Regulation of human endogenous retrovirus-K expression in melanomas by CpG methylation

The overall prognosis of patients with advanced melanoma is poor due to the lack of effective treatment. A key factor for successful therapy is an early detection of disease. Therefore, reliably detection methods and meaningful tumor markers are required. Expression of the human endogenous retrovirus (HERV)-K(HML-2) was found elevated in melanomas and it was shown that HERV-K supports the in vitro transition of melanoma cells from adherent to a more malignant, nonadherent phenotype. Furthermore, the detection of HERV-K-specific antibodies in melanoma patients was found to correlate with reduced survival. However, the reason for HERV-K expression in melanomas still remains unclear and its use as a tumor marker needs further investigation. Therefore, the tumor-specific transcriptional regulation of HERV-K expression in melanoma was studied in detail. Human melanoma cell lines were investigated for HERV-K expression using real-time PCR. Five cell lines showed very high levels of HERV-K mRNA as a result of increased promoter activity. This promoter activity was directly silenced by DNA methylation in reporter gene experiments. Higher levels of long terminal repeat (LTR) methylation in cells not expressing HERV-K compared with cells expressing HERV-K were found using methylation-sensitive PCR and bisulfite sequencing. Treatment of cell lines with the demethylating agent 5-aza-2′-deoxycytidine resulted in increased levels of HERV-K expression in cells previously not expressing HERV-K and it was shown that this increase is not the result of transcription factor activation. These results demonstrate that increased HERV-K expression in melanomas may be due to increased promoter activity and demethylation of the 5′LTR

Engineering Waveguide Nonlinear Effective Length via Low Index Thin Films

Novel photonic nanowires were fabricated using low-index materials and tested in the near-infrared spectrum to assess their nonlinear optical properties. In this work, we argue the need to redefine the standard nonlinear figure of merit in terms of nonlinear phase shift and optical transmission for a given propagation distance. According to this new metric, our devices largely outperform all established platforms for devices with a linear footprint in the range of 50 to 500 um, which is demonstrated to be an outstanding technological gap. For 85 fs pulses, with carrier wavelength at 1480nm and sub-uW power levels, a spectral broadening exceeding 80% of the initial bandwidth was recorded over a propagation length of just 50 um. Leveraging on CMOS-compatible processes and well-established materials such as silicon, silica, and indium tin oxide, our devices bring great promise for developing alternative all-optical devices with unparalleled nonlinear performances within the aforementioned range

Retinoic Acid and Its Derivatives in Skin

The retinoids are a group of compounds including vitamin A and its active metabolite all-trans-retinoic acid (ATRA). Retinoids regulate a variety of physiological functions in multiple organ systems, are essential for normal immune competence, and are involved in the regulation of cell growth and differentiation. Vitamin A derivatives have held promise in cancer treatment and ATRA is used in differentiation therapy of acute promyelocytic leukemia (APL). ATRA and other retinoids have also been successfully applied in a variety of dermatological conditions such as skin cancer, psoriasis, acne, and ichthyosis. Moreover, modulation of retinoic acid receptors and retinoid X (or rexinoid) receptors function may affect dermal cells. The studies using complex genetic models with various combinations of retinoic acid receptors (RARs) and retinoid X (or rexinoid) receptors (RXRs) indicate that retinoic acid and its derivatives have therapeutic potential for a variety of serious dermatological disorders including some malignant conditions. Here, we provide a synopsis of the main advances in understanding the role of ATRA and its receptors in dermatology

Deregulated expression of HDAC9 in B cells promotes development of lymphoproliferative disease and lymphoma in mice

