Seasonal Variations in the Culturable Mycobiome of Acropora loripes along a Depth Gradient.

Coral associated fungi are widespread, highly diverse and are part and parcel of the coral holobiont. To study how environmental conditions prevailing near the coral-host may affect fungal diversity, the culturable (isolated on potato dextrose agar) mycobiome associated with Acropora loripes colonies was seasonally sampled along a depth gradient in the Gulf of Aqaba. Fragments were sampled from both apparently healthy coral colonies as well as those exhibiting observable lesions. Based on phylogenetic analysis of 197 fungal sequences, Ascomycota were the most prevalent (91.9%). The abundance of fungi increased with increasing water depth, where corals sampled at 25 m yielded up to 70% more fungal colony forming units (CFUs) than those isolated at 6 m. Fungal diversity at 25 m was also markedly higher, with over 2-fold more fungal families represented. Diversity was also higher in lesioned coral samples, when compared to apparently healthy colonies. In winter, concurrent with water column mixing and increased levels of available nutrients, at the shallow depths, Saccharomytacea and Sporidiobolacea were more prevalent, while in spring and fall Trichocomacea (overall, the most prevalent family isolated throughout this study) were the most abundant taxa isolated at these depths as well as at deeper sampling sites. Our results highlight the dynamic nature of the culturable coral mycobiome and its sensitivity to environmental conditions and coral health.

Human iPSCs can be differentiated into notochordal cells that reduce intervertebral disc degeneration in a porcine model.

Introduction: As many as 80% of the adult population experience back pain at some point in their lifetimes. Previous studies have indicated a link between back pain and intervertebral disc (IVD) degeneration. Despite decades of research, there is an urgent need for robust stem cell therapy targeting underlying causes rather than symptoms. It has been proposed that notochordal cells (NCs) appear to be the ideal cell type to regenerate the IVD: these cells disappear in humans as they mature, are replaced by nucleus pulposus (NP) cells, and their disappearance correlates with the initiation of degeneration of the disc. Human NCs are in short supply, thus here aimed for generation of notochordal-like cells from induced pluripotent cells (iPSCs). Methods: Human iPSCs were generated from normal dermal fibroblasts by transfecting plasmids encoding for six factors: OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA. Then the iPSCs were treated with GSK3i to induce differentiation towards Primitive Streak Mesoderm (PSM). The differentiation was confirmed by qRT-PCR and immunofluorescence. PSM cells were transfected with Brachyury (Br)-encoding plasmid and the cells were encapsulated in Tetronic-tetraacrylate-fibrinogen (TF) hydrogel that mimics the NP environment (G'=1kPa), cultured in hypoxic conditions (2% O2) and with specifically defined growth media. The cells were also tested in vivo in a large animal model. IVD degeneration was induced after an annular puncture in pigs, 4 weeks later the cells were injected and IVDs were analyzed at 12 weeks after the injury using MRI, gene expression analysis and histology. Results: After short-term exposure of iPSCs to GSK3i there was a significant change in cell morphology, Primitive Streak Mesoderm (PSM) markers (Brachyury, MIXL1, FOXF1) were upregulated and markers of pluripotency (Nanog, Oct4, Sox2) were downregulated, both compared to the control group. PSM cells nucleofected with Br (PSM-Br) cultured in TF hydrogels retained the NC phenotype consistently for up to 8 weeks, as seen in the gene expression analysis. PSM-Br cells were co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs) which, with time, expressed the NC markers in higher levels, however the levels of expression in BM-MSCs alone did not change. Higher expression of NC and NP marker genes in human BM-MSCs was found to be induced by iNC-condition media (iNC-CM) than porcine NC-CM. The annular puncture induced IVD degeneration as early as 2 weeks after the procedure. The injected iNCs were detected in the degenerated discs after 8 weeks in vivo. The iNC-treated discs were found protected from degeneration. This was evident in histological analysis and changes in the pH levels, indicative of degeneration state of the discs, observed using qCEST MRI. Immunofluorescence stains show that their phenotype was consistent with the in vitro study, namely they still expressed the notochordal markers Keratin 18, Keratin 19, Noto and Brachyury. Conclusion: In the present study, we report a stepwise differentiation method to generate notochordal cells from human iPSCs. These cells not only demonstrate a sustainable notochordal cell phenotype in vitro and in vivo, but also show the functionality of notochordal cells and have protective effect in case of induced disc degeneration and prevent the change in the pH level of the injected IVDs. The mechanism of this effect could be suggested via the paracrine effect on resident cells, as it was shown in the in vitro studies with MSCs.

Aging affects B-cell antigen receptor repertoire diversity in primary and secondary lymphoid tissues.

The elderly immune system is characterized by reduced responses to infections and vaccines, and an increase in the incidence of autoimmune diseases and cancer. Age-related deficits in the immune system may be caused by peripheral homeostatic pressures that limit bone marrow B-cell production or migration to the peripheral lymphoid tissues. Studies of peripheral blood B-cell receptor spectratypes have shown that those of the elderly are characterized by reduced diversity, which is correlated with poor health status. In the present study, we performed for the first time high-throughput sequencing of immunoglobulin genes from archived biopsy samples of primary and secondary lymphoid tissues in old (74 ± 7 years old, range 61-89) versus young (24 ± 5 years old, range 18-45) individuals, analyzed repertoire diversities and compared these to results in peripheral blood. We found reduced repertoire diversity in peripheral blood and lymph node repertoires from old people, while in the old spleen samples the diversity was larger than in the young. There were no differences in somatic hypermutation characteristics between age groups. These results support the hypothesis that age-related immune frailty stems from altered B-cell homeostasis leading to narrower memory B-cell repertoires, rather than changes in somatic hypermutation mechanisms.

