The current status of prophylactic femoral intramedullary nailing for metastatic cancer.

The most common site for cancer to spread is bone. At post-mortem, bony metastases have been found in 70% of patients dying from breast and prostate cancer. Due to the prevalence of cancer, bone metastasis and the associated management represents a huge burden on NHS resources. In patients with metastasis, around 56% of these involve the lower limb long bones. Due to the huge forces placed upon long bones during weight bearing, there is a high risk of fracture through areas of metastasis. It is reported that 23% of pathological fractures occur in the femoral subtrochanteric region. This area is subjected to forces up to four times the body weight, resulting in poor union rate for these fractures, and significant morbidity associated with difficulty in mobilising, and in patient nursing. As cancer treatments improve, the life expectancy in this subgroup of patients is likely to increase. Therefore medium-to-long-term management of these fractures, beyond the palliative, will become essential. We aim to evaluate the current management for metastatic malignant femoral disease, with particular focus on the prophylactic augmentation of diseased femorii using intramedullary nails.

Role of salivary epithelial toll-like receptors 2 and 4 in modulating innate immune responses in chronic periodontitis.

BACKGROUND AND OBJECTIVE: Chronic periodontitis is initiated by sequential colonization with a broad array of bacteria and is perpetuated by an immune-inflammatory response to the changing biofilm. Host recognition of microbes is largely mediated by toll-like receptors (TLRs), which interact with conserved pathogen-associated molecular patterns. Based on ligand recognition, TLR-2 and TLR-4 interact with most periodontal pathogens. Extracrevicular bacterial reservoirs, such as the oral epithelial cells, contribute to the persistence of periodontitis. Human saliva is a rich source of oral epithelial cells that express functional TLRs. In this study we investigated the role of salivary epithelial cell (SEC) TLR-2 and TLR-4 in patients with generalized chronic periodontitis. MATERIAL AND METHODS: Unstimulated whole saliva (UWS) was collected from patients with generalized chronic periodontitis and from healthy individuals after obtaining informed consent. Epithelial cells isolated from each UWS sample were assessed for TLR-2, TLR-4, peptidoglycan recognition protein (PGRP)-3 and PGRP-4 by quantitative real-time PCR. In addition, the SECs were stimulated in vitro with microbial products for up to 24 h. The culture supernatant was assessed for cytokines by ELISA. RESULTS: Stimulation with TLR-2- or TLR-4-specific ligands induced cytokine secretion with differential kinetics and up-regulated TLR2 and TLR4 mRNAs, respectively, in cultures of SECs from patients with periodontitis. In addition, the SECs from patients with periodontitis exhibited reduced PGRP3 and PGRP4 mRNAs, the TLR-responsive genes with antibacterial properties. CONCLUSION: SECs derived from the UWS of patients with chronic periodontitis are phenotypically distinct and could represent potential resources for assessing the epithelial responses to periodontal pathogens in the course of disease progression and persistence.

Cancer in older patients: an analysis of elderly oncology.

Is it possible to define when someone is elderly? The worldwide population is growing not only in number but also in age; it is estimated that the population will increase to around 750 million by 2021. Two thirds of cancer occurs in the over 65 age groups. With an increasing elderly population, it can be derived that cancer will become a more prevalent condition. The burden of cancer on the medical profession will be even more apparent than before. In addition the elderly age group has different needs compared with younger oncology patients; there can be no 'rule of thumb' with the management of elderly illness. Factors such as frailty are significant when treating cancer in the older patients. The assessment of quality of life in older patients with cancer is also an important factor. Is it best for a patient to enjoy life as it is with cancer or aim for increased life expectancy by undertaking treatment with the threat of morbidity however severe during that period? The volume of scientific evidence currently available to support all the issues in geriatric oncology is greatly limited; almost all treatments designed for oncology are being tested in randomized clinical trials preferentially using younger cohorts of patients. Changes need to be made in order to further this field of medicine. Geriatric oncology is no longer a palliative field, as a healthy active life can now be expected by some older patients. The burden of oncology in the elderly will need to take a modern approach regarding the management of these patients.

Differential recognition of phosphorylated transactivation domains of p53 by different p300 domains.

Histone acetyltransferases form crucial links in transducing extrinsic signals to actual initiation of transcription. A multitude of stress signal integrations occur through the interaction of p300 with p53 phosphorylated at different residues of the transactivation domain. How such interactions activate different gene expression programs remains largely unknown. p300 contains at least five domains that are known to interact with p53, but their role in transcription regulation is not known. We measured the binding affinity of various phosphorylated transactivation domains towards several p53 binding domains of p300 by fluorescence anisotropy. The binding affinities of different phosphorylated transactivation domains of p53 towards different domains of p300 vary by several orders of magnitude, indicating that interactions of different post-translationally modified forms of p53 may occur through different domains of p300. Thus, different post-translationally modified p53 fragments may form transcription-initiating complexes of different configurations, leading to the activation of different promoters and pathways.