Histone deacetylase 9 (HDAC9) is expressed in B cells, and its overexpression has been observed in B-lymphoproliferative disorders, including B-cell non-Hodgkin lymphoma (B-NHL). We examined HDAC9 protein expression and copy number alterations in primary B-NHL samples, identifying high HDAC9 expression among various lymphoma entities andHDAC9copy number gains in 50% of diffuse large B-cell lymphoma (DLBCL). To study the role of HDAC9 in lymphomagenesis, we generated a genetically engineered mouse (GEM) model that constitutively expressed anHDAC9transgene throughout B-cell development under the control of the immunoglobulin heavy chain (IgH) enhancer (Eμ). Here, we report that theEμ-HDAC9GEM model develops splenic marginal zone lymphoma and lymphoproliferative disease (LPD) with progression towards aggressive DLBCL, with gene expression profiling supporting a germinal center cell origin, as is also seen in human B-NHL tumors. Analysis ofEμ-HDAC9tumors suggested that HDAC9 might contribute to lymphomagenesis by altering pathways involved in growth and survival, as well as modulating BCL6 activity and p53 tumor suppressor function. Epigenetic modifications play an important role in the germinal center response, and deregulation of the B-cell epigenome as a consequence of mutations and other genomic aberrations are being increasingly recognized as important steps in the pathogenesis of a variety of B-cell lymphomas. A thorough mechanistic understanding of these alterations will inform the use of targeted therapies for these malignancies. These findings strongly suggest a role for HDAC9 in B-NHL and establish a novel GEM model for the study of lymphomagenesis and, potentially, preclinical testing of therapeutic approaches based on histone deacetylase inhibitors

Mindfulness and skills-based eHealth intervention to reduce distress in cancer-affected patients in the Reduct trial: Intervention protocol of the make it training optimized.

Introduction Cancer-affected patients experience high distress due to various burdens. One way to expand psycho-oncological support is through digital interventions. This protocol describes the development and structure of a web-based psycho-oncological intervention, the Make It Training optimized. This intervention is currently evaluated in the Reduct trial, a multicenter randomized controlled trial. Methods The Make It Training optimized was developed in six steps: A patient need and demand assessment, development and acceptability analysis of a prototype, the formation of a patient advisory council, the revision of the training, implementation into a web app, and the development of a motivation and evaluation plan. Results Through a process of establishing cancer-affected patients' needs, prototype testing, and patient involvement, the Make It Training optimized was developed by a multidisciplinary team and implemented in a web app. It consists of 16 interactive self-guided modules which can be completed within 16 weeks. Discussion Intervention protocols can increase transparency and increase the likelihood of developing effective web-based interventions. This protocol describes the process and results of developing a patient-oriented intervention. Future research should focus on the further personalization of web-based psycho-oncological interventions and the potential benefits of combining multiple psychotherapeutic approaches

Candida albicans β-Glucan Differentiates Human Monocytes Into a Specific Subset of Macrophages

β-Glucan derived from cell walls of Candida albicans is a potent immune modulator. It has been shown to induce trained immunity in monocytes via epigenetic and metabolic reprogramming and to protect from lethal sepsis if applied prior to infection. Since β-glucan-trained monocytes have not been classified within the system of mononuclear phagocytes we analyzed these cells metabolically, phenotypically and functionally with a focus on monocyte-to-macrophage differentiation and compared them with naïve monocytes and other types of monocyte-derived cells such as classically (M1) or alternatively (M2) activated macrophages and monocyte-derived dendritic cells (moDCs). We show that β-glucan inhibits spontaneous apoptosis of monocytes independent from autocrine or paracrine M-CSF release and stimulates monocyte differentiation into macrophages. β-Glucan-differentiated macrophages exhibit increased cell size and granularity and enhanced metabolic activity when compared to naïve monocytes. Although β-glucan-primed cells expressed markers of alternative activation and secreted higher levels of IL-10 after lipopolysaccharide (LPS), their capability to release pro-inflammatory cytokines and to kill bacteria was unaffected. Our data demonstrate that β-glucan priming induces a population of immune competent long-lived monocyte-derived macrophages that may be involved in immunoregulatory processes

Suppression of MYC by PI3K/AKT/mTOR pathway inhibition in combination with all-trans retinoic acid treatment for therapeutic gain in acute myeloid leukaemia.

