Detection of chromosome 21 aneuploidy by fluorescent in situ hybridization

Fluorescentna in situ hibridizacija (FISH) je nova visoko osetljiva metoda koja omogućava vizuelnu identifikaciju hromozoma ili delova hromozoma na preparatima. Ova nova metoda molekularne citogenetike zasnovana je na specifičnoj hibridizaciji ciljne DNK fiksirane na pločici i DNK probe obično obeležene biotinom. Visoka učestalost trizomije 21 (1 slučaj na 650 do 1000 novorođene dece) i povezanost sa teškim psihomotornim zastojem kod njihovih nosilaca, učinile su da je Daunov sindrom najviše izučavana hromozomska aberacija kod čoveka. Cilj ovog rada bio je razvijanje i primena FISH metode za brzu dstekciju aneuploidije hromozoma 21. Rezultati koji su ovde prikazani ukazuju da FISH predstavlja metodu koja se može primeniti za brzu prenatalnu dijagnostiku aneuploidija.Fluorescent in situ hybridization (FISH) is a new highly sensitive method that enables visual identification of chromosomes or part of chromosomes on slides. This new molecular cytogenetic method is based on specific hybridization of the target DNA fixed on slide and DNA probe usually labelled with biotin. The frequency of trisomy 21 in the population (1 in 650 to 1000 live births) and association with psycho-motor delay, make Down's syndrome the most extensively studied human chromosome abnormality. The aim of this work was to develop and apply FISH for rapid detection of chromosome 21 aneuploidy. The results presented here indicate that FISH provides a method for rapid prenatal detection of aneuploidies

Starvation Induces Phase-Specific Changes in the Proteome of Mouse Small Intestine

Food deprivation results in metabolic, structural, and functional changes in the small intestine that influences gut mucosal integrity, epithelial cell proliferation, mucin synthesis, and other processes. The underlying mechanisms are still unclear, which lead to the study of molecular effects of short-term and long-term starvation in the intestine of mice. A comparative proteomics approach, combining two-dimensional gel electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, was used to identify intestinal proteins whose expression is changed under different starvation conditions (0, 12, 24, and 72 h). In total, the expression levels of 80 protein spots changed significantly between the different groups. The results demonstrate that after 12 h of starvation, mainly proteins involved in glycolysis and energy metabolism show decreased expression levels. Starvation for 24 h results in a down-regulation of proteins involved in protein synthesis and amino acid metabolism. Simultaneously, proteins with a protective role, e.g., reg I and II, glutathione peroxidase 3, and carbonic anhydrase 3, are clearly up-regulated. The last starvation phase (72 h) is characterized by increased ezrin expression, which may enhance villus morphogenesis critical for survival. Together, these results provide novel insights in the intestinal starvation response and may contribute to improved nutritional support during conditions characterized by malnutrition

Fasting induces a biphasic adaptive metabolic response in murine small intestine

BACKGROUND: The gut is a major energy consumer, but a comprehensive overview of the adaptive response to fasting is lacking. Gene-expression profiling, pathway analysis, and immunohistochemistry were therefore carried out on mouse small intestine after 0, 12, 24, and 72 hours of fasting. RESULTS: Intestinal weight declined to 50% of control, but this loss of tissue mass was distributed proportionally among the gut's structural components, so that the microarrays' tissue base remained unaffected. Unsupervised hierarchical clustering of the microarrays revealed that the successive time points separated into distinct branches. Pathway analysis depicted a pronounced, but transient early response that peaked at 12 hours, and a late response that became progressively more pronounced with continued fasting. Early changes in gene expression were compatible with a cellular deficiency in glutamine, and metabolic adaptations directed at glutamine conservation, inhibition of pyruvate oxidation, stimulation of glutamate catabolism via aspartate and phosphoenolpyruvate to lactate, and enhanced fatty-acid oxidation and ketone-body synthesis. In addition, the expression of key genes involved in cell cycling and apoptosis was suppressed. At 24 hours of fasting, many of the early adaptive changes abated. Major changes upon continued fasting implied the production of glucose rather than lactate from carbohydrate backbones, a downregulation of fatty-acid oxidation and a very strong downregulation of the electron-transport chain. Cell cycling and apoptosis remained suppressed. CONCLUSION: The changes in gene expression indicate that the small intestine rapidly looses mass during fasting to generate lactate or glucose and ketone bodies. Meanwhile, intestinal architecture is maintained by downregulation of cell turnove

