Dynamics of Endothelial Cell Diversity and Plasticity in Health and Disease.

Endothelial cells (ECs) are vital structural units of the cardiovascular system possessing two principal distinctive properties: heterogeneity and plasticity. Endothelial heterogeneity is defined by differences in tissue-specific endothelial phenotypes and their high predisposition to modification along the length of the vascular bed. This aspect of heterogeneity is closely associated with plasticity, the ability of ECs to adapt to environmental cues through the mobilization of genetic, molecular, and structural alterations. The specific endothelial cytoarchitectonics facilitate a quick structural cell reorganization and, furthermore, easy adaptation to the extrinsic and intrinsic environmental stimuli, known as the epigenetic landscape. ECs, as universally distributed and ubiquitous cells of the human body, play a role that extends far beyond their structural function in the cardiovascular system. They play a crucial role in terms of barrier function, cell-to-cell communication, and a myriad of physiological and pathologic processes. These include development, ontogenesis, disease initiation, and progression, as well as growth, regeneration, and repair. Despite substantial progress in the understanding of endothelial cell biology, the role of ECs in healthy conditions and pathologies remains a fascinating area of exploration. This review aims to summarize knowledge and concepts in endothelial biology. It focuses on the development and functional characteristics of endothelial cells in health and pathological conditions, with a particular emphasis on endothelial phenotypic and functional heterogeneity.

Immunotherapy of Clear-Cell Renal-Cell Carcinoma.

Clear-cell Renal-Cell Carcinoma (ccRCC) is the most common type of renal-cell carcinoma (RCC). In many cases, RCC patients manifest the first symptoms during the advanced stage of the disease. For this reason, immunotherapy appears to be one of the dominant treatments to achieve a resolution. In this review, we focus on the presentation of the main immune checkpoint proteins that act as negative regulators of immune responses, such as PD-1, CTLA-4, LAG-3, TIGIT, and TIM-3, and their respective inhibitors. Interleukin-2, another potential component of the treatment of ccRCC patients, has also been covered. The synergy between several immunotherapies is one of the main aspects that unites the conclusions of research in recent years. To date, the combination of several immunotherapies enhances the efficacy of a monotherapy, which often manifests important limitations. Immunotherapy aimed at restoring the anti-cancer immune response in ccRCC, involved in the recognition and elimination of cancer cells, may also be a valid solution for many other types of immunogenic tumors that are diagnosed in the final stages.

Returning to employment following allogeneic hematopoietic stem cell transplant: A major problem among survivors.

Quality of life (QoL) is an important aspect of cancer survivorship. One of the most acute problems that impact survivors in many aspects of activities of daily living and compromise their QoL is the inability to return to employment following successful cancer therapy. This is most prominent among survivors after allogeneic hematopoietic stem cell transplant (allo-HSCT). More than 50% of the survivors following allo-HSCT remain unemployed one year after the procedure. This problem extends beyond the initial few years; unemployment rates among those who underwent allo-HSCT during their childhoods or adolescence have remained high. The inability to return to employment imposes a financial burden. Survivors following allo-HSCT also experience a multitude of chronic psychosocial complications that may be both contributing and consequential to the inability to return to employment. However, many transplant programs and cancer centers do not have return-to-employment programs. In this review paper, we discuss the prevalence of unemployment following allo-HSCT. We examine the psychosocial symptoms experienced by survivors and how they may affect survivors' ability to return to employment. Finally, we propose a multi-disciplinary multi-pronged occupation-focused approach to address the complex and inter-related psychosocial symptoms to help alleviate the problem.

Septins in Infections: Focus on Viruses.

