Floc Formation Reduces the pH Stress Experienced by Microorganisms Living in Alkaline Environments.

The survival of microorganisms within a cementitious geological disposal facility for radioactive wastes heavily depends on their ability to survive the calcium-dominated, hyperalkaline conditions resulting from the dissolution of the cementitious materials. The results from this study show that the formation of flocs, composed of a complex mixture of extracellular polymeric substances (EPS), provides protection against alkaline pH values up to 13.0. The flocs were dominated by Alishewanella and Dietzia spp., producing a mannose-rich carbohydrate fraction incorporating extracellular DNA, resulting in Ca2+ sequestration. EPS provided a ∼10-µm thick layer around the cells within the center of the flocs, which were capable of growth at pH values of 11.0 and 11.5, maintaining internal pH values of 10.4 and 10.7, respectively. Microorganisms survived at a pH of 12.0, where an internal floc pH of 11.6 was observed, as was a reduced associated biomass. We observed limited floc survival (<2 weeks) at a pH of 13.0. This study demonstrates that flocs maintain lower internal pHs in response to the hyperalkaline conditions expected to occur within a cementitious geological disposal facility for radioactive wastes and indicates that floc communities within such a facility can survive at pHs up to 12.0.IMPORTANCE The role of extracellular polymeric substances (EPS) in the survival of microorganisms in hyperalkaline conditions is poorly understood. Here, we present the taxonomy, morphology, and chemical characteristics of an EPS-based microbial floc, formed by a consortium isolated from an anthropogenic hyperalkaline site. Short-term (<2 weeks) survival of the flocs at a pH of 13 was observed, with indefinite survival observed at a pH of 12.0. Measurements from micro-pH electrodes (10-µm-diameter tip) demonstrated that flocs maintain lower internal pHs in response to hyperalkaline conditions (pH 11.0, 11.5, and 12.0), demonstrating that floc formation and EPS production are survival strategies under hyperalkaline conditions. The results indicate how microbial communities may survive and propagate within the hyperalkaline environment that is expected to prevail in a cementitious geological disposal facility for radioactive wastes; the results are also relevant to the wider extremophile community.

Large-scale extraction and characterization of CD271+ multipotential stromal cells from trabecular bone in health and osteoarthritis: implications for bone regeneration strategies based on uncultured or minimally cultured multipotential stromal cells.

OBJECTIVE: To test the hypothesis that CD45(low)CD271+ bone marrow multipotential stromal cells (MSCs) are abundant in the trabecular bone niche and to explore their functional "fitness" in health and osteoarthritis (OA). METHODS: Following enzymatic extraction, MSC release was evaluated using colony-forming unit-fibroblast (CFU-F) and colony-forming unit-osteoblast assays, flow cytometry, and confocal microscopy. CD45(low)CD271+ cells isolated by fluorescence-activated cell sorting were enumerated and expanded under standard and clonal conditions. Their proliferative and osteogenic potencies were assessed in relation to donor age and compared with those of aspirated CD45(low)CD271+ cells. In vitro and in vivo MSC "aging" was measured using quantitative polymerase chain reaction-based telomere length analysis, and standard differentiation assays were utilized to demonstrate multipotentiality. RESULTS: Cellular isolates from trabecular bone cavities contained approximately 65-fold more CD45(low)CD271+ cells compared with aspirates (P < 0.0001) (median 1.89% [n = 39] and 0.029% [n = 46], respectively), concordant with increased CFU-F release. Aspirated and enzymatically released CD45(low)CD271+ cells had identical MSC phenotypes (approximately 100% CD73+CD105+CD13+, approximately 50-60% CD146+CD106+CD166+) and contained large proportions of highly clonogenic multipotential cells. In vitro osteogenic potency of freshly isolated CD45(low)CD271+ cells was comparable with, and often above, that of early-passage MSCs (8-14%). Their frequency and in vivo telomere status in OA bone were similar to those in bone from age-matched controls. CONCLUSION: Our findings show that CD45(low)CD271+ MSCs are abundant in the trabecular bone cavity and indistinguishable from aspirated CD45(low)CD271+ MSCs. In OA they display aging-related loss of proliferation but no gross osteogenic abnormality. These findings offer new opportunities for direct study of MSCs in musculoskeletal diseases without the requirement for culture expansion. They are also relevant for direct therapeutic exploitation of prospectively isolated, minimally cultured MSCs in trauma and OA.

