Characterization of texturized meat analogues containing native lupin flour and lupin protein concentrate/isolate.

Lupin is a nutritious, yet undervalued grain used as a fodder and food crop. In the present study, native lupin flour (LF), lupin protein concentrate (LPC), and lupin protein isolate (LPI) were combined (70% LPI:LPC blend ratios [30:70, 50:50, and 70:30] and 30% LF constant fraction), extruded at high moisture (45-55%), and shaped with a long cooling die (800 mm) to obtain texturized meat analogues (TMAs) with fibrous structures. The characteristics of TMAs (e.g., hardness, water hydration capacity) depended heavily on water content, blend ratios (LPI:LPC), and to a lesser extent, the long cooling die temperature. Color changes (i.e., L*, b*) were mostly attributed to variations in blend ratios (LPI:LPC). Microstructure analysis showed that TMAs with higher water content (55%) were more likely to have thinner walls and smaller void thickness. Fluorescence imagery revealed that TMAs with lower LPI content presented more homogeneous structures. These findings show that reasonable amounts (30% d.m.) of native lupin flour can be incorporated into meat analogues by maintaining a sufficiently high protein content (>50% d.m.) to trigger the formation of fibrous structures.

Functionality of oat fiber concentrate and faba bean protein concentrate in plant-based substitutes for minced meat.

Oat has been recognized for its health-promoting fiber, ß-glucan, while protein-rich faba bean has remained underutilized in Nordic countries despite its good nutritional quality. This research investigated the functionality of oat fiber concentrate and faba bean protein concentrate in plant-based substitutes for minced meat (SMs). The resulting product aimed at mimicking the mechanical and physicochemical characteristics of beef minced meat (BM) and its applications (i.e., fried and burger patty). In this regard, the mechanical properties (e.g., chewiness, Young's modulus) of original/fried SMs were comparable to or higher than those of original/fried BM. SM patties (45% SMs) were structurally weaker than beef burger patties (100% BM). The rheological analysis showed that the presence of oat fiber concentrate increased the gel-like properties of the blend, which correlated with the overall strength of original SMs (e.g., Young's modulus). The results suggested that SMs could be used as BM for the preparation of vegetarian meat-like products.

Lamin B1 regulates somatic mutations and progression of B-cell malignancies.

Somatic hypermutation (SHM) is a pivotal process in adaptive immunity that occurs in the germinal centre and allows B cells to change their primary DNA sequence and diversify their antigen receptors. Here, we report that genome binding of Lamin B1, a component of the nuclear envelope involved in epigenetic chromatin regulation, is reduced during B-cell activation and formation of lymphoid germinal centres. Chromatin immunoprecipitation-Seq analysis showed that kappa and heavy variable immunoglobulin domains were released from the Lamin B1 suppressive environment when SHM was induced in B cells. RNA interference-mediated reduction of Lamin B1 resulted in spontaneous SHM as well as kappa-light chain aberrant surface expression. Finally, Lamin B1 expression level correlated with progression-free and overall survival in chronic lymphocytic leukaemia, and was strongly involved in the transformation of follicular lymphoma. In summary, here we report that Lamin B1 is a negative epigenetic regulator of SHM in normal B-cells and a 'mutational gatekeeper', suppressing the aberrant mutations that drive lymphoid malignancy.

Genomic disruption of the histone methyltransferase SETD2 in chronic lymphocytic leukaemia.

Histone methyltransferases (HMTs) are important epigenetic regulators of gene transcription and are disrupted at the genomic level in a spectrum of human tumours including haematological malignancies. Using high-resolution single nucleotide polymorphism (SNP) arrays, we identified recurrent deletions of the SETD2 locus in 3% (8/261) of chronic lymphocytic leukaemia (CLL) patients. Further validation in two independent cohorts showed that SETD2 deletions were associated with loss of TP53, genomic complexity and chromothripsis. With next-generation sequencing we detected mutations of SETD2 in an additional 3.8% of patients (23/602). In most cases, SETD2 deletions or mutations were often observed as a clonal event and always as a mono-allelic lesion, leading to reduced mRNA expression in SETD2-disrupted cases. Patients with SETD2 abnormalities and wild-type TP53 and ATM from five clinical trials employing chemotherapy or chemo-immunotherapy had reduced progression-free and overall survival compared with cases wild type for all three genes. Consistent with its postulated role as a tumour suppressor, our data highlight SETD2 aberration as a recurrent, early loss-of-function event in CLL pathobiology linked to aggressive disease.