Human septins comprise a family of 13 genes that encode conserved GTP-binding proteins. They form nonpolar complexes, which assemble into higher-order structures, such as bundles, scaffolding structures, or rings. Septins are counted among the cytoskeletal elements. They interact with the actin and microtubule networks and can bind to membranes. Many cellular functions with septin participation have been described in the literature, including cytokinesis, motility, forming of scaffolding platforms or lateral diffusion barriers, vesicle transport, exocytosis, and recognition of micron-scale curvature. Septin dysfunction has been implicated in diverse human pathologies, including neurodegeneration and tumorigenesis. Moreover, septins are thought to affect the outcome of host-microbe interactions. Implication of septins has been demonstrated in fungal, bacterial, and viral infections. Knowledge on the precise function of a particular septin in the different steps of the virus infection and replication cycle is still limited. Published data for vaccinia virus (VACV), hepatitis C virus (HCV), influenza A virus (H1N1 and H5N1), human herpesvirus 8 (HHV-8), and Zika virus (ZIKV), all of major concern for public health, will be discussed here.

The posterior ridge of the greater tuberosity of the humerus: a suitable landmark for the posterior approach to the shoulder joint?

BACKGROUND: The purpose of this study was to evaluate the posterior ridge of the greater tuberosity, a palpable prominence during surgery, as a landmark for the posterior approach to the glenohumeral joint. METHODS: Twenty-five human cadaveric shoulders were dissected. In 5 cases, a full-thickness rotator cuff tear was present. The posterior surgical anatomy was defined, and the distance from the ridge to the interval between the infraspinatus (IS) and teres minor (TM) muscle, the distance from the ridge to the inferior border of the glenoid (IBG), and the distance between the IS-TM interval and the IBG were determined. RESULTS: In all specimens, a prominent ridge on the posterior greater tuberosity lateral to the articular margin could be identified. The IS-TM interval was located, on average, 3 mm proximal to this ridge. The IS-TM interval corresponded to a point 5 mm proximal to the IBG. In all shoulders, the ridge was located, on average, 8 mm proximal to the IBG. The plane of the IS-TM interval showed a vertically oblique direction. CONCLUSION: The posterior ridge of the greater tuberosity is a suitable landmark to locate the internervous plane between the IS and TM and should not be crossed distally. Unlike other landmarks, the ridge moves with the humeral head, making it is less dependent on the patient's size, sex, and arm position and the quality of the rotator cuff. The ridge is always located proximal to the insertion of the TM and IBG.

The interaction between the vastus medialis and vastus intermedius and its influence on the extensor apparatus of the knee joint.

PURPOSE: Although the vastus medialis (VM) is closely associated with the vastus intermedius (VI), there is a lack of data regarding their functional relationship. The purpose of this study was to investigate the anatomical interaction between the VM and VI with regard to their origins, insertions, innervation and function within the extensor apparatus of the knee joint. METHODS: Eighteen human cadaveric lower limbs were investigated using macro-dissection techniques. Six limbs were cut transversely in the middle third of the thigh. The mode of origin, insertion and nerve supply of the extensor apparatus of the knee joint were studied. The architecture of the VM and VI was examined in detail, as was their anatomical interaction and connective tissue linkage to the adjacent anatomical structures. RESULTS: The VM originated medially from a broad hammock-like structure. The attachment site of the VM always spanned over a long distance between: (1) patella, (2) rectus femoris tendon and (3) aponeurosis of the VI, with the insertion into the VI being the largest. VM units were inserted twice-once on the anterior and once on the posterior side of the VI. The VI consists of a complex multi-layered structure. The layers of the medial VI aponeurosis fused with the aponeuroses of the tensor vastus intermedius and vastus lateralis. Together, they form the two-layered intermediate layer of the quadriceps tendon. The VM and medial parts of the VI were innervated by the same medial division of the femoral nerve. CONCLUSION: The VM consists of multiple muscle units inserting into the entire VI. Together, they build a potential functional muscular complex. Therefore, the VM acts as an indirect extensor of the knee joint regulating and adjusting the length of the extensor apparatus throughout the entire range of motion. It is of clinical importance that, besides the VM, substantial parts of the VI directly contribute to the medial pull on the patella and help to maintain medial tracking of the patella during knee extension. The interaction between the VM and VI, with responsibility for the extension of the knee joint and influence on the patellofemoral function, leads readily to an understanding of common clinical problems found at the knee joint as it attempts to meet contradictory demands for both mobility and stability. Surgery or trauma in the anteromedial aspect of the quadriceps muscle group might alter a delicate interplay between the VM and VI. This would affect the extensor apparatus as a whole.