The Impact of Alkaliphilic Biofilm Formation on the Release and Retention of Carbon Isotopes from Nuclear Reactor Graphite.

14C is an important consideration within safety assessments for proposed geological disposal facilities for radioactive wastes, since it is capable of re-entering the biosphere through the generation of 14C bearing gases. The irradiation of graphite moderators in the UK gas-cooled nuclear power stations has led to the generation of a significant volume of 14C-containing intermediate level wastes. Some of this 14C is present as a carbonaceous deposit on channel wall surfaces. Within this study, the potential of biofilm growth upon irradiated and 13C doped graphite at alkaline pH was investigated. Complex biofilms were established on both active and simulant samples. High throughput sequencing showed the biofilms to be dominated by Alcaligenes sp at pH 9.5 and Dietzia sp at pH 11.0. Surface characterisation revealed that the biofilms were limited to growth upon the graphite surface with no penetration of the deeper porosity. Biofilm formation resulted in the generation of a low porosity surface layer without the removal or modification of the surface deposits or the release of the associated 14C/13C. Our results indicated that biofilm formation upon irradiated graphite is likely to occur at the pH values studied, without any additional release of the associated 14C.

The use of the reamer-irrigator-aspirator to harvest mesenchymal stem cells.

The scarcity of mesenchymal stem cells (MSCs) in iliac crest bone marrow aspirate (ICBMA), and the expense and time in culturing cells, has led to the search for alternative harvest sites. The reamer-irrigation-aspirator (RIA) provides continuous irrigation and suction during reaming of long bones. The aspirated contents pass via a filter, trapping bony fragments, before moving into a 'waste' bag from which MSCs have been previously isolated. We examined the liquid and solid phases, performed a novel digestion of the solid phase, and made a comparative assessment in terms of number, phenotype and differentiation capacity with matched ICBMA. The solid fraction from the filtrate was digested for 60 minutes at 37° C with collagenase. Enumeration was performed via the colony-forming unit fibroblast (CFU-F) assay. Passage (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages, and their phenotypes assessed using flow cytometry (CD33, CD34, CD45, CD73, CD90, and CD105). MSCs from the RIA phases were able to differentiate at least as well as those from ICBMA, and all fractions had phenotypes consistent with other established sources. The median number of colonies for the three groups was: ICBMA = 8.5 (2 to 86), RIA-liquid = 19.5 (4 to 90), RIA-solid = 109 (67 to 200) per 200 µl. The mean total yield of cells for the three groups was: ICBMA = 920 (0 to 4275), RIA-liquid = 114,983 (16,500 to 477,750), RIA-solid = 12,785 (7210 to 28 475). The RIA filtrate contains large numbers of MSCs that could potentially be extracted without enzymatic digestion and used for bone repair without prior cell expansion.

Haematopoietic repopulating activity in human cord blood CD133+ quiescent cells.

We have demonstrated previously that cord blood CD133(+) cells isolated in the G(0) phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity, in contrast to CD133(+)G(1) cells. Here, we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133(+)G(0) cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133(+)G(1) counterparts (13.5 and 4.1%, respectively). This suggests that stem cell activity resides in the CD133(+)G(0) population. In long-term BM cultures, the CD133(+)G(0) cells generate significantly more progenitors than the CD34(+)G(0) population (P<0.001) throughout the culture period. Furthermore, a comparison of CD133(+)G(0) versus CD133(+)G(1) cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G(0) cells. We conclude that CD133(+) cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G(1) counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols.