Curcumin Intake Affects miRNA Signature in Murine Melanoma with mmu-miR-205-5p Most Significantly Altered

Melanoma is the most aggressive form of skin cancer with estimated 48,000 deaths per year worldwide. The polyphenol curcumin derived from the plant Curcuma longa is well known for its anti-inflammatory and anti-cancerogenic properties. Accordingly, dietary intake of this compound may be suitable for melanoma prevention. However, how this compound affects basic cellular mechanisms in developing melanoma still remains elusive. Therefore, the aim of this study was to investigate for the first time the impact of oral curcumin administration on the miRNA signature of engrafting melanoma. For this purpose, the effects of a 4% curcumin diet were tested on melanoma, which were established by injection of murine B78H1 cells in the flank of C57BL/6 mice. Curcumin diet or standard chow (control) was administered two weeks prior to injection of tumor cells until termination of the experiment. High throughput chip-based array analysis was deployed to detect alterations in the miRNA signature of the tumors. Curcumin treatment significantly reduced the growth of the flank tumors. Furthermore the miRNA expression signature in tumors was substantially altered by curcumin intake with mmu-miR-205-5p over 100 times higher expressed when compared to controls. The expression levels of identified key miRNAs in the tumor samples were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). A comparable expression pattern of these miRNAs was also detected in other curcumin-treated melanoma cell lines under in vitro conditions. Putative targets of curcumin-induced up-regulated miRNAs were enriched in ‘o-glycan biosynthesis’, ‘endoplasmatic reticulum protein processing’ and different cancer-related pathways. Western Blot analyses revealed that of these targets anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were significantly down-regulated in curcumin-treated tumors. These findings demonstrate a profound alteration of the miRNA expression signature in engrafting curcumin-treated melanoma with mmu-miR-205-5p being up-regulated most significantly.     

High-Field Open versus Short-Bore Magnetic Resonance Imaging of the Spine: A Randomized Controlled Comparison of Image Quality

Background

The purpose of the present study was to compare the image quality of spinal magnetic resonance (MR) imaging performed on a high-field horizontal open versus a short-bore MR scanner in a randomized controlled study setup.

Methods

Altogether, 93 (80% women, mean age 53) consecutive patients underwent spine imaging after random assignement to a 1-T horizontal open MR scanner with a vertical magnetic field or a 1.5-T short-bore MR scanner. This patient subset was part of a larger cohort. Image quality was assessed by determining qualitative parameters, signal-to-noise (SNR) and contrast-to-noise ratios (CNR), and quantitative contour sharpness.

Results

The image quality parameters were higher for short-bore MR imaging. Regarding all sequences, the relative differences were 39% for the mean overall qualitative image quality, 53% for the mean SNR values, and 34–37% for the quantitative contour sharpness (P<0.0001). The CNR values were also higher for images obtained with the short-bore MR scanner. No sequence was of very poor (nondiagnostic) image quality. Scanning times were significantly longer for examinations performed on the open MR scanner (mean: 32±22 min versus 20±9 min; P<0.0001).

Conclusions

In this randomized controlled comparison of spinal MR imaging with an open versus a short-bore scanner, short-bore MR imaging revealed considerably higher image quality with shorter scanning times.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00715806","term_id":"NCT00715806"}}NCT00715806     

The Mitochondrial Genome of Arctica islandica; Phylogeny and Variation

Arctica islandica is known as the longest-lived non-colonial metazoan species on earth and is therefore increasingly being investigated as a new model in aging research. As the mitochondrial genome is associated with the process of aging in many species and bivalves are known to possess a peculiar mechanism of mitochondrial genome inheritance including doubly uniparental inheritance (DUI), we aimed to assess the genomic variability of the A. islandica mitochondrial DNA (mtDNA). We sequenced the complete mitochondrial genomes of A. islandica specimens from three different sites in the Western Palaearctic (Iceland, North Sea, Baltic Sea). We found the A. islandica mtDNA to fall within the normal size range (18 kb) and exhibit similar coding capacity as other animal mtDNAs. The concatenated protein sequences of all currently known Veneroidea mtDNAs were used to robustly place A. islandica in a phylogenetic framework. Analysis of the observed single nucleotide polymorphism (SNP) patterns on further specimen revealed two prevailing haplotypes. Populations in the Baltic and the North Sea are very homogenous, whereas the Icelandic population, from which exceptionally old individuals have been collected, is the most diverse one. Homogeneity in Baltic and North Sea populations point to either stronger environmental constraints or more recent colonization of the habitat. Our analysis lays the foundation for further studies on A. islandica population structures, age research with this organism, and for phylogenetic studies. Accessions for the mitochondrial genome sequences: KC197241 Iceland; KF363951 Baltic Sea; KF363952 North Sea; KF465708 to KF465758 individual amplified regions from different speciemen     

Exchange of Cytosolic Content between T Cells and Tumor Cells Activates CD4 T Cells and Impedes Cancer Growth

Background

T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described.

Methods/ Findings

Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC) cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes – even in CD4⁺ T cells and murine B cells – which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement.Electron microscopy disclosed 100-200nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL) model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice.

Conclusions

The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.     

Podoplanin Immunopositive Lymphatic Vessels at the Implant Interface in a Rat Model of Osteoporotic Fractures

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing.     

Associations between Lifetime Traumatic Events and Subsequent Chronic Physical Conditions: A Cross-National,Cross-Sectional Study

Background

Associations between lifetime traumatic event (LTE) exposures and subsequent physical ill-health are well established but it has remained unclear whether these are explained by PTSD or other mental disorders. This study examined this question and investigated whether associations varied by type and number of LTEs, across physical condition outcomes, or across countries.