Experience from an optional dissection course in a clinically-orientated concept to complement system-based anatomy in a reformed curriculum.

Profound anatomical knowledge is the basis for modern demands in medicine and surgery, but many countries worldwide including Australia and New Zealand have discontinued offering dissection courses to medical and dental students during the past decades. This educational project done in Australia aimed at enhancing basic and advanced anatomy teaching by engaging a sub-group of second-year undergraduate students of a compulsory prosection- and model-based anatomy course (n = 54/170) in an optional multimodal course, which should easily articulate with a vertical curriculum. With topographical cadaver dissections as core, peer student-teams prepared and peer-assessed anatomy lectures based on clinical topics, which were rated highly by the peers and teachers. Anatomical knowledge was tested by quizzes and a multiple-choice examination. Individual dissection skills were self- and teacher-assessed. A final course grade was assigned based on these assessments. The grades in the system-based compulsory course achieved by the attendees of the paralleling dissection course were compared with their peers attending other optional courses. After beginning of the semester, the students in the dissection course performed similar, significantly (P < 0.005) improved during the semester (78.5% vs. 69.9%, 70.1% vs. 64.1%), but in the integrated (including anatomy, biochemistry, physiology) final examination at the end of the year only tended to higher scores. As assessed through interviews and a voluntary questionnaire, all students of the optional dissection course liked these activities, which enhanced their learning experience. Thus, this concept elegantly integrates anatomical dissection with modern teaching demands and is feasible for implementation in modernized curricula. Anat Sci Educ 11: 32-43. © 2017 American Association of Anatomists.

Microglia at center stage: a comprehensive review about the versatile and unique residential macrophages of the central nervous system.

Microglia cells are the unique residential macrophages of the central nervous system (CNS). They have a special origin, as they derive from the embryonic yolk sac and enter the developing CNS at a very early stage. They play an important role during CNS development and adult homeostasis. They have a major contribution to adult neurogenesis and neuroinflammation. Thus, they participate in the pathogenesis of neurodegenerative diseases and contribute to aging. They play an important role in sustaining and breaking the blood-brain barrier. As innate immune cells, they contribute substantially to the immune response against infectious agents affecting the CNS. They play also a major role in the growth of tumours of the CNS. Microglia are consequently the key cell population linking the nervous and the immune system. This review covers all different aspects of microglia biology and pathology in a comprehensive way.

Potential Risk to the Superior Gluteal Nerve During the Anterior Approach to the Hip Joint: An Anatomical Study.

BACKGROUND: The anterior approach to the hip joint is widely used in pediatric and adult orthopaedic surgery, including hip arthroplasty. Atrophy of the tensor fasciae latae muscle has been observed in some cases, despite the use of this internervous approach. We evaluated the nerve supply to the tensor fasciae latae and its potential risk for injury during the anterior approach to the hip joint. METHODS: Cadaveric hemipelves (n = 19) from twelve human specimens were dissected. The course of the nerve branch to the tensor fasciae latae muscle, as it derives from the superior gluteal nerve, was studied in relation to the ascending branch of the lateral circumflex femoral artery where it enters the tensor fasciae latae. RESULTS: The nerve supply to the tensor fasciae latae occurs in its proximal half by divisions of the inferior branch of the superior gluteal nerve. The nerve branches were regularly coursing in the deep surface on the medial border of the tensor fasciae latae muscle. In seventeen of nineteen cases, one or two nerve branches entered the tensor fasciae latae within 10 mm proximal to the entry point of the ascending branch of the lateral circumflex femoral artery. CONCLUSIONS: Coagulation of the ascending branch of the lateral circumflex femoral artery and the placement of retractors during the anterior approach to the hip joint carry the potential risk for injury to the motor nerve branches supplying the tensor fasciae latae. CLINICAL RELEVANCE: During the anterior approach, the ligation or coagulation of the ascending branch of the lateral circumflex femoral artery should not be performed too close to the point where it enters the tensor fasciae latae. The nerve branches to the tensor fasciae latae could also be compromised by the extensive use of retractors, broaching of the femur during hip arthroplasty, or the inappropriate proximal extension of the anterior approach.

