Elemental Mapping of Cryosections from Cnidarian Nematocytes

The distribution of elements in stinging capsules containing cells called nematocytes is shown by pseudocoloured maps representing the X-ray intensity collected from freeze-dried cryosections. This method provides a distinct overview in addition to the quantitative evaluation of single X-ray spectra. Selected examples illustrate the elemental compartmentation in various cnidarian animals. In particular the matrix of capsules in Hydra vulgaris contains high amounts of K in comparison to the tubule, the surrounding capsule wall and the cytoplasm, whereas in Actinia eguina capsules have either high concentration of Ca or Mg, the latter accompanied by S

Microbiota-related Changes in Bile Acid & Tryptophan Metabolism are Associated with Gastrointestinal Dysfunction in a Mouse Model of Autism

peer-reviewedAutism spectrum disorder (ASD) is one of the most prevalent neurodevelopmental conditions worldwide. There is growing awareness that ASD is highly comorbid with gastrointestinal distress and altered intestinal microbiome, and that host-microbiome interactions may contribute to the disease symptoms. However, the paucity of knowledge on gut-brain axis signaling in autism constitutes an obstacle to the development of precision microbiota-based therapeutics in ASD. To this end, we explored the interactions between intestinal microbiota, gut physiology and social behavior in a BTBR T+ Itpr3tf/J mouse model of ASD. Here we show that a reduction in the relative abundance of very particular bacterial taxa in the BTBR gut – namely, bile-metabolizing Bifidobacterium and Blautia species, - is associated with deficient bile acid and tryptophan metabolism in the intestine, marked gastrointestinal dysfunction, as well as impaired social interactions in BTBR mice. Together these data support the concept of targeted manipulation of the gut microbiota for reversing gastrointestinal and behavioral symptomatology in ASD, and offer specific plausible targets in this endeavor.The APC Microbiome Institute is a research institute funded by Science Foundation Ireland (SFI) through the Irish Government's National Development Plan. J.F·C, T.G.D, C.S., S.A.J. and C.G.M.G. are supported by SFI (Grant Nos. SFI/12/RC/2273). S.A.J is also funded by SFI-EU 16/ERA-HDHL/3358. J.F·C, C.S. and T.G.D have research support from Mead Johnson, Cremo, 4D Pharma, Suntory Wellness, and Nutricia. J.F.C, C.S., T.G.D and G.C. have spoken at meetings sponsored by food and pharmaceutical companies

Acute Hypoxic Stress Affects Migration Machinery of Tissue O2-Adapted Adipose Stromal Cells

The ability of mesenchymal stromal (stem) cells (MSCs) to be mobilised from their local depot towards sites of injury and to participate in tissue repair makes these cells promising candidates for cell therapy. Physiological O2 tension in an MSC niche in vivo is about 4–7%. However, most in vitro studies of MSC functional activity are performed at 20% O2. Therefore, this study focused on the effects of short-term hypoxic stress (0.1% O2, 24 h) on adipose tissue-derived MSC motility at tissue-related O2 level. No significant changes in integrin expression were detected after short-term hypoxic stress. However, O2 deprivation provoked vimentin disassembly and actin polymerisation and increased cell stiffness. In addition, hypoxic stress induced the downregulation of ACTR3, DSTN, MACF1, MID1, MYPT1, NCK1, ROCK1, TIAM1, and WASF1 expression, the products of which are known to be involved in leading edge formation and cell translocation. These changes were accompanied by the attenuation of targeted and nontargeted migration of MSCs after short-term hypoxic exposure, as demonstrated in scratch and transwell migration assays. These results indicate that acute hypoxic stress can modulate MSC function in their native milieu, preventing their mobilisation from sites of injury

Characteristics of cbHSPCs-1 population at 20% O₂ and 5% O₂.

