Do bacteria shape our development? Crosstalk between intestinal microbiota and HPA axis

Contains fulltext : 179965.pdf (publisher's version ) (Closed access)The human body contains as many bacteria in the intestine as the total number of human body cells. These bacteria have a central position in human health and disease, and would also play a role in the regulation of emotions, behavior, and even higher cognitive functions. The Hypothalamic-Pituitary-Adrenal axis (HPA axis) is a major physiological stress system that produces cortisol. This hormone is involved in responding to environmental stress and also shapes many aspects of brain development. Both the HPA axis and the intestinal microbiota show rapid and profound developmental changes during the first years of life. Early environmental disturbances can affect the development of both systems. Early adversity, for example, is known to lead to later unbalances in both, as well as to psychopathological behavior and emotions. The goal of this theoretical review is to summarize current knowledge on the developmental crosstalk between the intestinal microbiota and the HPA axis, providing a basis for understanding the development and bidirectional communication between these two essential systems in human functioning.14 p

Effects of Gigapascal Level Pressure on Protein Structure and Function

Information on very high pressure (VHP) effects on proteins is limited and therefore effects of VHP on chemistry, structure and function of two model proteins in medical use were studied. VHP (8 GPa) application to l-asparaginase (L-ASNase) resulted in faster mobility on clear native gels. VHP induced generation of lower-MW forms of L-ASNase but VHP treatment did not deteriorate asparaginase activity. Electrophoretic patterns in native and denaturing gels were comparable for untreated and pressurized recombinant human growth hormone (rhGH). rhGH function, however, was deteriorated as shown by a bioassay. In L-ASNase and rhGH a series of protein modifications and amino acid exchanges (indicating cleavage of covalent bonds) were revealed that may probably lead to functional and conformational changes. The findings have implications in protein chemistry, structure, and function and are useful for designing biotechnological applications of protein products

Effects of Gigapascal Level Pressure on Protein Structure and Function

Information on very high pressure (VHP) effects on proteins is limited and therefore effects of VHP on chemistry, structure and function of two model proteins in medical use were studied. VHP (8 GPa) application to l-asparaginase (L-ASNase) resulted in faster mobility on clear native gels. VHP induced generation of lower-MW forms of L-ASNase but VHP treatment did not deteriorate asparaginase activity. Electrophoretic patterns in native and denaturing gels were comparable for untreated and pressurized recombinant human growth hormone (rhGH). rhGH function, however, was deteriorated as shown by a bioassay. In L-ASNase and rhGH a series of protein modifications and amino acid exchanges (indicating cleavage of covalent bonds) were revealed that may probably lead to functional and conformational changes. The findings have implications in protein chemistry, structure, and function and are useful for designing biotechnological applications of protein products

Effects of Gigapascal Level Pressure on Protein Structure and Function

Information on very high pressure (VHP) effects on proteins is limited and therefore effects of VHP on chemistry, structure and function of two model proteins in medical use were studied. VHP (8 GPa) application to l-asparaginase (L-ASNase) resulted in faster mobility on clear native gels. VHP induced generation of lower-MW forms of L-ASNase but VHP treatment did not deteriorate asparaginase activity. Electrophoretic patterns in native and denaturing gels were comparable for untreated and pressurized recombinant human growth hormone (rhGH). rhGH function, however, was deteriorated as shown by a bioassay. In L-ASNase and rhGH a series of protein modifications and amino acid exchanges (indicating cleavage of covalent bonds) were revealed that may probably lead to functional and conformational changes. The findings have implications in protein chemistry, structure, and function and are useful for designing biotechnological applications of protein products

Reliable Quantification of Protein Expression and Cellular Localization in Histological Sections

<div><p>In targeted therapy, patient tumors are analyzed for aberrant activations of core cancer pathways, monitored based on biomarker expression, to ensure efficient treatment. Thus, diagnosis and therapeutic decisions are often based on the status of biomarkers determined by immunohistochemistry in combination with other clinical parameters. Standard evaluation of cancer specimen by immunohistochemistry is frequently impeded by its dependence on subjective interpretation, showing considerable intra- and inter-observer variability. To make treatment decisions more reliable, automated image analysis is an attractive possibility to reproducibly quantify biomarker expression in patient tissue samples. We tested whether image analysis could detect subtle differences in protein expression levels. Gene dosage effects generate well-graded expression patterns for most gene-products, which vary by a factor of two between wildtype and haploinsufficient cells lacking one allele. We used conditional mouse models with deletion of the transcription factors <i>Stat5ab</i> in the liver as well <i>Junb</i> deletion in a T-cell lymphoma model. We quantified the expression of total or activated STAT5AB or JUNB protein in normal (<i>Stat5ab^+/+ or JunB^+/+</i>), hemizygous (<i>Stat5ab^+/Δ</i> or <i>JunB^+/Δ</i>) or knockout (<i>Stat5ab^Δ/Δ</i> or <i>JunB^Δ/Δ</i>) settings. Image analysis was able to accurately detect hemizygosity at the protein level. Moreover, nuclear signals were distinguished from cytoplasmic expression and translocation of the transcription factors from the cytoplasm to the nucleus was reliably detected and quantified using image analysis. We demonstrate that image analysis supported pathologists to score nuclear STAT5AB expression levels in immunohistologically stained human hepatocellular patient samples and decreased inter-observer variability.</p></div

Detection and quantification of transcription factor translocation into the nucleus in mouse livers.

