Microbial hydrogen sinks in the sand-bentonite backfill material for the deep geological disposal of radioactive waste.

The activity of subsurface microorganisms can be harnessed for engineering projects. For instance, the Swiss radioactive waste repository design can take advantage of indigenous microorganisms to tackle the issue of a hydrogen gas (H2) phase pressure build-up. After repository closure, it is expected that anoxic steel corrosion of waste canisters will lead to an H2 accumulation. This occurrence should be avoided to preclude damage to the structural integrity of the host rock. In the Swiss design, the repository access galleries will be back-filled, and the choice of this material provides an opportunity to select conditions for the microbially-mediated removal of excess gas. Here, we investigate the microbial sinks for H2. Four reactors containing an 80/20 (w/w) mixture of quartz sand and Wyoming bentonite were supplied with natural sulfate-rich Opalinus Clay rock porewater and with pure H2 gas for up to 108 days. Within 14 days, a decrease in the sulfate concentration was observed, indicating the activity of the sulfate-reducing bacteria detected in the reactor, e.g., from Desulfocurvibacter genus. Additionally, starting at day 28, methane was detected in the gas phase, suggesting the activity of methanogens present in the solid phase, such as the Methanosarcina genus. This work evidences the development, under in-situ relevant conditions, of a backfill microbiome capable of consuming H2 and demonstrates its potential to contribute positively to the long-term safety of a radioactive waste repository.

In-situ X-ray fluorescence to investigate iodide diffusion in opalinus clay: Demonstration of a novel experimental approach.

During the last two decades, the Mont Terri rock laboratory has hosted an extensive experimental research campaign focusing on improving our understanding of radionuclide transport within Opalinus Clay. The latest diffusion experiment, the Diffusion and Retention experiment B (DR-B) has been designed based on an entirely different concept compared to all predecessor experiments. With its novel experimental methodology, which uses in-situ X-ray fluorescence (XRF) to monitor the progress of an iodide plume within the Opalinus Clay, this experiment enables large-scale and long-term data acquisition and provides an alternative method for the validation of previously acquired radionuclide transport parameters. After briefly presenting conventional experimental methodologies used for field diffusion experiments and highlighting their limitations, this paper will focus on the pioneer experimental methodology developed for the DR-B experiment and give a preview of the results it has delivered thus far.

A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues.

Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases.

Minimal mechanical load and tissue culture conditions preserve native cell phenotype and morphology in tendon-a novel ex vivo mouse explant model.

Appropriate mechanical load is essential for tendon homeostasis and optimal tissue function. Due to technical challenges in achieving physiological mechanical loads in experimental tendon model systems, the research community still lacks well-characterized models of tissue homeostasis and physiological relevance. Toward this urgent goal, we present and characterize a novel ex vivo murine tail tendon explant model. Mouse tail tendon fascicles were extracted and cultured for 6 days in a load-deprived environment or in a custom-designed bioreactor applying low magnitude mechanical load (intermittent cycles to 1% strain, at 1 Hz) in serum-free tissue culture. Cells remained viable, as did collagen structure and mechanical properties in all tested conditions. Cell morphology in mechanically loaded tendon explants approximated native tendon, whereas load-deprived tendons lost their native cell morphology. These losses were reflected in altered gene expression, with mechanical loading tending to maintain tendon specific and matrix remodeling genes phenotypic of native tissue. We conclude from this study that ex vivo load deprivation of murine tendon in minimal culture medium results in a degenerative-like phenotype. We further conclude that onset of tissue degeneration can be suppressed by low-magnitude mechanical loading. Thus a minimal explant culture model featuring serum-free medium with low mechanical loads seems to provide a useful foundation for further investigations. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1383-1390, 2018.

Reconstructing a hydrogen-driven microbial metabolic network in Opalinus Clay rock.