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells.

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

Distal extension of the direct anterior approach to the hip poses risk to neurovascular structures: an anatomical study.

BACKGROUND: The anterior approach to the hip gained popularity for total hip arthroplasty in recent years. Distal extension of the anterior approach, sometimes needed intraoperatively, potentially endangers neurovascular structures to the quadriceps. The aim of this study was to determine the anatomical structures placed at risk by distal extension of the anterior approach to the hip. METHODS: Seventeen cadaveric hemipelves from twelve human specimens were dissected. The femoral nerve and its branches and the vessels arising from the lateral femoral circumflex artery were assessed in relation to the distal extension of the anterior approach. The damage caused by the introduction of a cerclage cable passer was also investigated. RESULTS: The area immediately distal to the intertrochanteric line is a common entry point for several nerve branches and is a useful distal landmark for surgeons to use to protect important neurovascular structures. The distal extension of the anterior approach compromises the nerve supply to the anterolateral portions of the quadriceps. Introduction of a cerclage cable passer through the anterior access also jeopardizes nerve branches to the vastus lateralis, lateral parts of the vastus intermedius, and branches of the lateral femoral circumflex artery. CONCLUSIONS: Distal extension of the direct anterior approach to the hip is challenging to accomplish without neurovascular injury to anterolateral parts of the quadriceps muscle group. In addition, important neurovascular structures are endangered with the introduction of a cable passer through the anterior approach. CLINICAL RELEVANCE: Distal extension of the direct anterior approach to the hip beyond the intertrochanteric line may compromise neurovascular structures supplying the quadriceps muscle.

Cisplatin induces differentiation of breast cancer cells.

Breast tumors are heterogeneous including cells with stem cell properties and more differentiated cells. This heterogeneity is reflected into the molecular breast cancer subtypes. Breast cancer stem cells are resistant to chemotherapy, thus recent efforts are focusing on identifying treatments that shift them toward a more differentiated phenotype, making them more susceptible to chemotherapy. We examined whether the drug cisplatin induces differentiation in breast cancer cell lines that represent different breast cancer subtypes. We used three cell lines representing triple-negative breast cancers, BT-549 and MDA-MB-231 (claudin-low), and MDA-MB-468 (basal-like), along with estrogen and progesterone receptor positive MCF-7 cells (luminal). Cisplatin was applied at 2.5, 5, 10, and 20 µM, and cell viability and proliferation were measured using MTS and BrdU assays, respectively. The effect of cisplatin on the cellular hierarchy was examined by flow cytometry, immunofluorescence and qRT-PCR. Cisplatin treatment of 10 and 20 µM reduced cell viability by 36-51% and proliferation capacity by 36-67%. Treatment with cisplatin resulted in 12-67% down-regulation of stem cell markers (CD49f, SSEA4) and 10-130% up-regulation of differentiation markers (CK18, SMA, ß-tubulin). At the mRNA level, CD49f was down-regulated whilst ß-tubulin was up-regulated in the claudin-low cell lines. SSEA4 protein expression decreased upon cisplatin treatment, but SSEA4 mRNA expression increased indicating a differential regulation of cisplatin at the post-transcriptional level. It is concluded that cisplatin reduces breast cancer cell survival and induces differentiation of stem/progenitor cell subpopulations within breast cancer cell lines. These effects indicate the potential of this drug to target specific chemotherapy-resistant cells within a tumor.