Analyzing Remodeling of Cardiac Tissue: A Comprehensive Approach Based on Confocal Microscopy and 3D Reconstructions.

Microstructural characterization of cardiac tissue and its remodeling in disease is a crucial step in many basic research projects. We present a comprehensive approach for three-dimensional characterization of cardiac tissue at the submicrometer scale. We developed a compression-free mounting method as well as labeling and imaging protocols that facilitate acquisition of three-dimensional image stacks with scanning confocal microscopy. We evaluated the approach with normal and infarcted ventricular tissue. We used the acquired image stacks for segmentation, quantitative analysis and visualization of important tissue components. In contrast to conventional mounting, compression-free mounting preserved cell shapes, capillary lumens and extracellular laminas. Furthermore, the new approach and imaging protocols resulted in high signal-to-noise ratios at depths up to 60 µm. This allowed extensive analyzes revealing major differences in volume fractions and distribution of cardiomyocytes, blood vessels, fibroblasts, myofibroblasts and extracellular space in control vs. infarct border zone. Our results show that the developed approach yields comprehensive data on microstructure of cardiac tissue and its remodeling in disease. In contrast to other approaches, it allows quantitative assessment of all major tissue components. Furthermore, we suggest that the approach will provide important data for physiological models of cardiac tissue at the submicrometer scale.

Genetic polymorphisms of twelve X-STRs of the investigator Argus X-12 kit and additional six X-STR centromere region loci in an Egyptian population sample.

Recently, many researchers have focused on analysis of different X-chromosomal STRs as they bear the potential to efficiently complement the analysis of autosomal and Y-chromosomal STRs in solving special complex kinship deficiency cases. In the current study we examined a sample of 250 unrelated Egyptian males with the Investigator Argus X-12 kit (Qiagen GmbH, Hilden, Germany) which detects 12 X-STR markers distributed over the entire X-chromosome as four closely linked clusters. Microvariant off ladder alleles as well as null alleles have been detected in some loci. Furthermore, discordant results were observed between the Investigator Argus X-12 and the Mentype(®) Argus X-8 kits (Biotype AG, Dresden, Germany). New primers were designed for loci DXS10101, DXS10146 and DXS10148 to correct the allele drop outs observed in these loci with the Investigator Argus X-12 kit. Additionally, DNA sequence analysis revealed the polymorphisms responsible for the allele drop outs. Furthermore, six additional X-STRs (DXS10161, DXS10159, DXS10162, DXS10163, DXS10164 and DXS10165) located in the centromere region at Xp11.21-Xq11.1 were examined in a single multiplex reaction. Allele and haplotype frequencies as well as different forensic statistical parameters of the 18 X-STR loci tested indicated that they are highly informative in different forensic applications in the Egyptian population. However, some modifications still need to be performed on the Investigator Argus X-12 kit before its use in forensic casework is validated.

Monitoring of WT1 expression in PB and CD34(+) donor chimerism of BM predicts early relapse in AML and MDS patients after hematopoietic cell transplantation with reduced-intensity conditioning.

Relapse of malignant disease remains the major complication in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) after hematopoietic cell transplantation (HCT) with reduced-intensity conditioning (RIC). In this study, we investigated the predictive value of disease-specific markers (DSMs), donor chimerism (DC) analysis of unsorted (UDC) or CD34(+) sorted cells and Wilms' tumor gene 1 (WT1) expression. Eighty-eight patients with AML or MDS were monitored after allogenic HCT following 2 Gy total-body irradiation with (n=84) or without (n=4) fludarabine 3 × 30 mg/m(2), followed by cyclosporin A and mycophenolate mofetil. DSMs were determined by fluorescence in situ hybridization (FISH) and WT1 expression by real-time polymerase chain reaction. Chimerism analysis was performed on unsorted or CD34(+) sorted cells, by FISH or short tandem repeat polymerase chain reaction. Twenty-one (24%) patients relapsed within 4 months after HCT. UDC, CD34(+) DC and WT1 expression were each significant predictors of relapse with sensitivities ranging from 53 to 79% and specificities of 82-91%. Relapse within 28 days was excluded almost entirely on the basis of WT1 expression combined with CD34(+) DC kinetics. Monitoring of WT1 expression and CD34(+) DC predict relapse of AML and MDS after RIC-HCT.