Nur77-deficiency in bone marrow-derived macrophages modulates inflammatory responses, extracellular matrix homeostasis, phagocytosis and tolerance

The nuclear orphan receptor Nur77 (NR4A1, TR3, or NGFI-B) has been shown to modulate the inflammatory response of macrophages. To further elucidate the role of Nur77 in macrophage physiology, we compared the transcriptome of bone marrow-derived macrophages (BMM) from wild-type (WT) and Nur77-knockout (KO) mice. In line with previous observations, SDF-1α (CXCL12) was among the most upregulated genes in Nur77-deficient BMM and we demonstrated that Nur77 binds directly to the SDF-1α promoter, resulting in inhibition of SDF-1α expression. The cytokine receptor CX3CR1 was strongly downregulated in Nur77-KO BMM, implying involvement of Nur77 in macrophage tolerance. Ingenuity pathway analyses (IPA) to identify canonical pathways regulation and gene set enrichment analyses (GSEA) revealed a potential role for Nur77 in extracellular matrix homeostasis. Nur77-deficiency increased the collagen content of macrophage extracellular matrix through enhanced expression of several collagen subtypes and diminished matrix metalloproteinase (MMP)-9 activity. IPA upstream regulator analyses discerned the small GTPase Rac1 as a novel regulator of Nur77-mediated gene expression. We identified an inhibitory feedback loop with increased Rac1 activity in Nur77-KO BMM, which may explain the augmented phagocytic activity of these cells. Finally, we predict multiple chronic inflammatory diseases to be influenced by macrophage Nur77 expression. GSEA and IPA associated Nur77 to osteoarthritis, chronic obstructive pulmonary disease, rheumatoid arthritis, psoriasis, and allergic airway inflammatory diseases. Altogether these data identify Nur77 as a modulator of macrophage function and an interesting target to treat chronic inflammatory diseas

Insulin-like growth factor binding protein 5 enhances survival of LX2 human hepatic stellate cells

ABSTRACT: BACKGROUND: Expression of insulin-like growth factor binding protein 5 (IGFBP5) is strongly induced upon activation of hepatic stellate cells and their transdifferentiation into myofibroblasts in vitro. This was confirmed in vivo in an animal model of liver fibrosis. Since IGFBP5 has been shown to promote fibrosis in other tissues, the aim of this study was to investigate its role in the progression of liver fibrosis. METHODS: The effect of IGFBP5 was studied in LX2 cells, a model for partially activated hepatic stellate cells, and in human primary liver myofibroblasts. IGFBP5 signalling was modulated by the addition of recombinant protein, by lentiviral overexpression, and by siRNA mediated silencing. Furthermore, the addition of IGF1 and silencing of the IGF1R was used to investigate the role of the IGF-axis in IGFBP5 mediated effects. RESULTS: IGFBP5 enhanced the survival of LX2 cells and myofibroblasts via a >50% suppression of apoptosis. This effect of IGFBP5 was not modulated by the addition of IGF1, nor by silencing of the IGF1R. Additionally, IGFBP5 was able to enhance the expression of established pro-fibrotic markers, such as collagen Ialpha1, TIMP1 and MMP1. CONCLUSION: IGFBP5 enhances the survival of (partially) activated hepatic stellate cells and myofibroblasts by lowering apoptosis via an IGF1-independent mechanism, and enhances the expression of profibrotic genes. Its lowered expression may, therefore, reduce the progression of liver fibrosi