Expression of the Pluripotency Transcription Factor OCT4 in the Normal and Aberrant Mammary Gland.

Breast cancers with lactating features, some of which are associated with pregnancy and lactation, are often poorly differentiated, lack estrogen receptor, progesterone receptor, and HER2 expression and have high mortality. Very little is known about the molecular mechanisms that drive uncontrolled cell proliferation in these tumors and confer lactating features. We have recently reported expression of OCT4 and associated embryonic stem cell self-renewal genes in the normal lactating breast and breastmilk stem cells (hBSCs). This prompted us to examine OCT4 expression in breast cancers with lactating features and compare it with that observed during normal lactation, using rare specimens of human lactating breast. In accordance with previous literature, the normal resting breast (from non-pregnant, non-lactating women) showed minimal OCT4 nuclear expression (0.9%). However, this increased in the normal lactating breast (11.4%), with further increase in lactating adenomas, lactating carcinomas, and pregnancy-associated breast cancer (30.7-48.3%). OCT4 was expressed in the epithelium and at lower levels in the stroma, and was co-localized with NANOG. Comparison of normal non-tumorigenic hBSCs with OCT4-overexpressing tumorigenic breast cell lines (OTBCs) demonstrated upregulation of OCT4, SOX2, and NANOG in both systems, but OTBCs expressed OCT4 at significantly higher levels than SOX2 and NANOG. Similar to hBSCs, OTBCs displayed multi-lineage differentiation potential, including the ability to differentiate into functional lactocytes synthesizing milk proteins both in vitro and in vivo. Based on these findings, we propose a hypothesis of normal and malignant transformation in the breast, which centers on OCT4 and its associated gene network. Although minimal expression of these embryonic genes can be seen in the breast in its resting state throughout life, a controlled program of upregulation of this gene network may be a potential regulator of the normal remodeling of the breast toward a milk-secretory organ during pregnancy and lactation. Deregulation of this gene network either within or outside pregnancy and lactation may lead to aberrant breast cell proliferation and malignant transformation, suggesting a role of these genes in both normal lactation and breast oncogenesis.

Adipose tissue can be generated in vitro by using adipocytes from human fat tissue mesenchymal stem cells seeded and cultured on fibrin gel sheet.

The current study has developed an innovative procedure to generate ex novo fat tissue by culturing adipocytes from human fat tissue mesenchymal stem cells (hFTMSCs) on fibrin gel sheet towards applications in medicine and cosmetology. Fibrin gel has been obtained by combining two components fibrinogen and thrombin collected by human peripheral blood. By this procedure it was possible to generate blocks of fibrin gel containing adipocytes within the gel that show similar features and consistency to human fat tissue mass. Results were assessed by histological staining methods, fluorescent immune-histochemistry staining as well photos by scanning electron microscopy (SEM) to demonstrate the adhesion and growth of cells in the fibrin gel. This result opens a real possibility for future clinical applications in the treatment of reconstructive and regenerative medicine where the use of stem cell may eventually be a unique solution or in the field of aesthetic medicine where autograft fat stem cells may grant for a safer and better outcome with long lasting results.

Breastmilk is a novel source of stem cells with multilineage differentiation potential.

