Shared and Compartment-Specific Processes in Nucleus Pulposus and Annulus Fibrosus During Intervertebral Disc Degeneration.

Elucidating how cell populations promote onset and progression of intervertebral disc degeneration (IDD) has the potential to enable more precise therapeutic targeting of cells and mechanisms. Single-cell RNA-sequencing (scRNA-seq) is performed on surgically separated annulus fibrosus (AF) (19,978; 26,983 cells) and nucleus pulposus (NP) (20,884; 24,489 cells) from healthy and diseased human intervertebral discs (IVD). In both tissue types, depletion of cell subsets involved in maintenance of healthy IVD is observed, specifically the immature cell subsets - fibroblast progenitors and stem cells - indicative of an impairment of normal tissue self-renewal. Tissue-specific changes are also identified. In NP, several fibrotic populations are increased in degenerated IVD, indicating tissue-remodeling. In degenerated AF, a novel disease-associated subset is identified, which expresses disease-promoting genes. It is associated with pathogenic biological processes and the main gene regulatory networks include thrombospondin signaling and FOXO1 transcription factor. In NP and AF cells thrombospondin protein promoted expression of genes associated with TGFß/fibrosis signaling, angiogenesis, and nervous system development. The data reveal new insights of both shared and tissue-specific changes in specific cell populations in AF and NP during IVD degeneration. These identified mechanisms and molecules are novel and more precise targets for IDD prevention and treatment.

LEA Proteins and the Evolution of the WHy Domain.

The late embryogenesis abundant (LEA) family is composed of a diverse collection of multidomain and multifunctional proteins found in all three domains of the tree of life, but they are particularly common in plants. Most members of the family are known to play an important role in abiotic stress response and stress tolerance in plants but are also part of the plant hypersensitive response to pathogen infection. The mechanistic basis for LEA protein functionality is still poorly understood. The group of LEA 2 proteins harbor one or more copies of a unique domain, the water stress and hypersensitive response (WHy) domain. This domain sequence has recently been identified as a unique open reading frame (ORF) in some bacterial genomes (mostly in the phylum Firmicutes), and the recombinant bacterial WHy protein has been shown to exhibit a stress tolerance phenotype in Escherichia coli and an in vitro protein denaturation protective function. Multidomain phylogenetic analyses suggest that the WHy protein gene sequence may have ancestral origins in the domain Archaea, with subsequent acquisition in Bacteria and eukaryotes via endosymbiont or horizontal gene transfer mechanisms. Here, we review the structure, function, and nomenclature of LEA proteins, with a focus on the WHy domain as an integral component of the LEA constructs and as an independent protein.

Arsenate uptake by Al nanoclusters and other Al-based sorbents during water treatment.

In many parts of the world, arsenic from geogenic and anthropogenic sources deteriorates the quality of drinking water resources. Effective methods of arsenic removal include adsorption and coagulation with iron- and aluminum-based materials, of which polyaluminum chloride is widely employed as coagulant in water treatment due to its low cost and high efficiency. We compared the arsenic uptake capacity and the arsenic bonding sites of different Al-based sorbents, including Al nanoclusters, polyaluminum chloride, polyaluminum granulate, and gibbsite. Extended X-ray absorption fine structure (EXAFS) spectroscopy revealed that As(V) forms bidentate-binuclear complexes in interaction with all Al-based removal agents. The octahedral configuration of nanoclusters and the distribution of sorption sites remain the same in all types of removal agents consisting of nano-scale Al oxyhydroxide particles. The obtained distances for As(V)-O and As(V)-Al agreed with previously published data and were found to be 1.69 ± 0.02 Å and 3.17-3.21 Å, respectively. Our study suggests that As(V) binds to Al nanoclusters as strongly as to Al oxide surfaces. The As sorption capacity of Al nanoclusters was found to be very similar to that of Al clusters in a polyaluminum chloride. The most efficient Al-based sorbents for arsenic removal were Al nanoclusters, followed by polyaluminum granulate.

Adsorption of arsenic on polyaluminum granulate.

The kinetics and efficiencies of arsenite and arsenate removal from water were evaluated using polyaluminum granulates (PAG) with high content of aluminum nanoclusters. PAG was characterized to be meso- and macroporous, with a specific surface area of 35 ± 1 m(2) g(-1). Adsorption experiments were conducted at pH 7.5 in deionized water and synthetic water with composition of As-contaminated groundwater in the Pannonian Basin. As(III) and As(V) sorption was best described by the Freundlich and Langmuir isotherm, respectively, with a maximum As(V) uptake capacity of ~200 µmol g(-1) in synthetic water. While As(III) removal reached equilibrium within 40 h, As(V) was removed almost entirely within 20 h. Micro X-ray fluorescence and electron microscopy revealed that As(III) was distributed uniformly within the grain, whereas As(V) diffused up to 81 µm into PAG. The results imply that As(V) is adsorbed 3 times faster while being transported 10(5) times slower than As(III) in Al hydroxide materials.

Polyaluminum chloride with high Al30 content as removal agent for arsenic-contaminated well water.