The Opalinus Clay formation will host geological nuclear waste repositories in Switzerland. It is expected that gas pressure will build-up due to hydrogen production from steel corrosion, jeopardizing the integrity of the engineered barriers. In an in situ experiment located in the Mont Terri Underground Rock Laboratory, we demonstrate that hydrogen is consumed by microorganisms, fuelling a microbial community. Metagenomic binning and metaproteomic analysis of this deep subsurface community reveals a carbon cycle driven by autotrophic hydrogen oxidizers belonging to novel genera. Necromass is then processed by fermenters, followed by complete oxidation to carbon dioxide by heterotrophic sulfate-reducing bacteria, which closes the cycle. This microbial metabolic web can be integrated in the design of geological repositories to reduce pressure build-up. This study shows that Opalinus Clay harbours the potential for chemolithoautotrophic-based system, and provides a model of microbial carbon cycle in deep subsurface environments where hydrogen and sulfate are present.

A minimalistic microbial food web in an excavated deep subsurface clay rock.

Clay rocks are being considered for radioactive waste disposal, but relatively little is known about the impact of microbes on the long-term safety of geological repositories. Thus, a more complete understanding of microbial community structure and function in these environments would provide further detail for the evaluation of the safety of geological disposal of radioactive waste in clay rocks. It would also provide a unique glimpse into a poorly studied deep subsurface microbial ecosystem. Previous studies concluded that microorganisms were present in pristine Opalinus Clay, but inactive. In this work, we describe the microbial community and assess the metabolic activities taking place within borehole water. Metagenomic sequencing and genome-binning of a porewater sample containing suspended clay particles revealed a remarkably simple heterotrophic microbial community, fueled by sedimentary organic carbon, mainly composed of two organisms: a Pseudomonas sp. fermenting bacterium growing on organic macromolecules and releasing organic acids and H2, and a sulfate-reducing Peptococcaceae able to oxidize organic molecules to CO(2). In Opalinus Clay, this microbial system likely thrives where pore space allows it. In a repository, this may occur where the clay rock has been locally damaged by excavation or in engineered backfills.

Disruption of Lrp4 function by genetic deletion or pharmacological blockade increases bone mass and serum sclerostin levels.

We identified previously in vitro LRP4 (low-density lipoprotein receptor-related protein 4) as a facilitator of the WNT (Wingless-type) antagonist sclerostin and found mutations disrupting this function to be associated with high bone mass in humans similar to patients lacking sclerostin. To further delineate the role of LRP4 in bone in vivo, we generated mice lacking Lrp4 in osteoblasts/osteocytes or osteocytes only. Lrp4 deficiency promoted progressive cancellous and cortical bone gain in both mutants, although more pronouncedly in mice deficient in osteoblast/osteocyte Lrp4, consistent with our observation in human bone that LRP4 is most strongly expressed by osteoblasts and early osteocytes. Bone gain was related primarily to increased bone formation. Interestingly, Lrp4 deficiency in bone dramatically elevated serum sclerostin levels whereas bone expression of Sost encoding for sclerostin was unaltered, indicating that osteoblastic Lrp4 retains sclerostin within bone. Moreover, we generated anti-LRP4 antibodies selectively blocking sclerostin facilitator function while leaving unperturbed LRP4-agrin interaction, which is essential for neuromuscular junction function. These antibodies increased bone formation and thus cancellous and cortical bone mass in skeletally mature rodents. Together, we demonstrate a pivotal role of LRP4 in bone homeostasis by retaining and facilitating sclerostin action locally and provide a novel avenue to bone anabolic therapy by antagonizing LRP4 sclerostin facilitator function.

Age-dependent regulation of tendon crimp structure, cell length and gap width with strain.

The black-and-white patterning of tendon fascicles when visualized by light microscopy, also known as crimp, is a well-known feature of fiber-forming collagens. However, not much is known about its development, function and response to strain. The objective of this study is to investigate the interaction of tenocyte and crimp morphology as well as their changes with increasing age and acute strain. In contrast to previous studies, which used indirect measures, such as polarized light, to investigate the crimp structure, this study visualizes internal crimp structure in three dimensions without freezing, sectioning, staining or fixing the tissue, via two-photon imaging of green fluorescent protein expressing cells within mouse tail tendon fascicles. This technique further allows straining of the live tissue while visualizing changes in crimp morphology and cell shape with increasing specimen length. Combining this novel microscopy technique with computational image and data analysis revealed a complex relationship between tenocytes and the extracellular matrix that evolves with increasing age. While the reduction of crimping with strain was observed as expected, most of the crimps were gone at 0-1% strain already. Even relatively low strains of 3% led to pronounced changes in the crimp structure after relaxation, particularly in the young animals, which could not be seen with bright-field imaging. Cell length and gap width increased with strain. However, while the cells were able to return to their original length even after high strains of 6%, the gaps between the cells widened, which may imply modified cell-cell communication after overstretching.

