Degradation of hydroxyapatite in vivo and in vitro requires osteoclastic sodium-bicarbonate co-transporter NBCn1

Dissolution of the inorganic bone matrix releases not only calcium and phosphate ions, but also bicarbonate. Electroneutral sodium-bicarbonate co-transporter (NBCn1) is expressed in inactive osteoclasts, but its physiological role in bone resorption has remained unknown. We show here that NBCn1, encoded by the SLC4A7 gene, is directly involved in bone resorption. NBCn1 protein was specifically found at the bone-facing ruffled border areas, and metabolic acidosis increased NBCn1 expression in rats in vivo. In human hematopoietic stem cell cultures, NBCn1 mRNA expression was observed only after formation of resorbing osteoclasts. To further confirm the critical role of NBCn1 during bone resorption, human hematopoietic stem cells were transduced with SLC4A7 shRNA lentiviral particles. Downregulation of NBCn1 both on mRNA and protein level by lentiviral shRNAs significantly inhibited bone resorption and increased intracellular acidification in osteoclasts. The lentiviral particles did not impair osteoclast survival, or differentiation of the hematopoietic or mesenchymal precursor cells into osteoclasts or osteoblasts in vitro. Inhibition of NBCn1 activity may thus provide a new way to regulate osteoclast activity during pathological bone resorption.     

Non-methane biogenic volatile organic compound emissions from boreal peatland microcosms under warming and water table drawdown

Boreal peatlands have significant emissions of non-methane biogenic volatile organic compounds (BVOCs). Climate warming is expected to affect these ecosystems both directly, with increasing temperature, and indirectly, through water table drawdown following increased evapotranspiration. We assessed the combined effect of warming and water table drawdown on the BVOC emissions from boreal peatland microcosms. We also assessed the treatment effects on the BVOC emissions from the peat soil after the 7-week long experiment. Emissions of isoprene, monoterpenes, sesquiterpenes, other reactive VOCs and other VOCs were sampled using a conventional chamber technique, collected on adsorbent and analyzed by GC–MS. Carbon emitted as BVOCs was less than 1% of the CO₂ uptake and up to 3% of CH₄ emission. Water table drawdown surpassed the direct warming effect and significantly decreased the emissions of all BVOC groups. Only isoprene emission was significantly increased by warming, parallel to the increased leaf number of the dominant sedge Eriophorum vaginatum. BVOC emissions from peat soil were higher under the control and warming treatments than water table drawdown, suggesting an increased activity of anaerobic microbial community. Our results suggest that boreal peatlands could have concomitant negative and positive radiative forcing effects on climate warming following the effect of water table drawdown. The observed decrease in CH₄ emission causes a negative radiative forcing while the increase in CO₂ emission and decrease in reactive BVOC emissions, which could reduce the cooling effect induced by the lower formation rate of secondary organic aerosols, both contribute to increased radiative forcing.     

TRIM37 defective in mulibrey nanism is a novel RING finger ubiquitin E3 ligase

Mulibrey nanism is an autosomal recessive prenatal-onset growth disorder characterized by dysmorphic features, cardiomyopathy, and hepatomegaly. Mutations in TRIM37 encoding a tripartite motif (TRIM, RING-B-box-coiled-coil)-family protein underlie mulibrey nanism. We investigated the ubiquitin ligase activity predicted for the RING domain of TRIM37 by analyzing its autoubiquitination. Full-length TRIM37 and its TRIM domain were highly polyubiquitinated when co-expressed with ubiquitin. Polyubiquitination was decreased in a mutant protein with disrupted RING domain (Cys35Ser;Cys36Ser) and in the Leu76Pro mutant protein, a disease-associated missense mutation affecting the TRIM domain of TRIM37. Bacterially produced GST-TRIM domain fusion protein, but not its Cys35Ser;Cys36Ser or Leu76Pro mutants, were polyubiquitinated in cell-free conditions, implying RING-dependent modification. Ubiquitin was also identified as an interaction partner for TRIM37 in a yeast two-hybrid screen. Ectopically expressed TRIM37 rapidly formed aggregates that were ubiquitin-, proteasome subunit-, and chaperone-positive in immunofluorescence analysis, defining them as aggresomes. The Cys35Ser;Cys36Ser mutant and the Leu76Pro and Gly322Val patient mutant proteins were markedly less prone to aggregation, implying that aggresomal targeting reflects a physiological function of TRIM37. These findings suggest that TRIM37 acts as a TRIM domain-dependent E3 ubiquitin ligase and imply defective ubiquitin-dependent degradation of an as-yet-unidentified target protein in the pathogenesis of mulibrey nanism.     