The mammary gland undergoes significant remodeling during pregnancy and lactation, which is fuelled by controlled mammary stem cell (MaSC) proliferation. The scarcity of human lactating breast tissue specimens and the low numbers and quiescent state of MaSCs in the resting breast have hindered understanding of both normal MaSC dynamics and the molecular determinants that drive their aberrant self-renewal in breast cancer. Here, we demonstrate that human breastmilk contains stem cells (hBSCs) with multilineage properties. Breastmilk cells from different donors displayed variable expression of pluripotency genes normally found in human embryonic stem cells (hESCs). These genes included the transcription factors (TFs) OCT4, SOX2, NANOG, known to constitute the core self-renewal circuitry of hESCs. When cultured in the presence of mouse embryonic feeder fibroblasts, a population of hBSCs exhibited an encapsulated ESC-like colony morphology and phenotype and could be passaged in secondary and tertiary clonogenic cultures. While self-renewal TFs were found silenced in the normal resting epithelium, they were dramatically upregulated in breastmilk cells cultured in 3D spheroid conditions. Furthermore, hBSCs differentiated in vitro into cell lineages from all three germ layers. These findings provide evidence that breastmilk represents a novel and noninvasive source of patient-specific stem cells with multilineage potential and establish a method for expansion of these cells in culture. They also highlight the potential of these cells to be used as novel models to understand adult stem cell plasticity and breast cancer, with potential use in bioengineering and tissue regeneration.

Microfluidic fabrication of cationic curcumin nanoparticles as an anti-cancer agent.

Curcumin nanoparticles of less than 50 nm in diameter are accessible using a continuous flow microfluidic rotating tube processor (RTP) under scalable conditions, at room temperature. A mixture of DDAB and Pluronic F127 renders higher stability of the curcumin nanoparticles in physiological pH 7.4 for up to eight hours. The nanoparticles have enhanced cytotoxicity in estrogens receptor negative and positive breast cancer cell lines compared with free curcumin.

Influence of metal ions on human lymphocytes and the generation of titanium-specific T-lymphocytes.

BACKGROUND: There is mounting evidence to suggest the involvement of the immune system by means of activation by metal ions released via biocorrosion, in the pathophysiologic mechanisms of aseptic loosening of orthopedic implants. However, the detailed mechanisms of how metal ions become antigenic and are presented to T-lymphocytes, in addition to how the local inflammatory response is driven, remain to be investigated. METHODS: Human T-lymphocytes were cultured in the presence of a variety of metal ions before investigating functional and phenotypic changes using flow cytometric analysis. Additionally, human monocyte-derived dendritic cells (mDC) loaded with metal ions were used as antigen-presenting cells and incubated with naive T-lymphocytes with the aim of generating titanium-specific T-lymphocytes. RESULTS: Using an autologous in vitro model, with mDC treated with Titanium (IV), we were able to induce Titanium (IV)-specific T-lymphocytes. These T-lymphocytes responded in a dose-related manner to Titanium (IV), while they did not cross-react with Titanium (III) or other metal ions, indicating that the new antigenic peptide complexes formed by Titanium (IV) are highly specific. CONCLUSION: This study showed that mDC exposed to Titanium (IV) are able to induce the generation of Titanium (IV)-specific T-lymphocytes, demonstrating the strong and specific antigenicity of Titanium (IV) ions released by biocorrosion.

CX3CR1 deficiency exacerbates neuronal loss and impairs early regenerative responses in the target-ablated olfactory epithelium.

The olfactory epithelium is a site of sustained adult neurogenesis where olfactory sensory neurons are continuously replaced from endogenous stem/progenitor cells. Epithelial macrophages have been implicated in the phagocytosis of degenerating cells but the molecular mechanisms allowing for their recruitment and activation while maintaining a neurogenic microenvironment are poorly understood. We have previously shown that the chemokine fractalkine (CX3CL1) is expressed by olfactory sensory neurons and ensheathing cells in the olfactory epithelium. In turn, the fractalkine receptor, CX3CR1, is expressed on macrophages and dendritic cells within the olfactory epithelium. We report that a selective cell death of olfactory sensory neurons in the epithelium of CX3CR1-deficient mice via target ablation (i.e. olfactory bulbectomy) results in an exacerbated loss of olfactory sensory neurons compared to wild-type mice. In addition, reduced proliferation of intraepithelial stem/progenitor cells was observed in lesioned CX3CR1-deficient mice, suggesting an impaired regenerative response. Importantly, a lack of CX3CL1-signaling caused increased recruitment of macrophages into the olfactory epithelium, which in turn contained higher levels of pro-inflammatory cytokines (e.g. TNF-α and IL-6) as determined by qPCR. We also present novel data showing that, relative to wild-type, CX3CR1-deficient macrophages have diminished phagocytic activity following stimulation with CX3CL1. Collectively, our data indicate that signaling through the CX3CR1 receptor modulates macrophage activity, resulting in an environment conducive to olfactory sensory neuron clearance and targeted replacement from endogenous stem/progenitor cells.