TGF-ß regulates sclerostin expression via the ECR5 enhancer.

Wnt signaling is critical for skeletal development and homeostasis. Sclerostin (Sost) has emerged as a potent inhibitor of Wnt signaling and, thereby, bone formation. Thus, strategies to reduce sclerostin expression may be used to treat osteoporosis or non-union fractures. Transforming growth factor-beta (TGF-ß) elicits various effects upon the skeleton both in vitro and in vivo depending on the duration and timing of administration. In vitro and in vivo studies demonstrate that TGF-ß increases osteoprogenitor differentiation but decreases matrix mineralization of committed osteoblasts. Because sclerostin decreases matrix mineralization, this study aimed to examine whether TGF-ß achieves such inhibitory effects via transcriptional modulation of Sost. Using the UMR106.01 mature osteoblast cell line, we demonstrated that TGF-ßTGF-ß(1)-ß(2)-ß(3) and Activin A increase Sost transcript expression. Pharmacologic inhibition of Alk4/5/7 in vitro and in vivo decreased endogenous Sost expression, and siRNA against Alk4 and Alk5 demonstrated their requirement for endogenous Sost expression. TGF-ß(1) targeted the Sost bone enhancer ECR5 and did not affect the transcriptional activity of the endogenous Sost promoter. These results indicate that TGF-ß(1) controls Sost transcription in mature osteoblasts, suggesting that sclerostin may mediate the inhibitory effect of TGF-ß upon osteoblast differentiation.

Environmental selenium research: from microscopic processes to global understanding.

Selenium is a natural trace element that is of fundamental importance to human health. The extreme geographical variation in selenium concentrations in soils and food crops has resulted in significant health problems related to deficient or excess levels of selenium in the environment. To deal with these kinds of problems in the future it is essential to get a better understanding of the processes that control the global distribution of selenium. The recent development of analytical techniques and methods enables accurate selenium measurements of environmental concentrations, which will lead to a better understanding of biogeochemical processes. This improved understanding may enable us to predict the distribution of selenium in areas where this is currently unknown. These predictions are essential to prevent future Se health hazards in a world that is increasingly affected by human activities.

Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator function.

Humans lacking sclerostin display progressive bone overgrowth due to increased bone formation. Although it is well established that sclerostin is an osteocyte-secreted bone formation inhibitor, the underlying molecular mechanisms are not fully elucidated. We identified in tandem affinity purification proteomics screens LRP4 (low density lipoprotein-related protein 4) as a sclerostin interaction partner. Biochemical assays with recombinant proteins confirmed that sclerostin LRP4 interaction is direct. Interestingly, in vitro overexpression and RNAi-mediated knockdown experiments revealed that LRP4 specifically facilitates the previously described inhibitory action of sclerostin on Wnt1/ß-catenin signaling. We found the extracellular ß-propeller structured domain of LRP4 to be required for this sclerostin facilitator activity. Immunohistochemistry demonstrated that LRP4 protein is present in human and rodent osteoblasts and osteocytes, both presumed target cells of sclerostin action. Silencing of LRP4 by lentivirus-mediated shRNA delivery blocked sclerostin inhibitory action on in vitro bone mineralization. Notably, we identified two mutations in LRP4 (R1170W and W1186S) in patients suffering from bone overgrowth. We found that these mutations impair LRP4 interaction with sclerostin and its concomitant sclerostin facilitator effect. Together these data indicate that the interaction of sclerostin with LRP4 is required to mediate the inhibitory function of sclerostin on bone formation, thus identifying a novel role for LRP4 in bone.

