Prenatal stress programs lipid metabolism enhancing cardiovascular risk in the female F1, F2, and F3 generation in the primate model common marmoset (Callithrix jacchus)

Background Many human diseases are modulated by intrauterine environment, which is called prenatal programming. This study investigated effects of prenatal glucocorticoids on the lipid metabolism of three filial generations of common marmosets. Methods Pregnant primates were treated with dexamethasone during pregnancy. Body weight and blood lipid parameters of adult female offspring (F1: n = 5, F2: n = 6, F3: n = 3) were compared with age‐related female controls (n = 12). Results F1, F2, and F3 offspring showed significantly lower percentage of plasma n3 fatty acids than controls. F2 and F3 presented higher cholesterol levels, with significantly more LDL cholesterol, significantly less HDL triglycerides and an enhanced cholesterol/HDL cholesterol ratio. Body weight was not significantly affected. Conclusions Prenatal dexamethasone led to higher amounts of cardiovascular risk factors and less protective parameters in female F1–F3 offspring. The intergenerational consequences suggest prenatal programming through epigenetic effects.     

Expression of BMP-receptor type 1A correlates with progress of osteoarthritis in human knee joints with focal cartilage lesions

Background aimsBone morphogenetic protein-2 (BMP-2) and its receptor type 1A (BMPR-1A) play significant roles in cartilage metabolism. The aim of this study was to evaluate a possible correlation between intra-articular expression of these proteins and the degree of osteoarthritis (OA) in human knees.MethodsBiopsies of synovia and debrided cartilage were taken in 15 patients undergoing autologous chondrocyte implantation. Expression of BMP-2 and BMPR-1A was evaluated semi-quantitatively by immunohistologic staining. These data were complemented by grading of cartilage lesions according to International Cartilage Repair Society (ICRS), defect size, duration of complaints, knee osteoarthritis scoring system (KOSS) and Henderson and Kellgren–Lawrence scores. General histologic stainings were used to determine Mankin, Pritzker and Krenn scores.ResultsThe expression of BMPR-1A but not of BMP-2 was significantly higher in cartilage biopsies taken in type 3 lesions with intact subchondral layer compared with type 4 defects (P < 0.05). In cartilage areas of bordering sectors, the intensity of immunohistologic staining of BMPR-1A was statistically significantly higher in mature cartilage compared with repair zones (P < 0.05). Expression of BMP-2 and its receptor 1A correlated in the cartilage biopsies (P < 0.02) but not in the synovia. The degree of OA was scored in all biopsies according to Mankin and Pritzker, and these scores correlated statistically significantly with BMPR-1A expression in the synovia (P < 0.05). In patients with an osteochondritis dissecans, the degree of OA was higher compared with other causes of chondromalacia, as evaluated by defect size, ICRS score, duration of complaints, Pritzker score and expression of BMPR-1A in cartilage (P < 0.05).ConclusionsThese data support the role of BMPR-1A as an indicator of OA progression in human knees with circumscribed cartilage lesions.     

Global microRNA level regulation of EGFR‐driven cell‐cycle protein network in breast cancer

The EGFR‐driven cell‐cycle pathway has been extensively studied due to its pivotal role in breast cancer proliferation and pathogenesis. Although several studies reported regulation of individual pathway components by microRNAs (miRNAs), little is known about how miRNAs coordinate the EGFR protein network on a global miRNA (miRNome) level. Here, we combined a large‐scale miRNA screening approach with a high‐throughput proteomic readout and network‐based data analysis to identify which miRNAs are involved, and to uncover potential regulatory patterns. Our results indicated that the regulation of proteins by miRNAs is dominated by the nucleotide matching mechanism between seed sequences of the miRNAs and 3′‐UTR of target genes. Furthermore, the novel network‐analysis methodology we developed implied the existence of consistent intrinsic regulatory patterns where miRNAs simultaneously co‐regulate several proteins acting in the same functional module. Finally, our approach led us to identify and validate three miRNAs (miR‐124, miR‐147 and miR‐193a‐3p) as novel tumor suppressors that co‐target EGFR‐driven cell‐cycle network proteins and inhibit cell‐cycle progression and proliferation in breast cancer.     