Polyaluminum chloride (PACl) is a well-established coagulant in water treatment with high removal efficiency for arsenic. A high content of Al(30) nanoclusters in PACl improves the removal efficiency over broader dosage and pH range. In this study we tested PACl with 75% Al(30) nanoclusters (PACl(Al30)) for the treatment of arsenic-contaminated well water by laboratory batch experiments and field application in the geothermal area of Chalkidiki, Greece, and in the Pannonian Basin, Romania. The treatment efficiency was studied as a function of dosage and the nanoclusters' protonation degree. Acid-base titration revealed increasing deprotonation of PACl(Al30) from pH 4.7 to the point of zero charge at pH 6.7. The most efficient removal of As(III) and As(V) coincided with optimal aggregation of the Al nanoclusters at pH 7-8, a common pH range for groundwater. The application of PACl(Al30) with an Al(tot) concentration of 1-5mM in laboratory batch experiments successfully lowered dissolved As(V) concentrations from 20 to 230 µg/L to less than 5 µg/L. Field tests confirmed laboratory results, and showed that the WHO threshold value of 10 µg/L was only slightly exceeded (10.8 µg/L) at initial concentrations as high as 2300 µg/L As(V). However, As(III) removal was less efficient (<40%), therefore oxidation will be crucial before coagulation with PACl(Al30). The presence of silica in the well water improved As(III) removal by typically 10%. This study revealed that the Al(30) nanoclusters are most efficient for the removal of As(V) from water resources at near-neutral pH.

Functional relevance of the IRF-1 promoter polymorphism rs2549009 on transcriptional activity in a native genomic environment.

Interferon regulatory factor-1 (IRF-1), a transcription regulator involved both in inducing and in mediating the effects of interferon, is encoded by a highly polymorphic gene in different ethnic populations. Some of these genetic variations have been described to be associated to disease traits in hepatitis C virus and in human immunodeficiency virus infection, including one single-nucleotide polymorphism rs2549009 within the promoter region. This study aimed at investigating the functional relevance of rs2549009 on IRF-1 transcriptional activity in peripheral blood mononuclear cells in its natural genomic environment. Haplotype-specific chromatin immunoprecipitation using antibodies directed against both the transcriptionally inactive and active RNA polymerase II (RNAPII) and allele-specific transcript quantification techniques were applied to ex vivo-derived samples from healthy heterozygous donors. Inactive serine 5 phosphorylated RNAPII was found to be preferentially bound to the rs2549009 A allele in all donors investigated. Active serine 2 phosphorylated (ser2-P) RNAPII, in contrast, was found to be precipitable, depending on the donor, preferentially either with the A or the G promoter variants or without any preference. The ratio of rs2549009 A/G promoter variants engaged by ser2-P RNAPII was closely related to the relative frequency of the respective IRF-1 transcripts, and relative allelic expression was found to be associated to total IRF-1 gene expression. These results provide evidence for a bidirectional IRF-1 gene expression imbalance that appears not to be solely controlled by rs2549009 in cis and may rely on a yet unidentified variant or haplotype or on environmental control in trans.

TLR3 gene polymorphisms and liver disease manifestations in chronic hepatitis C.

Phenotypes of liver disease due to chronic hepatitis C virus (HCV) infection show a wide range of variations in terms of histological manifestations and the clinical outcome. Sensing of viral double-stranded RNA (dsRNA) by Toll-like receptor 3 (TLR3) is likely involved in early pathogen detection and the host response to viral infection. This study analyzed epidemiological and clinical data from a total of 137 patients with chronic HCV infection with regard to two polymorphic positions within the TLR3 gene: rs5743305 (T/A) is located within the promoter region and might affect transcriptional activity, rs3775291 (C/T) is a non-synonymous single nucleotide polymorphism (SNP) located within exon 4 and the variant receptor has been shown to be functionally impaired. TLR3 promoter and the exon 4 variations were not found to be associated with TLR3 gene expression in peripheral blood mononuclear cells (PBMCs). In the liver, however, a tendency of higher TLR3 gene expression was found for exon 4 TT genotypes. Both variations were not found to be associated with clinical parameters of chronic disease. On the other hand, an analysis of the TLR3 exon 4 genotype distribution with respect to HCV subtype revealed an absence of TT genotype among HCV subtype 1a infected individuals. This study thus failed to reveal any association of the two SNPs under investigation with clinical parameters of chronic hepatitis C. However, data argue for a functional relevance of the exon 4 SNP in terms of conferring a different susceptibility towards HCV subtype infection.

Functional impact of endotoxin receptor CD14 polymorphisms on transcriptional activity.

The polymorphism rs2569190 within the CD14 endotoxin (lipopolysaccharide, LPS) receptor gene is associated with various disease conditions that are assumed to rely on endotoxin sensitivity. In vitro experiments suggest that the T allele sensitizes the host for exogenous or endogenous LPS via an enhanced CD14 expression. To prove the impact of this single nucleotide polymorphism in its natural genomic context in vivo, two parameters of gene transcription were analyzed in peripheral blood mononuclear cells (PBMC) from single healthy individuals: (a) recruitment of RNA polymerase II by haplotype-specific chromatin immunoprecipitation and (b) the relative amount of transcripts by allele-specific transcript quantification (ASTQ). RNA polymerase II was found to be twice as much bound to the most prevalent haplotype, C-T-C-G, the only one carrying a T at the position rs2569190 of interest. ASTQ employing two independent read-out assays revealed, however, similar transcript numbers originating from C-T-C-G and non-C-T-C-G haplotypes. Total CD14 mRNA levels from freshly isolated PBMC, moreover, were neither related to donors' geno- nor haplogenotypes. Our data argue for a functional impact of the rs2569190 polymorphism in terms of a stronger transcription initiation on T allele gene variants even if preferential allele-specific binding does not result in an increase in transcript numbers. Endotoxin sensitivity associated with this genetic variation appears not to rely solely on a cis-acting regulatory impact of rs2569190 on CD14 gene transcription in PBMC.