Physical characterization of thin semi-porous poly(L-lactic acid)/poly(ethylene glycol) membranes for tissue engineering.

This study examines physical properties of solvent-cast poly(L-lactic acid) (PLLA): poly(ethylene glycol) PEG membranes as a function of PEG molecular weight (MW) and incubation in vitro for 6 weeks. PEGs of MW 400, 1450 and 8000 were used. The morphological, thermal, mechanical and permeability properties of the membranes were studied prior to and after 3 and 6 weeks of incubation in phosphate-buffered saline (PBS) at 37 degrees C. The membranes showed a thickness of about 35+/-5 microm and were found to be semi-porous, with a non-porous surface as well as a porous surface with pore-diameters of 0.5-5 microm. The surface pore size was found to be a function of PEG MW used. All membranes were mechanically strong, with elastic moduli and tensile strength of 150-440 MPa and 7-36 MPa, respectively, all through the 6-week incubation period. The lower-MW PEGs plasticized PLLA based on high initial percent elongation; however, the effect was lost after 3 weeks of incubation in PBS. All membranes except those fabricated with PEG 8000 were impermeable for up to 6 weeks of incubation in PBS. Permeability studies showed that only PLLA:PEG 8000 membranes were permeable to methylene blue after 3 weeks of degradation.

Specific inhibition of p300-HAT alters global gene expression and represses HIV replication.

Reversible acetylation of histone and nonhistone proteins plays pivotal role in cellular homeostasis. Dysfunction of histone acetyltransferases (HATs) leads to several diseases including cancer, neurodegenaration, asthma, diabetes, AIDS, and cardiac hypertrophy. We describe the synthesis and characterization of a set of p300-HAT-specific small-molecule inhibitors from a natural nonspecific HAT inhibitor, garcinol, which is highly toxic to cells. We show that the specific inhibitor selectively represses the p300-mediated acetylation of p53 in vivo. Furthermore, inhibition of p300-HAT down regulates several genes but significantly a few important genes are also upregulated. Remarkably, these inhibitors were found to be nontoxic to T cells, inhibit histone acetylation of HIV infected cells, and consequently inhibit the multiplication of HIV.

Histone chaperones in chromatin dynamics: implications in disease manifestation.

Histone chaperones are the histone interacting factors that stimulate histone transfer reaction without being a part of the final product. They are involved in the histone storage, histone translocation to the nucleus, and histone exchange and histone deposition onto the DNA for replication dependent chromatin assembly. Interestingly, they have also been demonstrated to possess the histone removal activity. While the involvement of the histone chaperones in chromatin transcription is undisputed, the question of their local versus global involvement is under scrutiny. This review enumerates the role played by various histone chaperones in the establishment of chromatin structure and regulation of chromatin transcription. The role of histone chaperones in disease manifestation is not very clear, preliminary results with few histone chaperones suggest that expression and function of these factors dramatically alters in carcinogenesis. This review will also focus on the possible role of histone chaperones in cancer diagnosis and progression

Small molecule modulators in epigenetics: implications in gene expression and therapeutics.

Altered gene expression resulting from changes in the post-translational modification patterns of the histones and DNA is collectively termed epigenetics. Such changes are inherited albeit there are no alterations in the DNA sequence. Epigenetic regulation of gene expression is implemented by a wide repertoire of histone and DNA modifying enzymes including the acetyltransferases and deacetylases, the methyltransferases and kinases among others. Therefore, a regulation of these enzyme activities affords a tighter regulation of gene expression. Conversely, aberrant enzymatic activities lead to unregulated gene expression, resulting in several diseases such as RTS (loss of CBP HAT activity) and Spinal and Bulbar muscular atrophy (HATs and HMTases), apart from several forms of cancers, particularly myeloid leukemia (RAR-PML or RAR-PLZF fusion proteins resulting in the mistargeting of HDACs). Thus these enzymes have emerged as novel targets for the design of therapeutics. In this direction, several small molecule modulators (activators and inhibitors) of HATs, HDACs and HMTases are being reported in literature. This chapter introduces the different histone modifying enzymes involved in gene regulation, their connection to disease manifestation and focuses on the role of small molecule modulators in understanding enzyme function and also the design and the evolution of chromatin therapeutics

Human histone chaperone nucleophosmin enhances acetylation-dependent chromatin transcription.