<p>Floating cbHSPCs-1 were harvested on Day 7. CD45, CD34 and CD133 positive cells were evaluated with flow cytometry and the number of CFCs was estimated in MetoCult H4534. A. Representative histograms of cbHSPCs immunostaining. Isotypic control—black line, grey fill, positively stained cells—black line. B. Enrichment of cbHSPC-1 population with low differentiated hematopoietic precursors. The data are presented as M±S.E.M (n = 4). *—p<0,05, significant difference from 20% O_<b>2</b>. C. In vitro progenitor assays. (i) Total CFCs number. (ii) The number of early erythroid progenitors (BFU-E), granulocyte-macrophage progenitors (CFU-GM, CFU-G and CFU-M) and multi-potential granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) CFCs. The data are presented as M+S.E.M (n = 5). *—p<0,05, significant difference from 20% O_<b>2</b>.</p

Viability of adhered cbHSPCs after 72 hours in co-culture with ASCs.

<p>To evaluate the viability of attached cbHSPCs, ASC/cbHSPC co-cultures were trypsinised, and the cell suspension was stained with the Annexin V—FITC/PI kit according to the manufacturer’s instructions, before being analysed on an Epics XL flow cytometer. For analysis, only cells gated as CD45⁺/CD90^- were used. The data are presented as M+S.E.M. (n = 4).</p><p>Viability of adhered cbHSPCs after 72 hours in co-culture with ASCs.</p

Identification of cbMNCs, attached to ASC layer on Day 3 of co-culturing at 20% O₂ and 5% O₂.

<p>Unmanipulated cbMNCs were cocultured with ASCs at 20% and 5% O_<b>2</b>. After 72 hours all floating cbMNCs were washed out, ASCs with attached cord blood cells were fixed with cold methanol and stained with May-Grunwald-Giemsa for differentiatial analysis of haematopoietic precursors. A—20% O₂; B -5% O₂, general view. Arrows indicate ASCs. Bar—50 um. There were no marked difference between the appearance of co-cultures at different O_<b>2</b> concentrations. C-G—larger magnification of cbMNCs on ASCs. Bar—10 um. Mature blood cells: M-<i>monocyte or promonocyte</i>; E—erythrocyte; L- lymphocyte. cbHSPCs: NM and EM—<i>neutrophilic myelocyte & eosinophilic myelocyte;</i> NMm and EMm—<i>eosinophilic metamyelocyte & neutrophilic metamyelocyte;</i> HSPCs—morphologically unidentified cells, usually are considered as lymphoid elements.</p

Reseeding of cbHSPCs-1 at 20% O₂ and 5% O₂.

<p>Floating cbHSPCs-1 were harvested on Day 7 and reseeded on new ASC layer. On Day 11 cbHSPCs-1 were located in three compartments, as was described above for cbMNC-derived cbHSPCs. A, B. An accrued population of floating reseeded cbHSPCs-1 was visible in co-cultures. Phase contrast. Bar—50 um. C, D. Single and clastered phase-bright reseeded cbHSPCs-1 on the ASC surface and phase-dim cbHSPCs-1 beneath ASC layer. Phase contrast. Bar—50 um. White arrows—the phase-bright, red arrows—phase-dim cbHSPCs-1. E, F—CAFC areas in ASC/reseeded cbHSPCs-1 co-culture, Day 14. NAMC contrast. Bar—100 um. Red arrows indicate CAFCs beneath the ASC layer. White arrows—phase-bright cbHSPCs-1 on the ASC surface.</p

HSPCs at 20% O₂ and 5% O₂.

<p>cbMNCs were co-cultured with ASCs, washed out after 72 hours and ASCs with adhered cbMNCs-derived HSPCs continued culturing up to 14 days. A, B—single and clustered cbHSPCs, Day 7 in culture. CEM. Bar—5 um. C, D—newly-generated floating cbHSPCs (cbHSPCs-1), Day 7. Phase contrast. Bar—50 um. E, F—CAFC areas in ASC/cbHSPCs co-culture, Day 11. Phase contrast. Bar—100 um. Red arrows indicate phase-dim CAFCs beneath the ASC layer. White arrows indicate the phase-bright cbHSPCs on the ASC surface.</p