Does osteocytic SOST suppression mediate PTH bone anabolism?

Parathyroid hormone (PTH) has bone anabolic activity when administered intermittently, affecting cells of the osteoblastic lineage at various stages, yet much remains to be learned about precisely how PTH promotes osteoblastic bone formation. Recent discoveries revealed that PTH causes transcriptional suppression of the osteocyte marker gene SOST, which encodes the potent secreted bone formation inhibitor, sclerostin. This review addresses whether osteocytes, terminally differentiated cells of the osteoblastic lineage, which are entrapped within the mineralized bone matrix, contribute to PTH-induced bone formation responses via regulation of sclerostin levels, and discusses recent evidence on how the bone anabolic responses elicited by intermittent PTH treatment or by sclerostin inhibition overlap and diverge.

Hepatitis B virus X protein affects S phase progression leading to chromosome segregation defects by binding to damaged DNA binding protein 1.

UNLABELLED: Chronic hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC), but its role in the transformation process remains unclear. HBV encodes a small protein, known as HBx, which is required for infection and has been implicated in hepatocarcinogenesis. Here we show that HBx induces lagging chromosomes during mitosis, which in turn leads to formation of aberrant mitotic spindles and multinucleated cells. These effects require the binding of HBx to UV-damaged DNA binding protein 1 (DDB1), a protein involved in DNA repair and cell cycle regulation, and are unexpectedly attributable to HBx interfering with S-phase progression and not directly with mitotic events. HBx also affects S-phase and induces lagging chromosomes when expressed from its natural viral context and, consequently, exhibits deleterious activities in dividing, but not quiescent, hepatoma cells. CONCLUSION: In addition to its reported role in promoting HBV replication, the binding of HBx to DDB1 may induce genetic instability in regenerating hepatocytes and thereby contribute to HCC development, thus making this HBV-host protein interaction an attractive target for new therapeutic intervention.

Control of the SOST bone enhancer by PTH using MEF2 transcription factors.

UNLABELLED: Expression of the osteocyte-derived bone formation inhibitor sclerostin in adult bone requires a distant enhancer. We show that MEF2 transcription factors control this enhancer and mediate inhibition of sclerostin expression by PTH. INTRODUCTION: Sclerostin encoded by the SOST gene is a key regulator of bone formation. Lack of SOST expression is the cause for the progressive bone overgrowth disorders sclerosteosis and Van Buchem disease. We have previously identified a distant enhancer within the 52-kb Van Buchem disease deletion downstream of the SOST gene that is essential for its expression in adult bone. Furthermore, we and others have reported that SOST expression is suppressed by PTH. The aim of this study was to identify transcription factors involved in SOST bone enhancer activity and mediating PTH responsiveness. MATERIALS AND METHODS: Regulation of the SOST enhancer and promoter was studied by luciferase reporter gene assays. Transcription factor binding sites were mapped by footprint analysis and functional mutation analyses using transient transfections of osteoblast-like UMR-106 cells that exhibit endogenous SOST expression. Specific transcription factor binding was predicted by sequence analysis and shown by gel retardation assays and antibody-induced supershifts. Expression of myocyte enhancer factors 2 (MEF2) was detected by in situ hybridization, quantitative RT-PCR (qPCR), and immunohistochemistry. The role of MEF2s in SOST expression was assessed by reporter gene assays and siRNA-mediated RNA knockdown. RESULTS: PTH completely suppressed the transcriptional activity of the SOST bone enhancer but did not affect the SOST promoter. A MEF2 response element was identified in the bone enhancer. It was essential for transcriptional activation, bound MEF2 transcription factors, and mediated PTH responsiveness. Expression of MEF2s in bone was shown by qPCR, in situ hybridization, and immunohistochemistry. MEF2s and sclerostin co-localized in osteocytes. Enhancer activity was stimulated by MEF2C overexpression and inhibited by co-expression of a dominant negative MEF2C mutant. Finally, siRNA-mediated knockdown of MEF2A, C, and D suppressed endogenous SOST expression in UMR-106 osteoblast-like cells. CONCLUSIONS: These data strongly suggest that SOST expression in osteocytes of adult bone and its inhibition by PTH is mediated by MEF2A, C, and D transcription factors controlling the SOST bone enhancer. Hence, MEF2s are implicated in the regulation of adult bone mass.