Histone chaperones are a group of proteins that aid in the dynamic chromatin organization during different cellular processes. Here, we report that the human histone chaperone nucleophosmin interacts with the core histones H3, H2B, and H4 but that this histone interaction is not sufficient to confer the chaperone activity. Significantly, nucleophosmin enhances the acetylation-dependent chromatin transcription and it becomes acetylated both in vitro and in vivo. Acetylation of nucleophosmin and the core histones was found to be essential for the enhancement of chromatin transcription. The acetylated NPM1 not only shows an increased affinity toward acetylated histones but also shows enhanced histone transfer ability. Presumably, nucleophosmin disrupts the nucleosomal structure in an acetylation-dependent manner, resulting in the transcriptional activation. These results establish nucleophosmin (NPM1) as a human histone chaperone that becomes acetylated, resulting in the enhancement of chromatin transcription.

Implications of small molecule activators and inhibitors of histone acetyltransferases in chromatin therapy.

Histone acetylation is a diagnostic feature of transcriptionally active chromatin. The group of enzymes, histone acetyltransferases (HATs), involved in this crucial step of gene regulation, covalently modifies the N-terminal lysine residues of histones by the addition of an acetyl group from acetyl coenzyme A. Dysfunction of these enzymes is often associated with several diseases, ranging from neurodegenerative disorders to cancer. These enzymes thus are potential new targets for therapeutics. We have discovered few small molecule compounds, which target HATs and either activate or inhibit the enzyme potently. These compounds would be useful as biological switching molecules for probing into the role of HATs in gene regulation and cell cycle and may be useful as new chemical entities for the development of new drugs.

Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression.

Histone acetylation is a diagnostic feature of transcriptionally active genes. The proper recruitment and function of histone acetyltransferases (HATs) and deacetylases (HDACs) are key regulatory steps for gene expression and cell cycle. Functional defects of either of these enzymes may lead to several diseases, including cancer. HATs and HDACs thus are potential therapeutic targets. Here we report that garcinol, a polyisoprenylated benzophenone derivative from Garcinia indica fruit rind, is a potent inhibitor of histone acetyltransferases p300 (IC50 approximately 7 microm) and PCAF (IC50 approximately 5 microm) both in vitro and in vivo. The kinetic analysis shows that it is a mixed type of inhibitor with an increased affinity for PCAF compared with p300. HAT activity-dependent chromatin transcription was strongly inhibited by garcinol, whereas transcription from DNA template was not affected. Furthermore, it was found to be a potent inducer of apoptosis, and it alters (predominantly down-regulates) the global gene expression in HeLa cells.

Small molecule modulators of histone acetyltransferase p300.

Histone acetyltransferases (HATs) are a group of enzymes that play a significant role in the regulation of gene expression. These enzymes covalently modify the N-terminal lysine residues of histones by the addition of acetyl groups from acetyl-CoA. Dysfunction of these enzymes is often associated with the manifestation of several diseases, predominantly cancer. Here we report that anacardic acid from cashew nut shell liquid is a potent inhibitor of p300 and p300/CBP-associated factor histone acetyltranferase activities. Although it does not affect DNA transcription, HAT-dependent transcription from a chromatin template was strongly inhibited by anacardic acid. Furthermore, we describe the design and synthesis of an amide derivative N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) using anacardic acid as a synthon, which remarkably activates p300 HAT activity but not that of p300/CBP-associated factor. Although CTPB does not affect DNA transcription, it enhances the p300 HAT-dependent transcriptional activation from in vitro assembled chromatin template. However, it has no effect on histone deacetylase activity. These compounds would be useful as biological switching molecules for probing into the role of p300 in transcriptional studies and may also be useful as new chemical entities for the development of anticancer drugs.

Effect of phosphorylation on the structure and fold of transactivation domain of p53.

Several phosphorylations are known to occur in the N-terminal transactivation domain of human p53. To explore the structural effects of these phosphorylations, we have chemically synthesized the unphosphorylated p53-(1-39) and its three phosphorylated analogs, phosphorylated at Ser-15, Thr-18, and Ser-20. p53-(1-39) and its Ser-15 and Thr-18 phosphorylated analogs were tested for interaction with p300. The order of binding affinities was similar to that derived from biochemical experiments with the whole protein, indicating functional integrity of the domain. Differences in chemical shifts and coupling constants indicate significant structural changes upon phosphorylations. The single tryptophan in the unphosphorylated domain has an emission maximum and a Stern-Volmer constant that are characteristics of tryptophans situated in protein interiors. The diffusion constant is monomer-like, with an axial ratio of 1:7.5, indicating a significant degree of compaction. Upon phosphorylations, the emission maximum and diffusion constant change significantly toward values that indicate more open conformations. Binding of the hydrophobic probe bis-1-anilino-8-naphthalenesulfonate to the unphosphorylated and one of the phosphorylated domains is also significantly different, suggesting different conformations. We propose that phosphorylations switch the largely folded transactivation domain to more open conformations that interact with transcription factors such as p300/cAMP- responsive element-binding protein-binding protein, leading to enhancement of gene expression.