Bone marrow fibroblasts induce expression of PI3K/NF-kappaB pathway genes and a pro-angiogenic phenotype in CLL cells.

Microarray-based gene expression profiling (GEP) was used to study how stroma modulates the survival of CLL cells in an in vitro coculture model employing the murine fibroblast cell line M2-10B4. CLL cells cultured in direct contact with the stromal layer (STR) showed a significantly better survival than cells cultured in transwell (TW) inserts above the M2-10B4 cells. STR as compared to TW conditions induced a significant up-regulation of PI3K/NF-kappaB pro-survival pathway genes and mediated a pro-angiogenetic switch in the CLL cells by up-regulation of vascular endothelial growth factor (VEGF) and osteopontin (OPN) and down-regulation of the anti-angiogenetic molecule thrombospondin-1 (TSP-1).

Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways.

Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-kappaB and Wnt/beta-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGFbeta was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent beta-catenin was monitored in response to the inhibition of GSK3beta. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGFbeta-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach.

Gene expression signatures separate B-cell chronic lymphocytic leukaemia prognostic subgroups defined by ZAP-70 and CD38 expression status.

B-cell chronic lymphocytic leukaemia (B-CLL) is a heterogenous disease with a highly variable clinical course and analysis of zeta-associated protein 70 (ZAP-70) and CD38 expression on B-CLL cells allowed for identification of patients with good (ZAP-70-CD38-) and poor (ZAP-70+CD38+) prognosis. DNA microarray technology was employed to compare eight ZAP-70+CD38+ with eight ZAP-70-CD38- B-CLL cases. The expression of 358 genes differed significantly between the two subgroups, including genes involved in B-cell receptor signaling, angiogenesis and lymphomagenesis. Three of these genes, that is, immune receptor translocation-associated protein 4 (IRTA4)/Fc receptor homologue 2 (FcRH2), angiopoietin 2 (ANGPT2) and Pim2 were selected for further validating studies in a cohort of 94 B-CLL patients. IRTA4/FcRH2 expression as detected by flow cytometry was significantly lower in the poor prognosis subgroup as compared to ZAP-70-CD38- B-CLL cells. In healthy individuals, IRTA4/FcRH2 protein expression was associated with a CD19+CD27+ memory cell phenotype. ANGPT2 plasma concentrations were twofold higher in the poor prognosis subgroup (P<0.05). Pim2 was significantly overexpressed in poor prognosis cases and Binet stage C. Disease progression may be related to proangiogenic processes and strong Pim2 expression.

X chromosomal and autosomal loss of heterozygosity and microsatellite instability in human cervical carcinoma.

The study analyzes tumor material and normal tissue from 27 patients with pure squamous cell carcinoma of the uterine cervix for loss of heterozygosity (LOH) and microsatellite instability (MSI) on 14 autosomal and 11 X chromosomal loci. Overall, 4-40% of the informative cases showed LOH at autosomal regions with the highest frequency at 3p (21-40%) and a marked frequency at 2q35-q37.1 (12.5%) and 17p13.3 (10%), representing regions with putative tumor suppressor gene (TSG) function. The frequency of X chromosomal LOH ranged from 4% to 20%, with a maximum at Xq28 (20%) and Xq11.2-q12 (17%), again indicating alterations in TSG. A 12% LOH was seen at Xq21.33-q22.3, a region encoding a protein with a regulatory function in the cell cycle via cyclin-dependent kinases. MSI was detected in autosomal regions in up to 7% in regions linked to the X chromosome in up to 11%, probably indicating alterations of mismatch repair mechanisms. Our results and those obtained from the literature suggest that autosomal LOH and MSI in carcinomas of the cervix uteri are predominantly found at regions with putative TSG function. Beside TSG alterations, X chromosomal LOH is probably more strongly connected to disturbances in cell cycle regulation.

