Protective effects of lipocalin-2 (LCN2) in acute liver injury suggest a novel function in liver homeostasis

Lipocalin-2 is expressed under pernicious conditions such as intoxication, infection, inflammation and other forms of cellular stress. Experimental liver injury induces rapid and sustained LCN2 production by injured hepatocytes. However, the precise biological function of LCN2 in liver is still unknown. In this study, LCN2^?/? mice were exposed to short term application of CCl₄, lipopolysaccharide and Concanavalin A, or subjected to bile duct ligation. Subsequent injuries were assessed by liver function analysis, qRT-PCR for chemokine and cytokine expression, liver tissue Western blot, histology and TUNEL assay. Serum LCN2 levels from patients suffering from liver disease were assessed and evaluated. Acute CCl₄ intoxication showed increased liver damage in LCN2^?/? mice indicated by higher levels of aminotransferases, and increased expression of inflammatory cytokines and chemokines such as IL-1β, IL-6, TNF-α and MCP-1/CCL2, resulting in sustained activation of STAT1, STAT3 and JNK pathways. Hepatocytes of LCN2^?/? mice showed lipid droplet accumulation and increased apoptosis. Hepatocyte apoptosis was confirmed in the Concanavalin A and lipopolysaccharide models. In chronic models (4 weeks bile duct ligation or 8 weeks CCl₄ application), LCN2^?/? mice showed slightly increased fibrosis compared to controls. Interestingly, serum LCN2 levels in diseased human livers were significantly higher compared to controls, but no differences were observed between cirrhotic and non-cirrhotic patients. Upregulation of LCN2 is a reliable indicator of liver damage and has significant hepato-protective effect in acute liver injury. LCN2 levels provide no correlation to the degree of liver fibrosis but show significant positive correlation to inflammation instead.     

The Holstein Friesian Lethal Haplotype 5 (HH5) Results from a Complete Deletion of TBF1M and Cholesterol Deficiency (CDH) from an ERV-(LTR) Insertion into the Coding Region of APOB

BackgroundWith the availability of massive SNP data for several economically important cattle breeds, haplotype tests have been performed to identify unknown recessive disorders. A number of so-called lethal haplotypes, have been uncovered in Holstein Friesian cattle and, for at least seven of these, the causative mutations have been identified in candidate genes. However, several lethal haplotypes still remain elusive. Here we report the molecular genetic causes of lethal haplotype 5 (HH5) and cholesterol deficiency (CDH). A targeted enrichment for the known genomic regions, followed by massive parallel sequencing was used to interrogate for causative mutations in a case/control approach.MethodsTargeted enrichment for the known genomic regions, followed by massive parallel sequencing was used in a case/control approach. PCRs for the causing mutations were developed and compared to routine imputing in 2,100 (HH5) and 3,100 (CDH) cattle.ResultsHH5 is caused by a deletion of 138kbp, spanning position 93,233kb to 93,371kb on chromosome 9 (BTA9), harboring only dimethyl-adenosine transferase 1 (TFB1M). The deletion breakpoints are flanked by bovine long interspersed nuclear elements Bov-B (upstream) and L1ME3 (downstream), suggesting a homologous recombination/deletion event. TFB1M di-methylates adenine residues in the hairpin loop at the 3’-end of mitochondrial 12S rRNA, being essential for synthesis and function of the small ribosomal subunit of mitochondria. Homozygous TFB1M^-/- mice reportedly exhibit embryonal lethality with developmental defects. A 2.8% allelic frequency was determined for the German HF population. CDH results from a 1.3kbp insertion of an endogenous retrovirus (ERV2-1-LTR_BT) into exon 5 of the APOB gene at BTA11:77,959kb. The insertion is flanked by 6bp target site duplications as described for insertions mediated by retroviral integrases. A premature stop codon in the open reading frame of APOB is generated, resulting in a truncation of the protein to a length of only <140 amino acids. Such early truncations have been shown to cause an inability of chylomicron excretion from intestinal cells, resulting in malabsorption of cholesterol. The allelic frequency of this mutation in the German HF population was 6.7%, which is substantially higher than reported so far. Compared to PCR assays inferring the genetic variants directly, the routine imputing used so far showed a diagnostic sensitivity of as low as 91% (HH5) and 88% (CDH), with a high specificity for both (≥99.7%).ConclusionWith the availability of direct genetic tests it will now be possible to more effectively reduce the carrier frequency and ultimately eliminate the disorders from the HF populations. Beside this, the fact that repetitive genomic elements (RE) are involved in both diseases, underline the evolutionary importance of RE, which can be detrimental as here, but also advantageous over generations.     

P2X7-mediated ATP secretion is accompanied by depletion of cytosolic ATP

Purinergic Signalling - ATP and its metabolites are important extracellular signal transmitters acting on purinergic P2 and P1 receptors. Most cells can actively secrete ATP in response to a...     

Improvement of femoral bone-cement adhesion in cemented revision hip arthroplasty by application of an amphiphilic bonder in a dynamic femur expulsion testing in vitro ]

Cemented femoral stems have shown decreased longevity compared to cementless implants in hip revision arthroplasty. The aim of this study was to evaluate the effect of an amphiphilic bonder on bone cement stability in a biomechanical femur expulsion test. A simplified hip simulator test setup with idealised femur stem specimens was carried out. The stems were implanted into bovine femurs (group 1: no bonder, n=10; group 2: bonder including glutaraldehyde, n=10; group 3: bonder without glutaraldehyde, n=10). A dynamic loading (maximum load: 800 N; minimum load: 100 N; frequency: 3 Hz; 105 cycles) was performed. Subsequently, the stem specimens were expulsed axially out of their implant beds and maximum load at failure was recorded. The static controls showed a mean maximum load to failure of 4123 N in group 1, 8357.5 N in group 2 and 5830.8 N in group 3. After dynamic loading, the specimens of group 2 reached the highest load to failure (8191.5 N), followed by group 3 (5649.5 N) and group 1 (3462 N), respectively. In group 2, we observed nine periprosthetic fractures at a load of 8400 N without signs of interface loosening. Application of an amphiphilic bonder led to a significant improvement of bonding stability, especially when glutaraldehyde was added to the bonder. This technique might offer an increased longevity of cemented femur revision stems in total hip replacement.     

Investigation of alpha-synuclein fibril structure by site-directed spin labeling

The misfolding and fibril formation of alpha-synuclein plays an important role in neurodegenerative diseases such as Parkinson disease. Here we used electron paramagnetic resonance spectroscopy, together with site-directed spin labeling, to investigate the structural features of alpha-synuclein fibrils. We generated fibrils from a total of 83 different spin-labeled derivatives and observed single-line, exchange-narrowed EPR spectra for the majority of all sites located within the core region of alpha-synuclein fibrils. Such exchange narrowing requires the orbital overlap between multiple spin labels in close contact. The core region of alpha-synuclein fibrils must therefore be arranged in a parallel, in-register structure wherein same residues from different molecules are stacked on top of each other. This parallel, in-register core region extends from residue 36 to residue 98 and is tightly packed. Only a few sites within the core region, such as residues 62-67 located at the beginning of the NAC region, as well as the N- and C-terminal regions outside the core region, are significantly less ordered. Together with the accessibility measurements that suggest the location of potential beta-sheet regions within the fibril, the data provide significant structural constraints for generating three-dimensional models. Furthermore, the data support the emerging view that parallel, in-register structure is a common feature shared by a number of naturally occurring amyloid fibrils.