Arsenic removal from Bangladesh tube well water with filter columns containing zerovalent iron filings and sand.

Arsenic removal is often challenging due to high As(III), phosphate, and silicate concentrations and low natural iron concentrations. Application of zerovalent iron is promising, as metallic iron is widely available. However, removal mechanisms remained unclear and currently used removal units with iron have not been tested systematically, partly due to their large size and long operation time. This study investigated smaller filter columns with 3-4 filters, each containing 2.5 g of iron filings and 100-150 g of sand. At a flow rate of 1 L/h, these columns were able to treat 75-90 L of well water with 440 microg/L As, 1.8 mg/L P, 4.7 mg/L Fe, 19 mg/L Si, and 6 mg/L dissolved organic carbon (DOC) to below 50 microg/L As(tot), without addition of an oxidant. As(III) was oxidized in parallel to oxidation of corrosion-released Fe(II) by dissolved oxygen and sorbed on the forming hydrous ferric oxides (HFO). The open filter columns prevented anoxic conditions. DOC did not appear to interfere with arsenic removal. Manganese was reduced after a slight initial increase from 0.3 mg/L to below 0.1 mg/L. About 100 mg of Fe(0)/L of water was required, 3-5 times less than that for larger units with sand and iron turnings.

Transcriptional activation by bidirectional RNA polymerase II elongation over a silent promoter.

Transcriptional interference denotes negative cis effects between promoters. Here, we show that promoters can also interact positively. Bidirectional RNA polymerase II (Pol II) elongation over the silent human endogenous retrovirus (HERV)-K 18 promoter (representative of 2.5 x 10(3) similar promoters genomewide) activates transcription. In tandem constructs, an upstream promoter activates HERV-K 18 transcription. This is abolished by inversion of the upstream promoter, or by insertion of a poly(A) signal between the promoters; transcription is restored by poly(A) signal mutants. TATA-box mutants in the upstream promoter reduce HERV-K 18 transcription. Experiments with the same promoters in a convergent orientation produce similar effects. A small promoter deletion partially restores HERV-K 18 activity, consistent with activation resulting from repressor repulsion by the elongating Pol II. Transcriptional elongation over this class of intragenic promoters will generate co-regulated sense-antisense transcripts, or, alternatively initiating transcripts, thus expanding the diversity and complexity of the human transcriptome.

Oxidation and removal of arsenic (III) from aerated groundwater by filtration through sand and zero-valent iron.

Removing arsenic from contaminated groundwater in Bangladesh is challenging due to high concentrations of As(III), phosphate and silicate. Application of zero-valent iron as a promising removal method was investigated in detail with synthetic groundwater containing 500 microg/L As(III), 2-3mg/L P, 20mg/L Si, 8.2mM HCO3-, 2.5mM Ca2+, 1.6mM Mg2+ and pH 7.0. In a series of experiments, 1L was repeatedly passed through a mixture of 1.5 g iron filings and 3-4 g quartz sand in a vertical glass column (10mm diameter), allowing the water to re-aerate between each filtration. At a flow rate of 1L/h, up to 8 mg/L dissolved Fe(II) was released. During the subsequent oxidation of Fe(II) by dissolved oxygen, As(III) was partially oxidized and As(V) sorbed on the forming hydrous ferric oxides (HFO). HFO was retained in the next filtration step and was removed by shaking of the sand-iron mixture with water. Rapid phosphate removal provided optimal conditions for the sorption of As(V). Four filtrations lead to almost complete As(III) oxidation and removal of As(tot) to below 50 microg/L. In a prototype treatment with a succession of four filters, each containing 1.5 g iron and 60 g sand, 36 L could be treated to below 50 microg/L in one continuous filtration, without an added oxidant.