Aberrant activity of the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR [PAM]) pathway, as well as suppressed retinoic acid signalling, contribute to enhanced proliferation and the differentiation blockade of immature myeloid cells in acute myeloid leukaemia (AML). Inhibition of the PAM pathway was shown to affect especially mixed-lineage leukaemia-rearranged AML. Here, we sought to test a combined strategy using small molecule inhibitors against members of the PAM signalling pathway in conjunction with all-trans retinoic acid (ATRA) to target a larger group of different AML subtypes. We find that ATRA treatment in combination with inhibition of PI3K (ZSTK474), mTOR (WYE132) or PI3K/mTOR (BEZ235, dactolisib) drastically reduces protein levels of the proto-oncogene MYC. In combination with BEZ235, ATRA treatment led to almost complete eradication of cellular MYC, G1 arrest, loss of clonal capacity and terminal granulocytic differentiation. We demonstrate that PAM inhibitor/ATRA treatment targets MYC via independent mechanisms. While inhibition of the PAM pathway causes MYC phosphorylation at threonine 58 via glycogen synthase kinase 3 beta and subsequent degradation, ATRA reduces its expression. Here, we present an approach using a combination of known drugs to synergistically reduce aberrant MYC levels, thereby effectively blocking proliferation and enabling differentiation in various AML subtypes

Resorbable screws versus pins for optimal transplant fixation (SPOT) in anterior cruciate ligament replacement with autologous hamstring grafts: rationale and design of a randomized, controlled, patient and investigator blinded trial [ISRCTN17384369]

BACKGROUND: Ruptures of the anterior cruciate ligament (ACL) are common injuries to the knee joint. Arthroscopic ACL replacement by autologous tendon grafts has established itself as a standard of care. Data from both experimental and observational studies suggest that surgical reconstruction does not fully restore knee stability. Persisting anterior laxity may lead to recurrent episodes of giving-way and cartilage damage. This might at least in part depend on the method of graft fixation in the bony tunnels. Whereas resorbable screws are easy to handle, pins may better preserve graft tension. The objective of this study is to determine whether pinning of ACL grafts reduces residual anterior laxity six months after surgery as compared to screw fixation. DESIGN/ METHODS: SPOT is a randomised, controlled, patient and investigator blinded trial conducted at a single academic institution. Eligible patients are scheduled to arthroscopic ACL repair with triple-stranded hamstring grafts, conducted by a single, experienced surgeon. Intraoperatively, subjects willing to engage in this study will be randomised to transplant tethering with either resorbable screws or resorbable pins. No other changes apply to locally established treatment protocols. Patients and clinical investigators will remain blinded to the assigned fixation method until the six-month follow-up examination. The primary outcome is the side-to-side (repaired to healthy knee) difference in anterior translation as measured by the KT-1000 arthrometer at a defined load (89 N) six months after surgery. A sample size of 54 patients will yield a power of 80% to detect a difference of 1.0 mm ± standard deviation 1.2 mm at a two-sided alpha of 5% with a t-test for independent samples. Secondary outcomes (generic and disease-specific measures of quality of life, magnetic resonance imaging morphology of transplants and devices) will be handled in an exploratory fashion. CONCLUSION: SPOT aims at showing a reduction in anterior knee laxity after fixing ACL grafts by pins compared to screws

Abstract 1363: Inhibition of the PI3K/AKT/mTOR Pathway Leads to Down-Regulation of c-Myc and Overcomes Resistance to ATRA in Acute Myeloid Leukemia.