Graves' disease and Hashimoto's thyroiditis in monozygotic twins: case study as well as transcriptomic and immunohistological analysis of thyroid tissues.

OBJECTIVE: To report on the rare simultaneous occurrence of Graves' disease (GD) and Hashimoto's thyroiditis (HT) in monozygotic twins. DESIGN: We compared the pattern of thyroid tissue-derived cDNAs to gain insight into previous and ongoing immune destruction and reconstruction processes using microarrays. The results were confirmed by immunohistology and real-time PCR. RESULTS: Destruction of thyroid tissue in HT reduced levels of thyrocyte-related cDNAs and cDNAs encoding extracellular matrix components, but increased levels of proteases involved in extracellular matrix degradation compared with GD. Lymphocytic infiltrates forming ectopic follicles replaced the thyroid tissue almost completely in HT. Thus, lymphocyte-related cDNA levels were higher in HT than in GD. The same was true for many chemokines and their receptors, which not only enable migration towards the thyroid but also maintain the lymphocytic infiltrate. HT also showed increased levels of cDNAs encoding molecules related to apoptosis than did GD. Surprisingly, the Th1- and Th2-specific cytokine profiles suggested for HT and GD respectively could not be confirmed. cDNAs encoding factors and receptors involved in angiogenesis were increased in GD compared with HT. CONCLUSIONS: Comparison of gene expression reflects the cellular differences between the two types of autoimmune thyroid disease in twins with identical genetic and similar environmental background.

Y-SNP-genotyping - a new approach in forensic analysis.

Y-chromosomal DNA polymorphisms, especially Y-STRs are well established in forensic routine case work. The STRs are used for identification in paternity deficiency cases and stain analysis with complicate mixtures of male and female DNA. In contrast, Y-chromosomal SNPs are a new tool in forensic investigations. At present, Y-SNPs are mainly used in molecular anthropology for evolutionary studies. Nevertheless, these markers could also provide very useful information for the analysis of forensic cases. The aim of the presented study was to test Y-SNP-typing for stain analyses using different methods-SNaPshot and MALDI-TOF MS. Both methods are based on the principle of minisequencing. The selected Y-SNP markers are suited to define the most important European haplogroups.

Validation of the X-chromosomal STR DXS6809.

This paper presents sequence and population genetic data of the microsatellite marker DXS6809 (GDB 365492) obtained from a German population sample ( n=725 chromosomes). DXS6809 is a highly polymorphic X-linked tetranucleotide polymorphism presenting 12 alleles in our population. Sequencing of 77 PCR products covering 12 alleles (by length), characterised DXS6809 as a marker with a complex repeat sequence structure. A polymorphism information content (PIC) of 0.825 and a mean exclusion chance (MEC) of 0.815 were obtained. A deviation from the Hardy-Weinberg equilibrium (HWE) could not be detected and male and female samples exhibited a similar allele distribution. Kinship testing revealed a typical X-linked inheritance and 2 mutations were found in 394 meioses. DXS6809 is located 90.18 Mb, i.e. 102.3 cM, from the Xp-telomere (Xp-tel), corresponding to Xq21.33. The presented data qualify DXS6809 as a useful supplement to the known forensic ChrX marker panel.

Use of X-linked markers for forensic purposes.

In forensic science, X-chromosomal short tandem repeats (ChrX STRs) bear the potential to efficiently complement the analysis of other genetic markers (autosomal, Y-chromosomal or mitochondrial). We review the population genetic properties and forensic utility of selected ChrX markers, and discuss the problems and limitations arising with their practical use. Formulae required to assess the evidential power of individual markers in different contexts are summarised and applied to ChrX STRs of interest. Since linkage and linkage disequilibrium between markers affect the inferential interpretation of genotype data, practically relevant information regarding the co-localisation and haplotypic association of ChrX STRs is provided. Finally, two examples of complex kinship testing are presented which serve to highlight the particular importance of ChrX STRs for solving deficiency cases and cases involving blood relatives.