. In 5 patients with “advanced” stage a sustained CR was reported (13.1%). Improvement on PS was obtained in 17 patients (34.6%). No side effects occurred. SCDSF treatment confirmed its efficacy in patients with “advanced” HCC, in terms of PS and tumoral response.

Stem cell differentiation stage factors (SCDSF), taken from Zebrafish embryos during the stage in which totipotent stem cells are differentiating into pluripotent stem cells, have been shown to inhibit proliferation and induce apoptosis in colon tumors. In order to ascertain if these embryonic factors could synergistically/additively interact with 5- Fluorouracil (5-Fu), whole cell-count, flow-cytometry analysis and apoptotic parameters were recorded in human colon cancer cells (Caco2) treated with Zebrafish stem cell differentiation stage factors (SCDSF 3 μg/ml) in association or not with 5-Fu in the sub-pharmacological therapeutic range (0.01 mg/ml). Cell proliferation was significantly reduced by SCDSF, meanwhile SCDSF+5-Fu leads to an almost complete growth-inhibition. SCDSF produces a significant apoptotic effect, meanwhile the association with 5-FU leads to an enhanced additive apoptotic rate at both 24 and 72 hrs. SCDSF alone and in association with 5-Fu trigger both the extrinsic and the intrinsic apoptotic pathways, activating caspase-8, -3 and -7. SCDSF and 5-Fu alone exerted opposite effects on Bax and Bcl-xL proteins, meanwhile SCDSF+5-Fu induced an almost complete suppression of Bcl-xL release and a dramatic increase in the Bax/Bcl-xL ratio. These data suggest that zebrafish embryo factors could improve chemotherapy efficacy by reducing anti-apoptotic proteins involved in drug resistance processes.

Low cost and simplicity of cultivating bacteria make the E. coli expression system a preferable choice for production of therapeutic proteins both on a lab scale and in industry. In addition straightforward recombinant DNA technology offers engineering tools to produce protein molecules with modified features. The lack of posttranslational modification mechanisms in bacterial cells such as glycosylation, proteolytic protein maturation or limited capacity for formation of disulfide bridges may, to a certain extent, be overcome with protein engineering. Protein engineering is also often employed to improve protein stability or to modulate its biological action. More sophisticated modifications may be achieved by genetic fusions of two proteins. This article presents a variety of examples of genetic engineering of therapeutic proteins. It emphasizes the importance of designing a construct without any unnecessary amino acid residues.

Few animal model studies have been conducted in order to evaluate the impact of androgenic anabolic steroids (AAS) supraphysiological doses on the cardiovascular system and myocardial injury. Twenty-five male CD1 mice (8-10 weeks old; 35g initial body weight) were randomized into three AAS treated groups and two control groups. The AAS mice received intramuscular Nandrolone Decanoate (DECA-DURABOLIN), vehicled in arachidis oil, for 42 days, twice per week, with different dosages, studying plasma lipid analysis, cardiac histopathological features, cardiac β1 adrenergic receptor expression, and the effects of the myocardial expression of inflammatory mediators (IL-1β, TNF-α) on the induction of cardiomyocytes apoptosis (HSP70, TUNEL), using proteomic and immunohistochemical analysis. The mice had free movements in their animal rooms (two groups) or exercised by running on a motor-driven treadmill the others three groups. Recurring high dose AAS administration and physical training in mice produce significant increase in body weight and for total cholesterol. A moderate increase of the heart weight, cardiac hypertrophy and wide colliquative myocytolysis, were observed in high dose AAS administration and physical training group. The expression of HSP70 and inflammatory cytokine IL-1β increased in the three AAS-treated groups. TNF-α showed a more extensive expression in the AAS-high dose group. A significant apoptotic process randomly sparse in the myocardium was described. Our data support the hypothesis that the combined effects of vigorous training, anabolic steroid abuse and stimulation of the sympathetic nervous system, may predispose to myocardial injury.

The liver X receptors (LXRs) are key regulators of genes involved in cholesterol homeostasis. Natural ligands and activators of LXRs are oxysterols. Numerous steroidal and non-steroidal synthetic LXR ligands are under development as potential drugs for individuals suffering from lipid disorders. N,N-dimethyl-3β-hydroxycholenamide (DMHCA) is a steroidal ligand of LXRs that exerts anti-atherogenic effects in apolipoprotein E-deficient mice without causing negative side effects such as liver steatosis or hypertriglyceridemia. In this report, we investigated the consequences of DMHCA treatment on cholesterol homeostasis in vivo and in vitro. Despite its hydrophobicity, DMHCA is readily absorbed by C57BL/6 mice and taken up by intestinal cells, the lung, heart and kidneys, but is undetectable in the brain. DMHCA significantly reduces cholesterol absorption and uptake in duodenum and jejunum of the small intestine and in turn leads to a reduction of plasma cholesterol by 24%. The most striking finding of this study is that DMHCA inhibited the enzyme 3β-hydroxysterol-Δ24-reductase resulting in an accumulation of desmosterol in the plasma and in feces. Thus, the reduction of plasma cholesterol was due to a block in the final step of cholesterol biosynthesis. Taken together, DMHCA is an interesting compound with properties distinct from other LXR ligands and might be used to study desmosterol- mediated effects in cells and tissues.

Sanger sequencing revolutionized the field of genetics by becoming the standard approach to appraise a given region of the genome at base-level resolution. However, the relatively recent need to sequence entire genomes has driven innovative developments within the market-place to allow for sequencing technology to be faster, cheaper and more accurate. In this review, we will cover these recent developments from both a technical and cost perspective. Firstly, we will place sequencing in a historical context by describing how it first came to the attention of the scientific community. Next, we will address the current high-throughput technologies generally available, including Roche's 454, Illumina's Genome Analyzer, Applied BioSystem's SOLiD, Complete Genomics, Helios, Pacific Biosciences and IonTorrent. These ‘next generation’ technologies also allow for applications related to target region deep sequencing, epigenetics(ChIP-seq), transcriptome sequencing (RNA-seq), megagenomics. Thus, these technologies offer unprecedented opportunities to increase our understanding of the functions and dynamics of the human genome in the near future.