Acute Promyelocytic Leukemia (APL) accounts for 5% of all cases of acute myeloid leukemia (AML). This disease is highly curable with all-trans-retinoic acid (ATRA) based therapy. In non-APL AML, ATRA has limited activity, and little is known about mechanisms of ATRA resistance. The apparent selective efficacy of ATRA in PML/RARα-associated APL poses an important question as to whether the presence of this fusion protein renders APL uniquely susceptible. Two compelling arguments can be made to counter this view. First, experiments in vitro show that ATRA effectively differentiates HL-60 cell lines, which lack the PML/RARα fusion protein. Second, clinical studies with ATRA in previously untreated older AML patients (excluding APL) have reported clinical activity. These observations confirm the therapeutic potential of ATRA beyond APL. In this context, our group has previously identified the lysine demethylase LSD-1, as a therapeutic target to re-sensitize leukemic blasts to ATRA. A clinical investigation of ATRA combined with LSD-1 inhibition is currently underway (NCT02273102). It is likely that other defects leading to ATRA resistance will be similarly amenable to pharmacologic manipulation. Defects in the proto-oncogene c-Myc have been widely implicated in the initiation and maintenance of AML. Over-expression of c-Myc in leukemic blasts enhances clonogenic survival and blocks ATRA induced differentiation. We hypothesized that down-regulation of c-Myc might increase the anti-leukemic effects of ATRA in AML. To date, c-Myc has been an evasive target for direct pharmacologic inhibition however, inhibitors of the PI3K/AKT/mTOR pathway have been shown to indirectly lower levels of c-Myc in leukemic blasts. In the current study, we show that the pro-differentiation effects of ATRA are markedly potentiated when combined with agents that target PI3K/AKT/mTOR signalling. In AML cell lines and primary patient samples, we observed additive pro-differentiation effects when ATRA was combined with inhibitors of PI3K (ZSTK474) and mTOR complex proteins (Torin-1, WYE-125132). However, when combined with the bromodomain inhibitor NVP-BEZ235, a dual inhibitor of PI3K and mTOR, we observed synergistic induction of CD11b by FACS analysis. Combination studies revealed loss of cell viability, cell cycle arrest in G1 phase, and impaired clonogenic survival, which was more prominent for ATRA combination treatments than with any agent used alone (Figure 1). To assess the role of c-Myc in mediating these effects, we measured c-Myc protein levels and PI3K/AKt/mTOR pathway markers at different time-points following treatment with ATRA alone and in combination with the inhibitors described above (Figure 2). Our findings suggest that ATRA alone quickly down-regulates c-Myc (within 6 hours) through transcriptional repression. Disruption of the PI3K/AKT/mTOR pathway further down-regulates c-Myc (within 3 hours) through destabilization and enhanced degradation. ATRA combined with NVP-BEZ235 produced maximal c-Myc suppression, and led to more cell kill than any other combination tested. Detailed analysis of changes in the transcriptome in MV-411 cells following treatment with ATRA and NVP-BEZ235 revealed that both agents act jointly on the regulation of the same biological pathways and processes, but regulate different sets of genes within these pathways. Updated mechanism based studies will be presented. In conclusion, suppression of c-Myc levels through disruption of PI3K/AKT/mTOR signalling augments the anti-leukemic effects of ATRA. These data support the clinical investigation of ATRA combined with rapalogs or bromodomain inhibitors

Detection of Crosslinks within and between Proteins by LC-MALDI-TOFTOF and the Software FINDX to Reduce the MSMS-Data to Acquire for Validation

Lysine-specific chemical crosslinking in combination with mass spectrometry is emerging as a tool for the structural characterization of protein complexes and protein-protein interactions. After tryptic digestion of crosslinked proteins there are thousands of peptides amenable to MSMS, of which only very few are crosslinked peptides of interest. Here we describe how the advantage offered by off-line LC-MALDI-TOF/TOF mass spectrometry is exploited in a two-step workflow to focus the MSMS-acquisition on crosslinks mainly. In a first step, MS-data are acquired and all the peak list files from the LC-separated fractions are merged by the FINDX software and screened for presence of crosslinks which are recognized as isotope-labeled doublet peaks. Information on the isotope doublet peak mass and intensity can be used as search constraints to reduce the number of false positives that match randomly to the observed peak masses. Based on the MS-data a precursor ion inclusion list is generated and used in a second step, where a restricted number of MSMS-spectra are acquired for crosslink validation. The decoupling of MS and MSMS and the peptide sorting with FINDX based on MS-data has the advantage that MSMS can be restricted to and focused on crosslinks of Type 2, which are of highest biological interest but often lowest in abundance. The LC-MALDI TOF/TOF workflow here described is applicable to protein multisubunit complexes and using 14N/15N mixed isotope strategy for the detection of inter-protein crosslinks within protein oligomers