Visualising gold inside tumour cells following treatment with an antitumour gold(I) complex.

Gold(I) phosphine complexes, such as [Au(d2pype)(2)]Cl, (1, where d2pype is 1,2-bis(di-2-pyridyl phosphinoethane)), belong to a class of promising chemotherapeutic candidates that have been shown to be selectively toxic to tumourigenic cells, and may act via uptake into tumour cell mitochondria. For a more holistic understanding of their mechanism of action, a deeper knowledge of their subcellular distribution is required, but to date this has been limited by a lack of suitable imaging techniques. In this study the subcellular distribution of gold was visualised in situ in human breast cancer cells treated with 1, using nano-scale secondary ion mass spectrometry. NanoSIMS ion maps of (12)C(14)N(-), (31)P(-), (34)S(-) and (197)Au(-) allowed, for the first time, visualisation of cellular morphology simultaneously with subcellular distribution of gold. Energy filtered transmission electron microscopy (EFTEM) element maps for gold were also obtained, allowing for observation of nuclear and mitochondrial morphology with excellent spatial resolution, and gold element maps comparable to the data obtained with NanoSIMS. Following 2 h treatment with 1, the subcellular distribution of gold was associated with sulfur-rich regions in the nucleus and cytoplasm, supporting the growing evidence for the the mechanism of action of Au(I) compounds based on inhibition of thiol-containing protein families, such as the thioredoxin system. The combination of NanoSIMS and EFTEM has broader applicability for studying the subcellular distribution of other types of metal-based drugs.

Culture and differentiation of osteoblasts on coral scaffold from human bone marrow mesenchymal stem cells.

In this paper we describe an approach that aims to provide fundamental information towards a scientific, biomechanical basis for the use of natural coral scaffolds to initiate mesenchymal stem cells into osteogenic differentiation for transplant purposes. Biomaterial, such as corals, is an osteoconductive material that can be used to home human derived stem cells for clinical regenerative purposes. In bone transplantation, the use of biomaterials may be a solution to bypass two main critical obstacles, the shortage of donor sites for autografts and the risk of rejection with allograft procedures. Bone regeneration is often needed for multiple clinical purposes for instance, in aesthetic reconstruction and regenerative procedures. Coral graft Porites lutea has been used by our team for a decade in clinical applications on over a thousand patients with different bone pathologies including spinal stenosis and mandibular reconstruction. It is well accepted that human bone marrow (hBM) is an exceptional source of mesenchymal stem cells (MSCs), which may differentiate into different cell phenotypes such as osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes and neurons. Isolated MSCs from human bone marrow were induced into osteoblasts using an osteogenic medium enriched with two specific growth factors, FGF9 and vitamin D2. Part of the cultured MSCs were directly transferred and seeded onto coral scaffolds (Porites Lutea) and induced to differentiate into osteoblasts and part were cultured in flasks for osteocell culture. The data support the concept that hBM is a reliable source of MSCs which may be easily differentiated into osteoblasts and seeded into coral as an optimal device for clinical application. Within this project we have also discussed the biological nature of MSCs, their potential application for clinical transplantation and the prospect of their use in gene therapy.