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.     

Sulfur-36S stable isotope labeling of amino acids for quantification (SULAQ)

We introduce a universal metabolic labeling strategy using elemental heavy 36Sulfur (36S) called 36Sulfur stable isotope labeling of amino acids for quantification (SULAQ). In the proof of principle experiment, Pseudomonas putida KT2440 was grown in defined minimal medium with sodium benzoate or sodium succinate as the sole carbon and 32S- or 36S-sodium sulfate as the sole sulfur sources. Quantification using mass spectrometry resulted in 562 proteins with 1991 unique peptides. SULAQ technology can be a valuable alternative strategy for the quantitative comparisons in MS-based proteomics approaches characterizing bacterial and other biological samples in different growth conditions.     

Helicascolide C,a new lactone from an Indonesian marine algicolous strain of Daldinia eschscholzii (Xylariaceae,Ascomycota)

From an endophytic Daldinia eschscholzii strain isolated from the agar-producing red alga Gracilaria sp. SGR-1, collected from the coast of South Sulawesi, Indonesia, a new lactone helicascolide C (1) was obtained as colourless crystals from the ethyl acetate extract together with the related structurally known compound helicascolide A (2). The structure of the new compound 1 reveals a carbonyl group replacing an alcohol group of compound 2. The structure of 1 was elucidated by X-ray diffraction and spectral analyses. Compound 1 showed fungistatic activity against the phytopathogenic fungus Cladosporium cucumerinum.     

Synthesis, hydrolytic activation and cytotoxicity of etoposide prodrugs

Two 4'-propylcarbonoxy derivatives (2,3) of etoposide (1), a topoisomerase II inhibitor, were synthesized and evaluated as potential prodrugs for anticancer therapy. Their activation via hydrolysis mechanisms was determined as a function of pH in buffer solutions, in human serum and in the presence of carboxyl ester hydrolase. Cytotoxicity was determined on various tumor cell lines and compared to the parent compound. On cell lines exhibiting resistance to etoposide we observed an enhanced cytotoxicity of the prodrugs of up to three orders of magnitude.     

Comparison of different IMAC techniques used for enrichment of phosphorylated peptides.

Four commercially available immobilized metal ion affinity chromatography (IMAC) methods for phosphopeptide enrichment were compared using small volumes and concentrations of phosphopeptide mixtures with or without extra-added bovine serum albumin (BSA) nonphosphorylated peptides. Addition of abundant tryptic BSA peptides to the phosphopeptide mixture increases the demand for selective IMAC capture. While SwellGel gallium Discs, IPAC Metal Chelating Resin, and ZipTipMC Pipette Tips allow for the possibility of enriching phosphopeptides, the Gyrolab MALDI IMAC1 also presents the possibility of verifying existing phosphopeptides after a dephosphorylation step. Phosphate-containing peptides are identified through a mass shift between phosphorylated and dephosphorylated spectra of 80 Da (or multiples of 80 Da). This verification is useful if the degree of phosphorylation is low in the sample or if the ionization is unfavorable, which often is the case for phosphopeptides. A peptide mixture in which phosphorylated serine, threonine, and tyrosine were represented was diluted in steps and thereafter enriched using the four different IMAC methods prior to analyses with matrix assisted laser desorption/ionization mass spectrometry. The enrichment of phosphopeptides using SwellGel Gallium Discs or Gyrolab MALDI IMAC1 was not significantly affected by the addition of abundant BSA peptides added to the sample mixture, and the achieved detection limits using these techniques were also the lowest. All four of the included phosphopeptides were detected by MALDI-MS only after enrichment using the Gyrolab MALDI IMAC1 compact disc (CD) and detection down to low femtomole levels was possible. Furthermore, selectivity, reproducibility, and detection for a number of other phosphopeptides using the IMAC CD are reported herein. For example, two phosphopeptides sent out in a worldwide survey performed by the Proteomics Research Group (PRG03) of the Association of Biomolecular Resource Facilities (ABRF) were detected and verified by means of the 80 Da mass shift achieved by on-column dephosphorylation.