<p><b>A</b> IHC of STAT5AB expression in <i>Stat5ab^+/+</i> (n = 3), <i>Stat5ab^+/Δ</i> (n = 3) and <i>Stat5ab^Δ/Δ</i> (n = 3) livers of GH treated and control mice. Size bar: 50 µm. <b>B</b> Scattergram of the analyzed IHC samples used in A. Discrimination of hepatocytes due to their hematoxylin area and mean intensity depicted in gate 4. Image of gated (hepatocytes, red circled) and of non-hepatocytic cells, mainly Kupffer cells (green circled) in an IHC stained liver sample. <b>C</b> Nuclear and cytoplasmic mask of cells as they are recognized by HistoQuest. The bar graph shows the ratios of nuclear versus cytoplasmic STAT5AB expression levels. Error bars are S.D. Student's t-test was performed to demonstrate statistical significance. <b>D</b> Histograms of nuclear/cytoplasmic STAT5AB intensities are shown; <i>Stat5ab^+/+</i> (blue), <i>Stat5ab^+/Δ</i> (red) and <i>Stat5ab^Δ/Δ</i> (green). The corresponding values in the histograms (black values) showed the gradual decrease of the mean intensities. Based on the fact that <i>Stat5ab^Δ/Δ</i> mice have no hepatic STAT5AB their values in the histograms can be set as background intensity. After background subtraction the numbers displayed the expected 2∶1∶0 ratios (red values). <b>E</b> Whiskers-box plots depicting the quantification of STAT5AB positive hepatocytes (cytoplasmic C; nuclear N) from <i>Stat5ab^+/+</i> with and without GH stimulation using the HistoQuest software. The box indicates the interquartile range; the horizontal line in the box depicts the median. Whiskers indicate the data range. Student's t-test was used to demonstrate statistical significance.</p

Scheme for the applied differentiation protocol.

<p>In order to initiate human AFS cell differentiation to a Schwann cell phenotype AFS cells were first treated in serum free α-MEM with 1 mM β-mercaptoethanol (Diff. I) for 24 hours. Afterwards cells were incubated in α-MEM supplemented with 10% fetal bovine serum and 35 ng/ml retinoic acid (Diff. II) for 72 hours. Subsequently, cells were cultured in α-MEM containing 10% fetal bovine serum supplemented with 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor, 5 mM forskolin, 5 ng/mL platelet-derived growth factor-AA and 200 ng/mL recombinant human heregulin-beta1 (Diff. III) until day 15 of differentiation. Media was changed every 3 days, indicated by arrows. Pharmacologic (pharm.) treatment, consisting of rapamycin or statin, was applied together with Diff. III media.</p

mTOR signaling is active in differentiated AFS cells and important for the differentiation process.

<p>(A) AKT phosphorylation at Ser 473 and ribosomal protein S6 phosphorylation at Ser 240/244 were quantified at the indicated time points during differentiation with and without rapamycin treatment. (B) NGFR, a marker for early differentiated AFS cells (labeled in green), was co-stained with phosphorylated S6 at Ser 240/244 protein (labeled in red) with and without rapamycin treatment (nuclei labeled in blue). Scale bar represents 10 µm. (C) Accumulation of free cholesterol was monitored by filipin III staining. Scale bar represents 10 µm.</p

Rapamycin decreases Schwann cell markers, whereas statin induces Schwann cell markers.

<p>(A) During the last 72 hrs of differentiation, AFS cells were treated with 5 µM and 10 µM statin. After 15 days cDNA was generated and used for quantitative PCR of respective genes. The results are expressed as means ± SEM of three independent experiments. P<0,05 for * vs control treated cells. (B) AFS cells were differentiated for 15 days and since day 5 continuously treated either with 25 nM rapamycin or 1 µM of statin. Fixed cells were stained with indicated antibodies (labeled in red, nuclei in green). Scale bar represents 10 µm. (C) Western blotting of cells differentiated for 15 days and since day 5 continuously treated either with 25 nM rapamycin or 1 µM of statin. GFAP was detected at about 50 kDa, LDLR at 160 kDa and HMGCR as a double band at 90 kDa.</p

Human monoclonal amniotic fluid stem cells can be differentiated into a early Schwann cell phenotype.

<p>(A) AFS cells are small cells with omnidirectional protruding filopodia and upon differentiation to Schwann-like cells, at day 15 of treatment, cells exhibited an increase in cellular volume and an elongated cell morphology. Scale bar represents 50 µm. (B) Immunofluorescence staining of AFS cells differentiated for 15 days (dAFS) compared to undifferentiated AFS cells (AFS) and MCM1 neural crest-derived cells (control), for the Schwann cell markers NGFR, GFAP and S100b (labeled in red, nuclei labeled in green). Purity of cells is indicated as percent positive cells versus total amount of cells ± S.D. Scale bar represents 10 µm. (C) Quantitative RT-PCR of cDNA derived from AFS cells and from AFS cells subjected to Schwann cell differentiation after different time points was performed. Results are shown as fold change expression of respective genes compared to undifferentiated AFS cells. The results are expressed as means ± SEM of three independent experiments. P<0.05 for * vs undifferentiated AFS cells.</p