Varietal Differences of Prokupac, Evita and Čokot Zemun Based onTheir Anthocyanins Content in Grape Skin Extract

In this study, we have analyzed the anthocyanin composition of skin extracts of three red grape varieties Prokupac, Evita and Čokot Zemun in order to distinguish these cultivars based on their anthocyanin profile. Also, mechanical analysis of grape bunches and berries was performed. According to our results, seventeen anthocyanins were identified using LC/MS technique and quantitative differences were recorded using HPLC-DAD method. The highest content of total anthocyanins was obtained for Evita variety and the lowest one was recorded in Prokupac. Also, clear differences were observed in anthocyanins ratios. In comparison to Prokupac and Evita varieties, Čokot Zemun was characterized with a high content of coumaroyl derivatives of anthocyanin compounds, while high levels of acetylated derivatives were recorded in Prokupac. Data reported in this study represent a certain contribution to a database of mechanical properties and chemical composition of grape varieties originating from Balkan

Processed food classification: Conceptualisation and challenges

Background: Processed foods are typically praised/revered for their convenience, palatability, and novelty; however, their healthfulness has increasingly come under scrutiny. Classification systems that categorise foods according to their “level of processing” have been used to predict diet quality and health outcomes and inform dietary guidelines and product development. However, the classification criteria used are ambiguous, inconsistent and often give less weight to existing scientific evidence on nutrition and food processing effects; critical analysis of these criteria creates conflict amongst researchers.Scope and approach: We examine the underlying basis of food classification systems and provide a critical analysis of their purpose, scientific basis, and distinguishing features by thematic analysis of the category definitions.Key findings and conclusions: These classification systems were mostly created to study the relationship between industrial products and health. There is no consensus on what factors determine the level of food processing. We identified four defining themes underlying the classification systems: 1. Extent of change (from natural state); 2. Nature of change (properties, adding ingredients); 3. Place of processing (where/by whom); and 4. Purpose of processing (why, essential/cosmetic). The classification systems embody socio-cultural elements and subjective terms, including home cooking and naturalness. Hence, “processing” is a chaotic conception, not only concerned with technical processes. Most classification systems do not include quantitative measures but, instead, imply correlation between “processing” and nutrition. The concept of “whole food” and the role of the food matrix in relation to healthy diets needs further clarification; the risk assessment/management of food additives also needs debate

Fasting induces a biphasic adaptive metabolic response in murine small intestine

Abstract Background The gut is a major energy consumer, but a comprehensive overview of the adaptive response to fasting is lacking. Gene-expression profiling, pathway analysis, and immunohistochemistry were therefore carried out on mouse small intestine after 0, 12, 24, and 72 hours of fasting. Results Intestinal weight declined to 50% of control, but this loss of tissue mass was distributed proportionally among the gut's structural components, so that the microarrays' tissue base remained unaffected. Unsupervised hierarchical clustering of the microarrays revealed that the successive time points separated into distinct branches. Pathway analysis depicted a pronounced, but transient early response that peaked at 12 hours, and a late response that became progressively more pronounced with continued fasting. Early changes in gene expression were compatible with a cellular deficiency in glutamine, and metabolic adaptations directed at glutamine conservation, inhibition of pyruvate oxidation, stimulation of glutamate catabolism via aspartate and phosphoenolpyruvate to lactate, and enhanced fatty-acid oxidation and ketone-body synthesis. In addition, the expression of key genes involved in cell cycling and apoptosis was suppressed. At 24 hours of fasting, many of the early adaptive changes abated. Major changes upon continued fasting implied the production of glucose rather than lactate from carbohydrate backbones, a downregulation of fatty-acid oxidation and a very strong downregulation of the electron-transport chain. Cell cycling and apoptosis remained suppressed. Conclusion The changes in gene expression indicate that the small intestine rapidly looses mass during fasting to generate lactate or glucose and ketone bodies. Meanwhile, intestinal architecture is maintained by downregulation of cell turnover.</p