A systematic review of MRI studies of language development from birth to 2 years of age

Neurocognitive functions supporting language development start to develop well before first words are spoken during the first years of life. This process coincides with the initial growth spurt of the brain. While the core components of the language network are well characterized in adults and children, the initial neural correlates of language skills are still relatively unknown. We reviewed 10 studies identified via a systematic search that combined magnetic resonance imaging and language‐related measures in healthy infants from birth to 2 years of age. We aimed to describe the current knowledge as well as point out viable future directions for similar studies. Expectedly, the implicated cerebral areas included many established components of the language networks, including frontal and temporal regions. A volumetric leftward asymmetry of the brain was suggested as a determinant of language skills, yet with marked interindividual variation. Overall, temporal and frontal brain volumes associated positively with language skills. Positive associations were described between the maturation of language related white matter tracts and language skills. The language networks showed adult‐like structural similarities already in neonates, with weaker asymmetry compared to adults. In summary, we found some evidence that the language circuit described in older age groups is also associated to language skills during the first 2 years of life. However, across the reviewed studies there were no systematic neural correlates of language skills, which is partly explained by a modest number of studies, scattered representation of ages in measurements and the variance in the used methods.     

Precise construction of oligonucleotide–Fab fragment conjugate for homogeneous immunoassay using HaloTag technology

The use of oligonucleotide–protein conjugates enables the development of novel types of bioanalytical assays. However, convenient methods for producing covalent and stoichiometric oligonucleotide–protein conjugates are still rare. Here we demonstrate, for the first time, covalent conjugation of DNA oligonucleotide to Fab fragments with a 1:1 ratio using HaloTag self-labeling technology. The oligonucleotide coupling was carried out while the Fab was attached to protein G matrix, thereby enabling straightforward production of covalent conjugates. Furthermore, it allowed convenient purification of the product because the unreacted components were easily removed before the elution of the high-purity conjugate. The prepared conjugate was employed in a homogeneous immunoassay where prostate-specific antigen was used as a model analyte. Switchable lanthanide luminescence was used for detection, and the obtained limit of detection was 0.27 ng/ml. In the future, the developed method for covalent conjugation and successive purification in protein G column could also be applied for introducing other kinds of modifications to Fab fragments in a simple and site-specific manner.     

Identification of a Novel Bacterial Outer Membrane Interleukin-1Β-Binding Protein from Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control protein for IL-1β also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1β-binding surface-exposed lipoprotein that may be part of the bacterial IL-1β-sensing system.     

Daily impact score in long-term acceleration measurements of exercise

Mechanical loading increases and maintains bone mass and strength. Daily stress stimulus and osteogenic index theories have been suggested to describe the osteogenic potential of exercise, using exponential or logarithmic relationships, respectively, between loading numbers and magnitude. Inspired by these theories, the aim of this study was to develop and test a daily impact score (DIS) using long-term continuous acceleration measurements of exercise.Acceleration data were collected during a previous exercise trial, in which the subjects (healthy women, 35–40 years, N=34 in the high-impact exercise group and N=30 in the control group) wore a body movement monitor on their waist during the 12-month study. DIS was calculated from the 12-month average daily acceleration distributions in two ways: DIS_Exp adopted from the daily stress stimulus and DIS_Log simplified from the osteogenic index. Areal bone mineral density (aBMD) at the proximal femur and cortical bone geometry at the mid-femur were measured at baseline and 12 months.DIS calculated in either of the ways was significantly higher in the exercise group than in the control group. DIS_Exp and DIS_Log were strongly correlated (R=0.982). Both DIS_Exp and DIS_Log were significantly associated with 12-month aBMD changes at the hip (R up to 0.550, p<0.01) and geometry changes at the mid-femur (R up to 0.472, p<0.05) in the exercise group.DIS calculated either from exponential or logarithmic relationship can be used in acceleration-based measurements of daily exercise. DIS was positively related with changes in hip aBMD and mid-femur bone geometry after 12 months of exercise.     

Glutamine synthetase and glucose-6-phosphate isomerase are adhesive moonlighting proteins of Lactobacillus crispatus released by epithelial cathelicidin LL-37

Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His(6)-GS, His(6)-GPI, His(6)-enolase, and His(6)-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His(6)-GS and His(6)-GPI proteins bound to type I collagen, and His(6)-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His(6)-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.