High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution.

Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10-2. By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.

Matrix stiffness determines the fate of nucleus pulposus-derived stem cells.

Intervertebral disc (IVD) degeneration and consequent low-back pain present a major medical challenge. Nucleus pulposus-derived stem cells (NP-SCs) may lead to a novel therapy for this severe disease. It was recently shown that survival and function of mature NP cells are regulated in part by tissue stiffness. We hypothesized that modification of matrix stiffness will influence the ability of cultured NP-SCs to proliferate, survive, and differentiate into mature NP cells. NP-SCs were subcultured in three-dimensional matrices of varying degrees of stiffness as measured by the material's shear storage modulus. Cell survival, activity, and rate of differentiation toward the chondrogenic or osteogenic lineage were analyzed. NP-SCs were found to proliferate and differentiate in all matrices, irrespective of matrix stiffness. However, matrices with a low shear storage modulus (G' = 1 kPa) promoted significantly more proliferation and chondrogenic differentiation, whereas matrices with a high modulus (G' = 2 kPa) promoted osteogenic differentiation. Imaging performed via confocal and scanning electron microscopes validated cell survival and highlighted stiffness-dependent cell-matrix interactions. These results underscore the effect of the matrix modulus on the fate of NP-SCs. This research may facilitate elucidation of the complex cross-talk between NP-SCs and their surrounding matrix in healthy as well as pathological conditions.

Immunoglobulin gene repertoire diversification and selection in the stomach - from gastritis to gastric lymphomas.

Chronic gastritis is characterized by gastric mucosal inflammation due to autoimmune responses or infection, frequently with Helicobacter pylori. Gastritis with H. pylori background can cause gastric mucosa-associated lymphoid tissue lymphoma (MALT-L), which sometimes further transforms into diffuse large B-cell lymphoma (DLBCL). However, gastric DLBCL can also be initiated de novo. The mechanisms underlying transformation into DLBCL are not completely understood. We analyzed immunoglobulin repertoires and clonal trees to investigate whether and how immunoglobulin gene repertoires, clonal diversification, and selection in gastritis, gastric MALT-L, and DLBCL differ from each other and from normal responses. The two gastritis types (positive or negative for H. pylori) had similarly diverse repertoires. MALT-L dominant clones (defined as the largest clones in each sample) presented higher diversification and longer mutational histories compared with all other conditions. DLBCL dominant clones displayed lower clonal diversification, suggesting the transforming events are triggered by similar responses in different patients. These results are surprising, as we expected to find similarities between the dominant clones of gastritis and MALT-L and between those of MALT-L and DLBCL.

Automated analysis of immunoglobulin genes from high-throughput sequencing: life without a template.

BACKGROUND: Immunoglobulin (that is, antibody) and T cell receptor genes are created through somatic gene rearrangement from gene segment libraries. Immunoglobulin genes are further diversified by somatic hypermutation and selection during the immune response. Studying the repertoires of these genes yields valuable insights into immune system function in infections, aging, autoimmune diseases and cancers. The introduction of high throughput sequencing has generated unprecedented amounts of repertoire and mutation data from immunoglobulin genes. However, common analysis programs are not appropriate for pre-processing and analyzing these data due to the lack of a template or reference for the whole gene. RESULTS: We present here the automated analysis pipeline we created for this purpose, which integrates various software packages of our own development and others', and demonstrate its performance. CONCLUSIONS: Our analysis pipeline presented here is highly modular, and makes it possible to analyze the data resulting from high-throughput sequencing of immunoglobulin genes, in spite of the lack of a template gene. An executable version of the Automation program (and its source code) is freely available for downloading from our website: http://immsilico2.lnx.biu.ac.il/Software.html.

Archaea in the Gulf of Aqaba.

Using a polyphasic approach, we examined the presence of Archaea in the Gulf of Aqaba, a warm marine ecosystem, isolated from major ocean currents and subject to pronounced seasonal changes in hydrography. Catalyzed reported deposition FISH analyses showed that Archaea make up to >20% of the prokaryotic community in the Gulf. A spatial separation between the two major phyla of Archaea was observed during summer stratification. Euryarchaeota were found exclusively in the upper 200 m, whereas Crenarchaeota were present in greater numbers in layers below the summer thermocline. 16S rRNA gene-based denaturing gradient gel electrophoresis confirmed this depth partitioning and revealed further diversity of Crenarchaeota and Euryarchaeota populations along depth profiles. Phylogenetic analysis showed pelagic Crenarchaeota and Euryarchaeota to differ from coral-associated Archaea from the Gulf, forming distinct clusters within the Marine Archaea Groups I and II. Endsequencing of fosmid libraries of environmental DNA provided a tentative identification of some members of the archaeal community and their role in the microbial community of the Gulf. Incorporation studies of radiolabeled leucine and bicarbonate in the presence of different inhibitors suggest that the archaeal community participates in autotrophic CO(2) uptake and contributes little to the heterotrophic activity.