Negative Regulation of Bone Formation by the Transmembrane Wnt Antagonist Kremen-2

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders

Results of WICOVIR Gargle Pool PCR Testing in German Schools Based on the First 100,000 Tests

Background: Opening schools and keeping children safe from SARS-CoV-2 infections at the same time is urgently needed to protect children from direct and indirect consequences of the COVID-19 pandemic. To achieve this goal, a safe, efficient, and cost-effective SARS-CoV-2 testing system for schools in addition to standard hygiene measures is necessary. Methods: We implemented the screening WICOVIR concept for schools in the southeast of Germany, which is based on gargling at home, pooling of samples in schools, and assessment of SARS-CoV-2 by pool rRT-PCR, performed decentralized in numerous participating laboratories. Depooling was performed if pools were positive, and results were transmitted with software specifically developed for the project within a day. Here, we report the results after the first 13 weeks in the project. Findings: We developed and implemented the proof-of-concept test system within a pilot phase of 7 weeks based on almost 17,000 participants. After 6 weeks in the main phase of the project, we performed >100,000 tests in total, analyzed in 7,896 pools, identifying 19 cases in >100 participating schools. On average, positive children showed an individual CT value of 31 when identified in the pools. Up to 30 samples were pooled (mean 13) in general, based on school classes and attached school staff. All three participating laboratories detected positive samples reliably with their previously established rRT-PCR standard protocols. When self-administered antigen tests were performed concomitantly in positive cases, only one of these eight tests was positive, and when antigen tests performed after positive pool rRT-PCR results were already known were included, 3 out of 11 truly positive tests were also identified by antigen testing. After 3 weeks of repetitive WICOVIR testing twice weekly, the detection rate of positive children in that cohort decreased significantly from 0.042 to 0.012 (p = 0.008). Interpretation: Repeated gargle pool rRT-PCR testing can be implemented quickly in schools. It is an effective, valid, and well-received test system for schools, superior to antigen tests in sensitivity, acceptance, and costs

Spatial Applications as Social Knowledge Provider. An Approach for Regional Virtual Communities

Abstract: Existing applications in visualization of blogs and online conversations have largely focused on the discovery of social networks and knowledge flows in the online space. At the same time, another class of visual spatial applications allows us to uncover the relationships between people and real space. This can be used to infer how individual and collective behaviour is not only a function of social contexts but also spatially situated in real physical environments. This paper presents a socio-technical approach and a prototype public tool integrating these two aspects in a synergetic way: (1) using social software to facilitate community forming in specific regional, spatial application scenarios and (2) eliciting new knowledge from community-base interaction processes in spatial settings. The prototypical implementation in a case-study in Upper Austria demonstrates how the developed sociotechnical tool enables new forms of cross-municipal communication and interaction which blend together physical, informational and social dimension of space

Decreased biomechanical stability of bones from <i>Col1a1-Krm2</i> transgenic mice.

<p>(A) µCT scanning of the vertebral bodies L6 from 24 weeks old female wildtype and <i>Col1a1-Krm2</i> transgenic littermates (scale bars, 1 mm). (B) Microcompression testing revealed reduced biomechanical stability (Fmax, maximal force until bone failure). Bars represent mean ± SD (n = 6). Asterisks indicate statistically significant differences. (C) µCT scanning of the femora showing reduced cortical thickness and impaired mineralization (scale bars, 500 µm). (D) Cortical thickness (C.Th.) and bone mineral density (vBMD) are decreased in <i>Col1a1-Krm2</i> transgenic mice compared to wildtype littermates. Bars represent mean ± SD (n = 6). Asterisks indicate statistically significant differences. (E) Three-point bending assays reveal reduced biomechanical competence of transgenic femora. Bars represent mean ± SD (n = 12). Asterisks indicate statistically significant differences. (F) Xray analysis of a 30 weeks old male <i>Col1a1-Krm2</i> transgenic mouse with a spontaneous tibia fracture (arrow) compared to a non-transgenic littermate (scale bars, 2 mm).</p

Increased bone formation in <i>Krm2</i>-deficient mice.

<p>(A) Von Kossa/van Gieson staining of non-decalcified vertebral body sections from 24 weeks old female wildtype (<i>Krm2^+/+</i>) and <i>Krm2</i>-deficient (<i>Krm2^−/−</i>) mice (scale bars, 1 mm). (B) Fluorescent micrographs showing a higher number of calcein-labelled surfaces in <i>Krm2</i>-deficient vertebral bodies (scale bars, 200 µm). (C) Histomorphometric quantification of the trabecular bone volume and osteoclast surface per bone surface (OcS/BS). (D) Histomorphometric quantification of the osteoblast surface per bone surface (ObS/BS) and the bone formation rate. (E) Von Kossa/van Gieson staining of non-decalcified tibia sections from 24 weeks old female wildtype and <i>Krm2</i>-deficient mice (scale bars, 1 mm). (F) Histomorphometric quantification of the trabecular bone volume and the trabecular number. All bars represent mean ± SD (n = 6). Asterisks indicate statistically significant differences.</p

<i>Krm2</i> expression in osteoblasts.

<p>(A) RT-PCR expression analysis of <i>Dkk</i>, <i>Krm</i> and <i>Rspo</i> genes in primary osteoblasts (Obl. d5, non-mineralized, Obl. d25, mineralized) and various tissues of 6 weeks old mice. (B) RT-PCR expression analysis of <i>Krm</i> genes in non-differentiated MC3T3-E1 cells and tissues of newborn mice. (C) Immunohistochemistry on human bone sections reveals that KRM2 is present on osteoblasts lining the trabecular bone surface (arrows, scale bars, 100 µm). The bottom panel shows staining of osteoclasts (scale bars, 20 µm). (D) DNA transfection in MC3T3-E1 cells using expression plasmids for Wnt1, Wnt2 or Wnt3, Dkk1 and/or Krm2 at the indicated combinations. Bars represent mean ± SD of three independent experiments (n = 9). Asterisks indicate statistically significant changes.</p

Impaired bone formation in <i>Col1a1-Krm2</i> transgenic mice.

<p>(A) Toluidine blue staining of non-decalcified vertebral body sections from 6 weeks old female wildtype and <i>Col1a1-Krm2</i> transgenic littermates revealing that the normal appearance of cuboidal osteoblasts (arrows) covering trabecular bone surfaces is only observed in wildtype controls (scale bars, 50 µm). (B) Histomorphometric quantification showing that the number of osteoblasts (ObN/BPm, osteoblast number per bone perimeter) is not significantly decreased in sections from transgenic mice. (C) Fluorescent micrographs showing that overall calcein labeling is reduced in vertebral bodies of 6 weeks old female <i>Col1a1-Krm2</i> transgenic mice (top, scale bars, 1 mm), as is the distance between the labeled surfaces at endosteal bone surfaces of the tibia (bottom, scale bars, 20 µm). (D) Histomorphometric quantification of the bone formation rate (BFR/BS, bone formation rate per bone surface) in female wildtype and <i>Col1a1-Krm2</i> transgenic littermates. (E) Northern Blot expression analysis for <i>Col1a1</i>, <i>Bglap</i> and <i>Ibsp</i> using femur RNA of 6 weeks old female wildtype and transgenic mice. (F) Serum levels of osteocalcin and activities of alkaline phosphatase in 6 weeks old female wildtype and transgenic mice. All bars represent mean ± SD (n = 6). Asterisks indicate statistically significant differences.</p