Methods

Cross-sectional, face-to-face household surveys of adults (18+) were conducted in 14 countries (n = 38, 051). The Composite International Diagnostic Interview assessed lifetime LTEs and DSM-IV mental disorders. Chronic physical conditions were ascertained by self-report of physician''s diagnosis and year of diagnosis or onset. Survival analyses estimated associations between the number and type of LTEs with the subsequent onset of 11 physical conditions, with and without adjustment for mental disorders.

Findings

A dose-response association was found between increasing number of LTEs and odds of any physical condition onset (OR 1.5 [95% CI: 1.4–1.5] for 1 LTE; 2.1 [2.0–2.3] for 5+ LTEs), independent of all mental disorders. Associations did not vary greatly by type of LTE (except for combat and other war experience), nor across countries. A history of 1 LTE was associated with 7/11 of the physical conditions (ORs 1.3 [1.2–1.5] to 1.7 [1.4–2.0]) and a history of 5+ LTEs was associated with 9/11 physical conditions (ORs 1.8 [1.3–2.4] to 3.6 [2.0–6.5]), the exceptions being cancer and stroke.

Conclusions

Traumatic events are associated with adverse downstream effects on physical health, independent of PTSD and other mental disorders. Although the associations are modest they have public health implications due to the high prevalence of traumatic events and the range of common physical conditions affected. The effects of traumatic stress are a concern for all medical professionals and researchers, not just mental health specialists.     

An RNAi-Based Approach to Down-Regulate a Gene Family In Vivo

Genetic redundancy poses a major problem to the analysis of gene function. RNA interference allows the down-regulation of several genes simultaneously, offering the possibility to overcome genetic redundancy, something not easily achieved with traditional genetic approaches. Previously we have used a polycistronic miR155-based framework to knockdown expression of three genes of the early B cell factor family in cultured cells. Here we develop the system further by generating transgenic mice expressing the RNAi construct in vivo in an inducible manner. Expression of the transgene from the strong CAG promoter is compatible with a normal function of the basal miRNA/RNAi machinery, and the miR155 framework readily allows inducible expression from the Rosa26 locus as shown by Gfp. However, expression of the transgene in hematopoietic cells does not lead to changes in B cell development and neuronal expression does not affect cerebellar architecture as predicted from genetic deletion studies. Protein as well as mRNA levels generated from Ebf genes in hetero- and homozygous animals are comparable to wild-type levels. A likely explanation for the discrepancy in the effectiveness of the RNAi construct between cultured cells and transgenic animals lies in the efficiency of the sequences used, possibly together with the complexity of the transgene. Since new approaches allow to overcome efficiency problems of RNAi sequences, the data lay the foundation for future work on the simultaneous knockdown of several genes in vivo.     

Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).     

MRI Plaque Imaging Detects Carotid Plaques with a High Risk for Future Cerebrovascular Events in Asymptomatic Patients

Purpose

The aim of this study was to investigate prospectively whether MRI plaque imaging can identify patients with asymptomatic carotid artery stenosis who have an increased risk for future cerebral events. MRI plaque imaging allows categorization of carotid stenosis into different lesion types (I–VIII). Within these lesion types, lesion types IV–V and VI are regarded as rupture-prone plaques, whereas the other lesion types represent stable ones.

Methods

Eighty-three consecutive patients (45 male (54.2%); age 54–88 years (mean 73.2 years)) presenting with an asymptomatic carotid stenosis of 50–99% according to ECST-criteria were recruited. Patients were imaged with a 1.5-T scanner. T1-, T2-, time-of-flight-, and proton-density weighted studies were performed. The carotid plaques were classified as lesion type I–VIII. Clinical endpoints were ischemic stroke, TIA or amaurosis fugax. Survival analysis and log rank test were used to ascertain statistical significance.

Results

Six out of 83 patients (7.2%) were excluded: 4 patients had insufficient MR image quality; 1 patient was lost-to-follow-up; 1 patient died shortly after the baseline MRI plaque imaging. The following results were obtained by analyzing the remaining 77 patients. The mean time of follow-up was 41.1 months.During follow-up, n = 9 (11.7%) ipsilateral ischemic cerebrovascular events occurred. Only patients presenting with the high-risk lesion types IV–V and VI developed an ipsilateral cerebrovascular event versus none of the patients presenting with the stable lesion types III, VII, and VIII (n = 9 (11.7%) vs. n = 0 (0%) during follow-up). Event-free survival was higher among patients with the MRI-defined stable lesion types (III, VII, and VIII) than in patients with the high-risk lesion types (IV–V and VI) (log rank test P<0.0001).

Conclusions

MRI plaque imaging has the potential to identify patients with asymptomatic carotid stenosis who are particularly at risk of developing future cerebral ischemia. MRI could improve selection criteria for invasive therapy in the future.     

Expression,Purification and Low-Resolution Structure of Human Vitamin C Transporter SVCT1 (SLC23A1)

Expression and purification of human membrane proteins for structural studies represent a great challenge. This is because micro- to milligram amounts of pure isolated protein are required. To this aim, we successfully expressed the human vitamin C transporter-1 (hSVCT1; SLC23A1) in Xenopus laevis oocytes and isolated highly pure protein in microgram amounts. Recombinant hSVCT1 was functional when expressed in oocytes and glycosylated. Structural analysis of purified hSVCT1 by transmission electron microscopy and single particle analysis unveiled its shape, dimensions and low-resolution structure as well as the existence of a major monomeric and minor dimeric population. Chemical crosslinking of isolated oocyte membranes containing expressed hSVCT1 indicated similar oligomeric states of hSVCT1 in lipid bilayers. This work reports the first purification and structural analysis of a human SVCT protein and opens the way for future functional and structural studies using purified hSVCT1.